Methods for assessing a health condition in a dog based on one or more biomarkers and methods for addressing mortality risks identified by a health condition - Patents.com

Abstract

The present invention is a method for assessing the risk of mortality and / or healthy life expectancy probability of a dog, said method comprising the step of measuring the value of one or more biomarkers in one or more samples obtained from the dog, said one or more biomarkers being selected from white blood cell count, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum globulins, serum calcium, platelet count, and / or red blood cell count.

Description

Technical Field

[0001]

[0001] The present invention relates to a method for evaluating the health status of dogs, particularly the mortality risk and / or the probability of a longer healthy lifespan, based on biomarkers.

Background Art

[0002]

[0002] The ability to evaluate information regarding the health of dogs is desirable for providing information about the health status and well-being of dogs. For example, being able to evaluate information regarding the health of dogs can also be useful for determining whether a dog will benefit from an intervention based on diet or nutritional supplements.

[0003]

[0003] Chronological age is known to be a major indicator of health status, and an increase in chronological age is associated with a decline in health. However, due to genetics, nutrition, and lifestyle habits, an individual may age slower or faster than their chronological age. Therefore, chronological age may not necessarily reflect the aging rate or the risk of health decline of an individual. On the other hand, the biological age of an individual (e.g., based on scales of clinical biochemistry and cell biology) can be different compared to other individuals of the same chronological age. Methods for evaluating biological age can be useful for identifying individuals who are at risk of age-related disorders earlier than expected based on their chronological age (see, for example, International Publication No. 2019 / 165064).

[0004]

[0004] However, there is a need for further methods for evaluating information regarding the health of dogs.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

[0006]

[0005] The present invention relates to a method for quantifying the health status of dogs based on biomarkers, particularly blood biomarkers. The method of the present invention enables the calculation of the mortality risk and / or the probability of a healthy lifespan of a dog by evaluating the phenotypic age of the dog.

[0007]

[0006] Existing methods for calculating the health status of dogs using blood biomarkers determine the biological age based on the correlation between the blood biomarker and the calendar age. Calculating the biological age of an animal can involve evaluating the difference in the values of one or more biomarkers compared to the predicted values of the biomarker at a given calendar age. Thus, such methods are based on using the calendar age as a major indicator of overall health.

[0008]

[0007] In contrast, the present invention takes into account the direct predictive value of the described biomarkers in terms of mortality risk and / or probability of a healthy lifespan. By way of example, a given biomarker may not be directly correlated with the calendar age but may indicate a specific pathological condition, and thus may indicate an increased mortality risk and / or an increased probability of a reduced healthy lifespan. Thus, the method of the present invention can be described as identifying the phenotypic age (PhenoAge) of the dog. Thus, the biomarkers of the present invention are not necessarily correlated with the calendar age and are related to the difference between the phenotypic age and the calendar age of the dog.

[0009]

[0008] The present invention provides a method for evaluating the mortality risk and / or the probability of a healthy lifespan of a dog, the method comprising the step of measuring the values of one or more biomarkers in one or more samples obtained from the dog.

[0010]

[0009] In one aspect, the present invention provides a method for evaluating the risk of death and / or the probability of healthy lifespan in dogs. The method includes the step of measuring the values of one or more biomarkers in one or more samples obtained from a dog, and the one or more biomarkers are selected from the number of white blood cells, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum globulin, serum calcium, platelet count, and / or red blood cell count.

[0011]

[0010] The inventors have shown that each of these biomarkers correlates individually with the phenotypic age and thus serves as a predictor of the risk of death and / or the probability of healthy lifespan.

[0012]

[0011] Evaluating the risk of death can refer to assessing the likelihood that a dog will survive for a longer or shorter period compared to, for example, equivalent dogs of the same chronological age, gender, and breed. Thus, the method of the present invention can evaluate, for example, the healthy period or the probability of healthy lifespan of a dog compared to equivalent dogs of the same chronological age, gender, and breed. Preferably, the risk of death can be equivalent to the probability of healthy lifespan of the dog, a decrease in the risk of death can be equivalent to an increase in the long-term healthy lifespan probability of the dog, or an increase in the risk of death can be equivalent to a decrease in the long-term healthy lifespan probability of the dog. The risk of death can be expressed as the difference between the phenoage and the chronological age of the dog (phenoage advance). For example, an increase in phenoage compared to chronological age can indicate an increase in the risk of death of the dog, whereas a decrease in phenoage compared to chronological age can indicate a decrease in the risk of death of the dog.

[0013]

[0012] In one embodiment, the method of the present invention may include the step of measuring the white blood cell count in one or more samples obtained from a dog. The method of the present invention may further include the step of measuring the value of one or more biomarkers selected from serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum globulin, serum calcium, platelet count, and / or red blood cell count in one or more samples.

[0014]

[0013] In one embodiment, the method of the present invention may include the step of measuring the serum albumin value in one or more samples obtained from a dog. The method of the present invention may further include the step of measuring the value of one or more biomarkers selected from white blood cell count, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum globulin, serum calcium, platelet count, and / or red blood cell count in one or more samples.

[0015]

[0014] In one embodiment, the method of the present invention may include the step of measuring the serum alkaline phosphatase value in one or more samples obtained from a dog. The method of the present invention may further include the step of measuring the value of one or more biomarkers selected from white blood cell count, serum albumin, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum globulin, serum calcium, platelet count, and / or red blood cell count in one or more samples.

[0016]

[0015] In one embodiment, the method of the present invention may include the step of measuring the serum creatine kinase value in one or more samples obtained from a dog. The method of the present invention may further include the step of measuring the value of one or more biomarkers selected from white blood cells, serum albumin, serum alkaline phosphatase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum globulin, serum calcium, platelet count, and / or red blood cell count in one or more samples.

[0017]

[0016] In one embodiment, the method of the present invention may include the step of measuring the hemoglobin value in one or more samples obtained from a dog. The method of the present invention may further include the step of measuring the value of one or more biomarkers selected from white blood cells, serum albumin, serum alkaline phosphatase, serum creatine kinase, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum globulin, serum calcium, platelet count, and / or red blood cell count in one or more samples.

[0018]

[0017] In one embodiment, the method of the present invention may include the step of measuring the hematocrit value in one or more samples obtained from a dog. The method of the present invention may further include the step of measuring the value of one or more biomarkers selected from white blood cells, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum globulin, serum calcium, platelet count, and / or red blood cell count in one or more samples.

[0019]

[0018] In one embodiment, the method of the present invention may include a step of measuring the mean corpuscular hemoglobin value in one or more samples obtained from a dog. The method may further include a step of measuring the value of one or more biomarkers selected from white blood cells, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, serum glucose, mean corpuscular volume, serum globulin, serum calcium, platelet count, and / or red blood cell count in the one or more samples.

[0020]

[0019] In one embodiment, the method of the present invention may include a step of measuring the serum glucose value in one or more samples obtained from a dog. The method of the present invention may further include a step of measuring the value of one or more biomarkers selected from white blood cells, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin concentration, mean corpuscular volume, serum globulin, serum calcium, platelet count, and / or red blood cell count in the one or more samples.

[0021]

[0020] In one embodiment, the method of the present invention may include a step of measuring the mean corpuscular volume in one or more samples obtained from a dog. The method of the present invention may further include a step of measuring the value of one or more biomarkers selected from white blood cells, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin concentration, serum glucose, serum globulin, serum calcium, platelet count, and / or red blood cell count in the one or more samples.

[0022]

[0021] In one embodiment, the method of the present invention may include the step of measuring serum globulin levels in one or more samples obtained from a dog. The method of the present invention may further include the step of measuring the levels of one or more biomarkers selected from white blood cells, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum calcium, platelet count, and / or red blood cell count in one or more samples.

[0023]

[0022] In one embodiment, the method of the present invention may include the step of measuring serum calcium levels in one or more samples obtained from a dog. The method of the present invention may further include the step of measuring the levels of one or more biomarkers selected from white blood cells, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum globulin, platelet count, and / or red blood cell count in one or more samples.

[0024]

[0023] In one embodiment, the method of the present invention may include the step of measuring platelet count in one or more samples obtained from a dog. The method of the present invention may further include the step of measuring the levels of one or more biomarkers selected from white blood cells, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum globulin, serum calcium, and / or red blood cell count in one or more samples.

[0025]

[0024] In one embodiment, the method of the present invention may include the step of measuring the number of red blood cells in one or more samples obtained from a dog. The method of the present invention may further include the step of measuring the values of one or more biomarkers selected from white blood cell count, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum globulin, serum calcium, and / or platelet count in one or more samples.

[0026]

[0025] In one embodiment, the one or more biomarkers may include white blood cell count, serum albumin, and serum alkaline phosphatase. Advantageously, it has been concluded that this combination of three biomarkers provides a significant prediction of the risk of death and / or the probability of healthy life expectancy. The predictive ability can be further improved by incorporating one or more additional biomarkers selected from serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum globulin, serum calcium, platelet count, and red blood cell count.

[0027]

[0026] In one embodiment, the one or more samples are derived from blood, such as a plasma sample.

[0028]

[0027] In one embodiment, the present invention provides a method for evaluating the risk of death and / or the probability of healthy life expectancy of a dog, the method comprising: a. measuring the values of the following biomarkers, white blood cell count, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, and serum globulin, in one or more samples obtained from a dog; and b. evaluating the phenotypic age (phenoage) of the dog using formula (1).

Number

[0029]

[0030] In the formula, xb is the sum of multiplying each value of the biomarker, gender, and breed by its respective coefficient according to formula (2).

Number

[0030] Gender is coded as a numerical value with female being 0 and male being 1. Breed is coded as a numerical value with small breed being 0 and medium breed being 1. The phenotypic age is used to evaluate the dog's risk of death and / or probability of healthy lifespan.

[0031]

[0034] Preferably, evaluating that the dog's phenoage exceeds the calendar age indicates a higher risk of death. Preferably, evaluating that the dog's phenoage is less than the calendar age indicates a reduced risk of death. Preferably, evaluating that the dog's phenoage exceeds the calendar age indicates a decrease in the long-term healthy lifespan probability. Preferably, evaluating that the dog's phenoage is less than the calendar age indicates an increase in the long-term healthy lifespan probability.

[0032]

[0035] The present invention further relates to a method of selecting a lifestyle or diet plan for a dog, the method comprising: a. performing the method described in the first aspect of the present invention; b. selecting an appropriate lifestyle or diet plan based on the risk of death and / or probability of healthy lifespan evaluated in step a.

[0033]

[0038] The present invention further provides a method for evaluating the effectiveness of a lifestyle or diet plan for reducing the risk of death and / or increasing the probability of a healthy lifespan in dogs, the method comprising: a. performing the method described in the first aspect of the present invention; b. applying a change in lifestyle or diet plan to the dog; c. after the period during which the change in lifestyle or diet plan is applied, performing the method described in the first aspect of the present invention; and d. determining whether there has been a change in the risk of death and / or the probability of a healthy lifespan in the dog between step a and step c.

[0034]

[0039] The present invention further provides a dietary intervention for use in reducing the risk of death and / or increasing the probability of a healthy lifespan in dogs, the dietary intervention being performed on dogs whose risk of death has been evaluated by the method of the present invention.

[0035]

[0040] The present invention provides for the use of a dietary intervention for reducing the risk of death evaluated for a dog and / or increasing the probability of a healthy lifespan in the dog, the dietary intervention being performed on a dog whose risk of death and / or probability of a healthy lifespan has been evaluated by the method of the present invention.

[0036]

[0041] The present invention further provides a computer-readable medium comprising instructions that, when executed, cause one or more processors to perform the method of the present invention.

[0037]

[0042] The present invention also provides a computer system for evaluating the risk of death and / or the probability of a healthy lifespan of a dog, the computer system comprising the steps of: given the values of one or more biomarkers of a sample from a dog, evaluating the risk of death and / or the probability of a healthy lifespan of the dog, wherein the one or more biomarkers are selected from the number of white blood cells, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume; serum globulin, serum calcium, platelet count, and / or the number of red blood cells, and generating a report of the risk of death and / or the probability of a healthy lifespan of the dog, and is programmed to perform one or more of the above steps.

[0038]

[0043] The present invention further provides a computer program product, the computer program product comprising computer-executable instructions for causing a programmable computer to evaluate the risk of death and / or the probability of a healthy lifespan of a dog given the values of one or more biomarkers from the dog, wherein the one or more biomarkers are selected from the number of white blood cells, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume; serum globulin, serum calcium, platelet count, and / or the number of red blood cells.

[0039]

[0044] Advantageously, embodiments of the present invention in which the values of multiple biomarkers are measured / evaluated enable the evaluation of the risk of death and / or the probability of a healthy lifespan based on markers of multiple organ systems and functions. Thus, the use of multiple biomarkers advantageously encompasses various possible organ dysfunctions.

[0040]

[0045] By evaluating the risk of death or probability of survival of a dog, it becomes possible to examine several aspects of the animal's well-being. First, the measurement / evaluation can predict whether this animal is more likely to require an intervention based on diet or nutritional supplements. Second, the measurement / evaluation can be used to examine the effectiveness of an intervention based on diet or nutritional supplements against aging. A specific set of biomarkers is also provided that can be examined to evaluate the risk of death or probability of survival of an animal and that is routinely examined in veterinary clinics or practices.

Brief Description of the Drawings

[0041]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Best Mode for Carrying Out the Invention

[0042]

[0053] Next, various preferred features and embodiments of the present invention will be described by way of non-limiting examples. It should be understood by those skilled in the art that all features of the present invention disclosed herein can be combined without departing from the scope of the present invention disclosed.

[0043]

[0054] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0044]

[0055] As used herein, the terms "comprising", "comprises", and "comprised of" are synonymous with "including", "includes", or "containing", "contains", and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. The terms "comprising", "comprises", and "comprised of" also include the term "consisting of".

[0045]

[0056] A numerical range includes the numbers defining the range.

[0046]

[0057] The publications discussed herein are provided solely because they were publicly available prior to the filing date of this application. No admission is to be construed that any of the documents cited herein constitutes prior art to the claims appended hereto.

[0047]

[0058] The methods and systems disclosed herein can be used by veterinarians, medical professionals, laboratory technicians, pet care providers, and the like.

[0048]

[0059] Subject

[0049]

[0060] The method is directed to canine subjects. Thus, the subject of the present invention is a dog.

[0050]

[0061] Preferably, the dog may be a dog having a body condition score (BCS) of at least 7 (e.g., 9-point scale by the American Animal Hospital Association (AAHA); AAHA guidelines; using the Nutritional Assessment Body and Muscle Condition Score).

[0051]

[0062] Breed

[0052]

[0063] The method of the present invention can also utilize information regarding the breed of the dog. The dog may be classified, for example, as a toy dog breed, small dog breed, medium dog breed, large dog breed, or giant dog breed. Preferably, the breed of the dog may be classified based on the weight of the dog. Preferably, the breed of the dog may be classified based on the average weight of the dog for a given breed.

[0053]

[0064] The dog may be classified based on genetic information obtained for the dog by DNA sequencing or SNP detection. For example, the dog may be classified based on the clade of the breed to which it belongs.

[0054]

[0065] In addition to, or instead of, the foregoing, the dog may be a hybrid. The hybrid dog may be classified based on the weight of the dog. For example, the hybrid dog can be classified as small, medium, large, or giant.

[0055]

[0066] In addition to, or instead of, the mongrel dog may be classified based on genetic information such as, for example, a DNA sequence, SNP, haplotype, or haplogroup. For example, the mongrel dog can be classified based on the closest breed or clade by genetic analysis.

[0056]

[0067] In addition to, or instead of, genetic information known to be related to breed identity, such as, for example, a DNA sequence, SNP, haplotype, or haploblock, can be used to classify mongrel or purebred dogs.

[0057]

[0068] In addition to, or instead of, genetic information known to be related to lifespan, such as, for example, a DNA sequence, SNP, haplotype, or haploblock, can be used to classify mongrel or purebred dogs.

[0058]

[0069] In addition to, or instead of, mongrel or purebred dogs can be classified as a robust type or an athletic type based on morphometric measurements (e.g., both are incorporated herein by reference in their entirety, U.S. Patent No. 8,091,509 entitled "Method for improving dog food" by Perez-Camargo et al. or U.S. Patent Application Publication No. 2017 / 0042194 entitled "Methods using morphometric measurements of a small dog to improving food for the small dog" by Bouthegourd et al.).

[0059]

[0070] Preferably, the dog may be classified as a small breed or a medium breed. Preferably, the classification is determined by the average weight of an adult dog of this breed. Preferably, a breed with an average weight of less than 10 kg is classified as a small breed and / or a breed with an average weight exceeding 10 kg is classified as a medium breed.

[0060]

[0071] Gender

[0061]

[0072] Preferably, the gender of the dog may be classified as male or female.

[0062]

[0073] Calendar age

[0063]

[0074] The calendar age may be defined as the amount of time elapsed from the birth of the subject to a given date. The calendar age may be expressed in years, months, days, etc.

[0064]

[0075] Preferably, the method of the present invention can be applied to dogs of any calendar age. In certain embodiments, the dog may be at least about 2 years old. Preferably, the dog may be at least about 2 years old, at least about 3 years old, at least about 4 years old, at least about 5 years old, at least about 6 years old, at least about 7 years old, at least about 8 years old, at least about 9 years old, or at least about 10 years old.

[0065]

[0076] Preferably, the dog may be at least about 7 years old.

[0066]

[0077] Sample

[0067]

[0078] The present invention includes the step of measuring the value of one or more biomarkers in one or more samples obtained from a subject.

[0068]

[0079] Preferably, the sample is derived from blood. The sample may contain a blood fraction or may be whole blood. The sample preferably includes plasma or serum. Techniques for collecting samples from a subject are well known in the art.

[0069]

[0080] An appropriate sample can be selected based on the biomarker to be measured. By way of example, if the biomarker is measured using a complete blood count (cbc) as part of, for example, a standard clinical complete blood count, a whole blood sample should be used.

[0070]

[0081] The method of the present invention may be performed on one or more samples obtained from a subject. For example, the method of the present invention may be performed using a first sample obtained at a given time point and a second sample obtained after a time interval after the first sample was obtained. The method of the present invention may be performed two or more times on samples obtained from the same dog over a period of time. For example, the samples may be obtained repeatedly once a month, once a year, or once every two years. Preferably, the samples may be obtained approximately once a year (e.g., during an annual veterinary health examination). This can be useful, for example, in evaluating the effect of a particular treatment, such as a dietary intervention or a change in exercise therapy, or the effect of a change in lifestyle habits.

[0071]

[0082] In one embodiment, the value of one or more biomarkers may be measured before a change in lifestyle habits (e.g., an intervention with a dietary product or a change in exercise therapy). In another embodiment, the value of one or more biomarkers may be measured, for example, before and after an intervention with a dietary product or a change in exercise therapy. The value of the biomarker may also be measured, for example, at predetermined time points during an intervention period with a dietary product or during a period of change in exercise therapy. These predetermined time points may be, for example, periodic during an intervention period with a dietary product or during a period of change in exercise therapy, for example, daily or every three days, or may vary depending on the subject being examined.

[0072]

[0083] Measurement of the value of one or more biomarkers in a sample

[0073]

[0084] The biomarkers used in the present invention can be measured using standard methods in the art and are typically measured as part of a standard blood test to evaluate the disease state of an animal. For example, biomarkers are generally measured as part of a standard complete blood count (cbc) and a standard clinical blood chemistry analysis.

[0074]

[0085] A complete blood count provides information about blood cells and their characteristics, such as red blood cells, white blood cells, and platelets. An exemplary complete blood count can include an automated process that uses flow cytometry or a Coulter counter to measure the number of cells in the blood. Such an automated system may also be able to measure other blood biomarker readings depending on their complexity in addition to measuring the cell count. Such systems can simultaneously measure blood cell counts, as well as red blood cell volume, hemoglobin value, mean corpuscular hemoglobin value, and hematocrit. For example, IDEXX Laboratories offers a blood analyzer that can measure white blood cell count (WBC), red blood cell count (RBC), platelet count (PLT), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) (IDEXX Laboratories Inc., ProCyte Dx Hematology Analyzer).

[0075]

[0086] Values of other biomarkers not related to blood cells, such as serum proteins / enzymes and / or molecular biomarkers, can be measured using chemical tests, particularly using an automated chemistry analyzer system. These methods can utilize a colorimetric-based approach for quantification. For example, IDEXX Laboratories offers an automated chemistry analyzer that can quantify serum albumin, serum alkaline phosphatase, serum creatine kinase, serum glucose, serum globulin, and serum calcium (IDEXX Laboratories Inc., Catalyst One Chemistry Analyzer).

[0076]

[0087] Thus, the method for measuring the value of a biomarker used in the present invention may include an assay that results in a change in spectrophotometry (e.g., a chemical change or an antibody-binding change that results in a detectable signal at a specific wavelength). Such assays can be highly automated and efficient and form the basis of many routine veterinary health diagnostics.

[0077]

[0088] Preferably, the value of the biomarker may be evaluated after an overnight fast and measured according to standard veterinary clinical practice.

[0078]

[0089] The value of an individual biomarker in a sample can be measured or evaluated by any suitable method known in the art. For example, mass spectrometry (MS), antibody detection methods such as enzyme-linked immunosorbent assay (ELISA), a matrix by a non-antibody protein (e.g., a matrix by fibronectin), radioimmunoassay (RIA), or aptamers can be used. Other spectroscopic methods, chromatographic methods, labeling techniques, or quantitative chemical methods may also be used.

[0079]

[0090] Antibodies suitable for use in the above methods are known in the art and / or can also be produced using known techniques. Suitable assay methods for detecting antibody values include, but are not limited to, immunoassay methods such as enzyme-linked immunosorbent assay, radioimmunoassay, Western blot, and immunoprecipitation.

[0080]

[0091] Biomarker

[0081]

[0092] White blood cell count

[0082]

[0093] White blood cells, also denoted as leukocytes, are a type of cells found in the blood. These cells have various immune-related functions depending on their subtypes: monocytes, lymphocytes, neutrophils, basophils, and eosinophils. White blood cells contain a nucleus and have variable cell shapes depending on the subtype. The white blood cell count is the number of these types of cells per volume of blood.

[0083]

[0094] Methods for measuring the white blood cell count, typically expressed as thousands of cells per microliter (10^3 / μL), are known in the art. The measurement of the white blood cell count can be performed manually on a blood smear using staining and microscopy techniques, but can also be performed as part of an automated complete blood count (CBC). IDEXX Laboratories offers automated blood analyzers capable of measuring the white blood cell count.

[0084]

[0095] Preferably, an increase in the white blood cell count may be associated with a negative effect on the reduction of the risk of death. Thus, an increase in the white blood cell count may be associated with an increase in the risk of death.

[0085]

[0096] Serum albumin

[0086]

[0097] Serum albumin is a globular protein found in the blood. Serum albumin is a 65 kDa protein consisting of three homologous domains. Albumin regulates colloid osmotic pressure, prevents the diffusion of fluid (loss of fluid) from the blood to the tissues, and functions as a transport protein for fatty acids, bilirubin, heme, heavy metals, hormones, and certain drugs. Albumin is very abundant in the blood, accounting for 25 - 50% by weight of total plasma proteins, and is produced by the liver. Abnormally high or low values of albumin in the blood may indicate liver or kidney disease.

[0087]

[0098] Methods for measuring serum albumin values, typically expressed in grams per deciliter (g / dL), are well known in the art and include zone electrophoresis, dye-binding assays including bromocresol green (BCG) or bromocresol purple (BCP), and ELISA methods. For example, Eagle Biosciences offers an ELISA-based assay for canine serum albumin (Eagle Biosciences Inc., product code SKU: CAE49-K01). Stokol et al. described the use of an automated system using a BCG-binding assay for measuring serum albumin values in dogs in a clinical setting (Vet Clin Pathol. 2001;30(4):170-176). Additionally, IDEXX Laboratories offers an automated chemistry analyzer capable of measuring serum albumin values as part of a combined blood test.

[0088]

[0099] Preferably, an increase in serum albumin value may be associated with a positive effect on reducing the risk of death. Thus, an increase in serum albumin value may be associated with a reduction in the risk of death.

[0089]

[0100] Serum alkaline phosphatase

[0090]

[0101] Alkaline phosphatase (ALP) is an enzyme that plays an important role in liver metabolism and skeletal development. Alkaline phosphatase is an 86 kDa homodimeric protein. High levels of this protein in the blood may indicate liver damage or bone disease.

[0091]

[0102] Methods for measuring serum alkaline phosphatase values, typically expressed in units per liter (U / L), are well known in the art and most often consist of quantifying enzyme activity by a colorimetric assay. Automated chemistry analyzers capable of measuring serum alkaline phosphatase values are available from IDEXX Laboratories.

[0092]

[0103] Preferably, an increase in serum alkaline phosphatase levels may be associated with a negative effect on the reduction of the risk of death. Thus, an increase in serum alkaline phosphatase levels may be associated with an increase in the risk of death.

[0093]

[0104] Serum creatine kinase

[0094]

[0105] Creatine kinase (CK) is an enzyme mainly found in muscle. This enzyme converts creatine and ATP into phosphocreatine for use in rapid energy production during muscle contraction. High levels of this enzyme in the blood may indicate muscle damage.

[0095]

[0106] Methods for measuring creatine kinase levels in blood, typically expressed in international units per liter (IU / L), are well known in the art and include, for example, using an enzyme assay to quantify creatine kinase levels. An automated chemistry analyzer provided by IDEXX Laboratories is an example of a commercially available tool for measuring serum creatine kinase levels.

[0096]

[0107] Preferably, an increase in creatine kinase levels may be associated with a negative effect on the reduction of the risk of death. Thus, an increase in creatine kinase levels may be associated with an increase in the risk of death.

[0097]

[0108] Hemoglobin

[0098]

[0109] Hemoglobin is a transport protein in red blood cells. Hemoglobin consists of a tetramer of two α-chains and two β-chains. Each peptide chain binds to a heme group consisting of a porphyrin ring to which an iron ion is bound. The heme group can reversibly bind oxygen, enabling hemoglobin to function as an oxygen transport carrier protein.

[0099]

[0110] Methods for measuring hemoglobin values, typically expressed in grams per deciliter (g / dL), are well known in the art. The International Committee for Standardization in Haematology has described a standardized method that utilizes spectrophotometric measurement of cyanmethemoglobin (Br J Haematol. 1967 Apr;13:71-5), and this approach can be used in commercially available automated chemical analyzers such as those provided by IDEXX Laboratories.

[0100]

[0111] Preferably, an increase in hemoglobin value may be associated with a positive effect on reducing the risk of death. Thus, an increase in hemoglobin may be associated with a reduction in the risk of death.

[0101]

[0112] Hematocrit

[0102]

[0113] Hematocrit is the volume percentage of red blood cells in the blood. Hematocrit values outside the normal range may indicate a disease or condition in which the proportion of red blood cells in the blood is higher or lower than normal. High hematocrit may indicate a condition such as dehydration, while low hematocrit may indicate anemia, hemolysis, or decreased red blood cell production.

[0103]

[0114] Methods for measuring hematocrit, typically expressed as a percentage value (%) of blood volume, are well known in the art. The measurement can be done manually using the packed cell volume by centrifuging the blood in a microhematocrit tube. Alternatively, hematocrit can be calculated from the mean corpuscular volume and the red blood cell count, both of which can be directly measured by a modern blood analyzer in a standard complete blood count (CBC).

[0104]

[0115] Preferably, an increase in hematocrit value may be associated with a positive effect on reducing the risk of death. Therefore, an increase in hematocrit may be associated with a reduction in the risk of death.

[0105]

[0116] Mean corpuscular hemoglobin

[0106]

[0117] Mean corpuscular hemoglobin (MCH) is the average amount of hemoglobin per red blood cell. MCH values outside the normal range may indicate certain diseases such as macrocytic anemia or hypochromic anemia.

[0107]

[0118] Methods for measuring MCH, typically expressed in picograms (pg), are well known in the art and generally involve calculations from observed values of hemoglobin and red blood cell counts that can be measured during a complete blood count (CBC) using a blood analyzer as described above.

[0108]

[0119] Preferably, an increase in MCH may be associated with a positive effect on reducing the risk of death. Therefore, an increase in MCH may be associated with a reduction in the risk of death.

[0109]

[0120] Serum glucose

[0110]

[0121] Serum glucose is a measure of the amount of glucose present in the blood. Blood glucose levels are regulated by hormones such as insulin, which keeps the glucose level within the normal range. If the glucose level in the blood is outside the normal value, it may indicate a disease, such as true diabetes.

[0111]

[0122] Methods for measuring serum glucose levels, typically expressed in milligrams per deciliter (mg / dL), are well known in the art. Most glucose assays are photometric, and there are many commercially available devices. For example, Acon Pharmaceuticals offers a veterinary glucose monitoring system (Acon Phramaceuticals Inc., CentriVet® GK), and Carradini et al. have described the use of a continuous glucose monitoring system device in dogs (Abbott Laboratories, FreeStyle Libre).

[0112]

[0123] Preferably, an increase in serum glucose levels may be associated with a negative effect on the reduction of the risk of death. Thus, an increase in serum glucose levels may be associated with an increase in the risk of death.

[0113]

[0124] Mean corpuscular volume

[0114]

[0125] The mean red cell volume (Mean red cell volume or mean corpuscular volume (MCV)) is a measure of the average volume of red blood cells in the blood. MCV can classify possible anemia into microcytic anemia, normocytic anemia, or macrocytic anemia, and is a diagnostic criterion that can help identify the underlying disease or disorder. High MCV may indicate disorders such as vitamin B12 deficiency, while low MCV may indicate disorders such as iron deficiency.

[0115]

[0126] Methods for calculating the mean red cell volume, typically expressed in femtoliters (fL), are well known in the art. The mean red cell volume can be calculated from measurements of other hematocrit values and red blood cell counts, but state-of-the-art hematology analyzers (e.g., the ProCyte Dx Hematology Analyzer from IDEXX Laboratories) can directly measure MCV as part of a complete blood count (CBC).

[0116]

[0127] Preferably, an increase in MCV may be associated with a positive effect on reducing the risk of death. Thus, an increase in MCV may be associated with a reduction in the risk of death.

[0117]

[0128] Serum globulin

[0118]

[0129] Serum globulin is a measure of the concentration of globular proteins in the blood. Globular proteins are mainly secreted by the liver and, to a lesser extent, by immune cells. Albumin is the most abundant serum globulin. The remaining serum globulins can be separated into multiple fractions based on their behavior in electrophoresis separation. Immunoglobulins are an important part of the immune system and are secreted by immune cells. Examples of other serum globulins are immune system proteins such as complement, hormones, and carrier proteins such as ferritin. Changes in the total serum globulin protein value can indicate a specific condition or disease. A general increase in serum globulin can indicate an infectious disease and an inflammatory immune response, whereas a decrease in value can indicate bleeding, gastrointestinal disease, or severe malnutrition.

[0119]

[0130] Methods for measuring serum globulin values, typically expressed in grams per deciliter (g / dL), are well known in the art. For example, Tothova et al. (Veterinarni Medicina, 2016, 61:475-496) describe chemical and physical methods, and automated chemical analyzers available from IDEXX can also measure serum globulin values.

[0120]

[0131] Preferably, an increase in serum globulin value may be associated with a negative effect on reducing the risk of death. Thus, an increase in serum globulin value may be associated with an increase in the risk of death.

[0121]

[0132] Serum calcium

[0122]

[0133] Serum calcium is a measure of the total concentration of calcium in the blood. Calcium in the blood can be ionized, complexed, or protein-bound. Calcium is required in the body for a variety of intracellular and extracellular functions, including muscle contraction and blood clotting, and is a major component of bone. A calcium value that is too high can be due to certain cancers or bone disorders, and a calcium value that is too low can be due to kidney disease, pancreatitis, or decreased serum albumin.

[0123]

[0134] Methods for measuring serum calcium values, typically expressed in milligrams per deciliter (mg / dL), are well known in the art. F. Gran described a colorimetric method for measuring calcium in serum (Acta Physiologica Scandinavica; 1960, 49: 192-197), and the latest automated chemical analyzers are capable of measuring serum calcium values.

[0124]

[0135] Preferably, an increase in serum calcium values can be associated with a positive effect on reducing the risk of death. Thus, an increase in serum calcium values can be associated with a reduction in the risk of death.

[0125]

[0136] Platelet count

[0126]

[0137] Platelets, also known as thrombocytes, are small cells that are components of blood. Platelets are small cells without a nucleus and are produced from the cytoplasm of bone marrow cells known as megakaryocytes. Platelets assist in the clotting process that stops bleeding at the site of blood vessel injury. Platelet numerical values above or below the normal range can indicate a disorder or disease. In particular, a decrease in platelet count can be caused by certain infections, cancers, immune system disorders, or pancreatitis.

[0127]

[0138] Methods for measuring platelet counts, typically expressed as thousands of cells per microliter (10^3 / μL), are well known in the art. Platelet counts can be performed manually on blood smears using staining and microscopic observation, but are generally performed as part of an automated complete blood count (CBC).

[0128]

[0139] Preferably, an increase in platelet count may be associated with a negative effect on reducing the risk of death. Thus, an increase in platelet count may be associated with an increase in the risk of death.

[0129]

[0140] Red blood cell count

[0130]

[0141] Red blood cells, also known as red blood corpuscles, are the most abundant cells present in the blood. Red blood cells do not contain a nucleus and instead consist mainly of hemoglobin contained within the cell membrane, and hemoglobin maximizes the oxygen-carrying capacity of red blood cells. Red blood cell counts above or below normal values indicate a disorder or disease. Low red blood cell counts may indicate hemolysis, blood loss, or decreased red blood cell production, which can result from multiple causes. High red blood cell counts may be due to dehydration or an increase in red blood cell production, indicating a relative increase in red blood cells per unit volume of blood compared to normal.

[0131]

[0142] Methods for measuring red blood cell counts, typically expressed as thousands of cells per microliter (10^3 / μL), are well known in the art. Red blood cell counts can be performed manually on blood smears by microscopic observation, but are generally performed as part of an automated complete blood count (CBC).

[0132]

[0143] Preferably, an increase in red blood cell count may be associated with a positive effect on reducing the risk of death. Thus, an increase in red blood cell count may be associated with a decrease in the risk of death.

[0133]

[0144] Combination of biomarkers

[0134]

[0145] In the method of the present invention, individual biomarkers may have predictive values, while on the other hand, the accuracy and / or predictive ability of the method of the present invention can be improved by combining the values obtained from a plurality of biomarkers.

[0135]

[0146] Accordingly, the method of the present invention may include the step of measuring the values of at least two biomarkers from the biomarkers defined herein. For example, the method of the present invention may include the step of measuring the values of two or more biomarkers selected from white blood cell count, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin amount, serum glucose, mean corpuscular volume, serum globulin, serum calcium, platelet count, and / or red blood cell count in one or more samples.

[0136]

[0147] The term "one or more biomarkers", as used herein, may include 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 11, at least 12, or at least 13 biomarkers.

[0137]

[0148] The term "one or more biomarkers" as used herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 biomarkers as described herein.

[0138]

[0149] Preferably, the method of the present invention may include the step of measuring the values of white blood cell count, serum albumin, and serum alkaline phosphatase in one or more samples. Advantageously, it has been concluded that this combination of three biomarkers provides a significant prediction of the risk of death and / or the probability of healthspan. The predictive ability can be further improved by incorporating one or more additional biomarkers selected from serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum globulin, serum calcium, platelet count, and red blood cell count.

[0139]

[0150] Preferably, the method of the present invention may include the step of measuring the respective values of white blood cell count, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum globulin, serum calcium, platelet count, and red blood cell count in one or more samples.

[0140]

[0151] Comparison with a reference or control

[0141]

[0152] The method of the present invention may further include the step of comparing the values of the individual biomarkers in the test sample with one or more reference values or control values. The reference values may be associated with a predetermined risk of death and / or the probability of healthspan. In some embodiments, the reference values are pre-obtained values for a subject or group of subjects for which the death outcome is known. The reference values may be based on the chronological age, breed, sex, and / or the average value, such as the average value or median value, from a group of subjects for which the death outcome is known. Preferably, the reference values may be based on the chronological age, breed, sex, and the average value, such as the average value or median value, from a group of subjects for which the death outcome is known.

[0142]

[0153] Combination of further measured values and / or characteristics with biomarker values

[0143]

[0154] Preferably, the method of the present invention further comprises combining the values of one or more biomarkers with one or more of the chronological age, breed, and / or gender of the dog. By combining this information, an improved model for the risk of death and / or probability of healthy lifespan of the dog is provided.

[0144]

[0155] In a preferred embodiment, the values of one or more biomarkers as defined herein are measured in a sample from a dog, and those values are combined with the chronological age, breed, and gender of the dog to assess the risk of death and / or probability of healthy lifespan of the dog.

[0145]

[0156] Preferably, the risk of death and / or probability of healthy lifespan is expressed as a phenotypic age (phenoage) and is given by the following formula.

Equation

[0146]

[0157] Wherein, xb is the sum of multiplying the values of each of the biomarker, gender, and breed by their respective coefficients according to Equation (2).

Equation

[0147] Gender is coded as a numerical value with females as 0 and males as 1, and breed is coded as a numerical value with small breeds as 0 and medium breeds as 1.

[0148]

[0160] The coefficient values of the various parameters typically depend on the units of measurement of all the variables in the model. Thus, as will be understood by those skilled in the art, the exact values of the respective coefficient values will vary depending on, for example, the number and nature of the different parameters used in the model and the nature of the training data provided. Accordingly, conventional statistical methods can be applied to the training data set to arrive at the coefficient values for use in the above formula. Such methods include, for example, calculating two Gompertz functions for a training set (e.g., when the status (alive or dead) of the dog is known), a function that models survival as a function of the selected biomarker, chronological age, breed class (small dog or medium dog), and gender (Model 1), and a second function that considers only chronological age, breed class, and gender (Model 2). These models can be fitted using the flexsurv package (v 2.1) in the R software environment.

[0149]

[0161] Preferably, a negative coefficient for a given biomarker means that the higher the value of the biomarker, the more positive the effect on the reduction of the risk of death, and / or the lower the value of the biomarker, the more negative the effect on the reduction of the risk of death. Preferably, a positive coefficient for a given biomarker means that the higher the value of the biomarker, the more negative the effect on the reduction of the risk of death, and / or the lower the value of the biomarker, the more positive the effect on the reduction of the risk of death.

[0150]

[0162] The phenotypic age can be defined as the time variable ("chronological age") at which the probability of survival of the animal given by Model 2 is equal to the probability of survival at those chronological ages given by Model 1.

[0151]

[0163] The phenotypic age of a dog (i.e., phenoage) may be expressed in years, months, days, etc.

[0152]

[0164] Preferably, the risk of death and / or the probability of healthy life expectancy are expressed as the difference between the phenoage and the chronological age of the dog. This difference may be referred to as the phenoage progression of the dog.

[0153]

[0165] For example, an increase in phenoage compared to chronological age may indicate an increase in the risk of death of the dog. For example, a decrease in phenoage compared to chronological age may indicate a decrease in the risk of death of the dog. By way of illustration, the inventors have concluded that the difference (phenoage progression) between phenoage and chronological age is associated with a significant increase in the risk of death, and the magnitude of the effect was calculated to be a hazard ratio of 1.75 for a 1-year increase in phenoage compared to chronological age (see Example 3). In other words, the inventors have concluded that when the phenoage increases by 1 year compared to the chronological age, the risk of death at any given point in life associated with this increases by 75%.

[0154]

[0166] Stratification of subjects

[0155]

[0167] The risk of death and / or the probability of healthy life expectancy evaluated by the method of the present invention can also be compared with one or more predetermined thresholds. Using such thresholds, the subjects may be stratified into categories indicating the evaluated risk of death and / or the probability of healthy life expectancy, for example, low, medium, or high evaluated risk of death. The degree of deviation from the threshold is useful for identifying which subjects will benefit most from a particular intervention. In this way, dietary interventions and lifestyle modifications can be optimized. The evaluated risk of death and / or the probability of healthy life expectancy may be presented in units of a numerical score or percentage, for example, indicating the evaluated risk of death and / or the probability of healthy life expectancy compared to a control or reference population.

[0156]

[0168] Method for selecting / monitoring the lifestyle or diet plan of a subject

[0157]

[0169] In a further aspect, the present invention provides a method of selecting a lifestyle or diet plan for a subject. Modification of the lifestyle may be any of the modifications described herein, such as, for example, a dietary intervention and / or a change in exercise therapy. Modification of the lifestyle may also be the administration of a therapeutic modality.

[0158]

[0170] The lifestyle or diet plan can be applied to a dog over any suitable period. After such a period, the risk of death and / or the probability of healthy life of the dog can be re-evaluated using the method of the present invention to assess the effectiveness of the lifestyle or diet plan for reducing the risk of death of the dog and / or increasing the probability of healthy life. By way of example, the lifestyle or diet plan may be applied for at least 2 weeks, at least 4 weeks, at least 8 weeks, at least 16 weeks, at least 32 weeks, or at least 64 weeks. The lifestyle or diet plan may be applied for at least 3 months, at least 6 months, at least 12 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months.

[0159]

[0171] The modification is preferably a dietary intervention as described herein. The term "dietary intervention" means an external factor applied to a subject that changes the subject's diet. More preferably, the dietary intervention includes at least providing a diet product or diet plan or dietary supplement.

[0160]

[0172] The dietary intervention may be a diet, a diet plan, a dietary supplement, or a dietary supplement plan.

[0161]

[0173] The dietary intervention or dietary product described in this specification may be any suitable diet plan, for example, a calorie-restricted diet, a senior diet, a low-protein diet, a phosphorus diet, a potassium-supplemented diet, a polyunsaturated fatty acid (PUFA)-supplemented diet, an antioxidant-supplemented diet, a vitamin B-supplemented diet, a liquid diet, a selenium-supplemented diet, an omega 3-6 ratio diet, or a diet supplemented with a nicotinamide precursor such as carnitine, branched-chain amino acids or derivatives, nucleotides, nicotinamide mononucleotide (MNM) or nicotinamide riboside (NR), or any combination of the above.

[0162]

[0174] Preferably, the dietary intervention or dietary product may be a calorie-restricted diet, a senior diet, or a low-protein diet. Preferably, the dietary intervention or dietary product may be a calorie-restricted diet. Preferably, the dietary intervention product or dietary product may be a low-protein diet.

[0163]

[0175] The dietary intervention can also be determined based on the dog's baseline maintenance energy requirement (MER). Preferably, the MER may be the amount of food that stabilizes the dog's body weight (change less than 5% over 3 weeks).

[0164]

[0176] As an example, it is generally understood that young growing dogs benefit from a high-energy / high-protein diet. However, since older dogs may have lower energy requirements, the diet can be appropriately modified. In particular, many manufacturers produce "senior" ranges of dog food that are low in calories, high in fiber, but have appropriate amounts of protein and fat for older dogs.

[0165]

[0177] Preferably, the calorie-restricted diet may include about 60%, about 65%, about 75%, or about 80% of the dog's MER. Preferably, the calorie-restricted diet may include about 60% or about 75% of the dog's MER.

[0166]

[0178] Preferably, the low-protein diet may contain less than 20% protein (% dry matter). For example, the low-protein diet may contain less than 15% or less than 10% protein (% dry matter).

[0167]

[0179] These diets are generally recommended based on the dog's chronological age. For example, a dog may be recommended to switch to a senior diet around 7 or 8 years old. However, in the context of the present invention, an assessment of an increase in the risk of death and / or a decrease in the probability of a healthy lifespan compared to what is expected given the dog's chronological age may enable the decision to switch the dog's diet to a senior diet at an earlier age. In contrast, dogs with a reduced risk of death and / or an increased probability of a healthy lifespan compared to their chronological age may be able to continue a high-energy / high-protein diet for a longer period.

[0168]

[0180] Dietary interventions may include foods, nutraceuticals, and / or beverages that contain nutrients and / or bioactive substances that mimic the benefits of calorie restriction (CR) without restricting the daily calorie intake. For example, the foods, nutraceuticals, and / or beverages may contain functional raw materials having benefits such as CR. Preferably, the foods, nutraceuticals, and / or beverages may contain autophagy inducers. Preferably, the foods, nutraceuticals, and / or beverages may contain fruits and / or nuts (or extracts). Suitable examples include, but are not limited to, pomegranate, strawberry, blackberry, camu camu, walnut, chestnut, pistachio, pecan. Preferably, the foods, nutraceuticals, and / or beverages may contain probiotics with or without fruit extracts or nut extracts.

[0169]

[0181] Also, the modification of the subject's lifestyle includes indicating to the subject the need to change the lifestyle, for example, instructing to exercise more. Similar to dietary interventions, an assessment of an increase in the risk of death and / or a decrease in the probability of a healthy lifespan in dogs compared to what is expected given the chronological age may enable the decision to switch the dog to a more appropriate exercise program.

[0170]

[0182] Modifying the target lifestyle also includes recommending a treatment modality or treatment plan. The treatment modality or treatment plan may be, for example, a modality useful for treating and / or preventing arthritis, dental diseases, endocrine disorders, heart diseases, diabetes, liver diseases, kidney diseases, prostate disorders, cancer, and behavioral or cognitive disorders. Preferably, preventive therapy can also be administered to dogs identified as being at risk of such disorders due to an increased risk of death (phenoage) and / or based on specific biomarkers known to be associated with disease-related pathways. In other embodiments, dogs evaluated as being at risk of a particular condition (due to an increased risk of death (phenoage) and / or based on specific biomarkers known to be associated with disease-related pathways) may be monitored more regularly so that diagnosis and treatment can be initiated as early as possible.

[0171]

[0183] Thus, the present invention can advantageously also enable the identification of dogs that are expected to respond particularly well to a given intervention (e.g., lifestyle or diet plan). Thus, the intervention can be applied in a more targeted manner to dogs expected to respond.

[0172]

[0184] The present invention further provides a method for evaluating the effectiveness of a lifestyle or diet plan for reducing the risk of death in dogs and / or increasing the probability of a healthy lifespan in dogs, the method comprising: a. evaluating the first risk of death and / or the probability of a healthy lifespan of a dog according to the method of the first aspect of the present invention; b. applying a lifestyle or diet plan to the dog; c. after the period of applying the lifestyle or diet plan to the dog, evaluating the second risk of death and / or the probability of a healthy lifespan of the dog according to the method of the first aspect of the present invention; d. determining whether there has been a change between the first risk of death and / or the probability of a healthy lifespan of the dog and the second risk of death and / or the probability of a healthy lifespan of the dog after the period according to the lifestyle or diet plan.

[0173]

[0189] The present invention also provides a method for evaluating the effectiveness of a lifestyle or diet plan for reducing the assessed risk of death and / or increasing the probability of a healthy lifespan in dogs, the method comprising: a. applying to a dog a lifestyle or diet plan selected according to a method according to a first aspect of the present invention, and selecting a suitable lifestyle or diet plan based on the assessed risk of death and / or probability of a healthy lifespan; b. after a period of applying the lifestyle or diet plan to the dog, evaluating a second risk of death and / or probability of a healthy lifespan of the dog by carrying out the method of the first aspect of the present invention; c. determining whether there has been a change in the risk of death and / or probability of a healthy lifespan of the dog after a period of following the lifestyle or diet plan.

[0174]

[0193] The present invention further provides a method for evaluating the effectiveness of a lifestyle or diet plan for reducing the risk of death and / or increasing the probability of a healthy lifespan in dogs, the method comprising: a. selecting for a dog a lifestyle or diet plan according to a method according to a first aspect of the present invention, and selecting a suitable lifestyle or diet plan based on the assessed risk of death and / or probability of a healthy lifespan; b. applying the lifestyle or diet plan to the dog; c. after a period of applying the lifestyle or diet plan to the dog, evaluating a second risk of death and / or probability of a healthy lifespan of the dog according to the method of the first aspect of the present invention; d. determining whether there has been a change in the risk of death of the dog between step a and step c.

[0175]

[0198] If the second (or subsequent) assessed risk of death in the dog is lower compared to the first (or earlier) assessed risk of death in the dog after a period of applying the lifestyle or diet plan, this indicates that the lifestyle or diet plan is effective in reducing the risk of death in the dog.

[0176]

[0199] The risk of death and / or probability of healthy lifespan of a dog may be evaluated before and after a lifestyle or diet plan is applied to the dog. The risk of death and / or probability of healthy lifespan of a dog may also be evaluated at predetermined times during the application of the lifestyle or diet plan. These predetermined times may be periodic during the lifestyle or diet plan, for example, daily or every three days, weekly, every two weeks, monthly, every two months, every six months, annually, or every two years, etc. The predetermined times may vary depending on the subject to be examined. Preferably, the lifestyle or diet plan may be applied to the dog during a period before the first risk of death and / or probability of healthy lifespan is evaluated. However, the effectiveness of the lifestyle or diet plan for reducing the risk of death and / or increasing the probability of healthy lifespan may be continuously monitored by evaluating the risk of death and / or probability of healthy lifespan at two or more predetermined times during the application of the lifestyle or diet plan.

[0177]

[0200] Use of dietary intervention

[0178]

[0201] In one aspect, the present invention provides a dietary intervention for use in reducing the risk of death and / or increasing the probability of healthy lifespan of a dog, the dietary intervention being performed on a dog whose risk of death and / or probability of healthy lifespan has been evaluated by the method of the present invention.

[0179]

[0202] In another aspect, the present invention provides the use of a dietary intervention for reducing the predicted risk of death and / or increasing the probability of healthy lifespan for a dog, the dietary intervention being performed on a dog whose risk of death and / or probability of healthy lifespan has been evaluated by the method of the present invention.

[0180]

[0203] As described herein, the dietary intervention may be a food product or a diet plan or a dietary supplement.

[0181]

[0204] Computer program product

[0182]

[0205] The method of the present invention may be implemented using a computer. Therefore, the method may be implemented in silico.

[0183]

[0206] The methods described herein may be implemented as a computer program operating on general-purpose hardware such as one or more computer processors. In some embodiments, the functions described herein may be implemented by a device such as a smartphone, tablet terminal, or personal computer.

[0184]

[0207] In one aspect, the present invention provides a computer program product comprising computer-executable instructions for causing a programmable computer to evaluate the risk of death and / or the probability of a healthy lifespan of a dog, as described herein.

[0185]

[0208] In another aspect, the present invention provides a computer program product comprising computer-executable instructions for causing a device to evaluate the risk of death and / or the probability of a healthy lifespan of a dog, given one or more biomarker values from a user, wherein the biomarker is selected from one or more biomarkers defined herein. Preferably, the biomarker value is a fasting value. The computer program product may also be provided with additional parameters or characteristics for the dog. As described herein, the additional parameters or characteristics may include chronological age, breed, and gender.

[0186]

[0209] In one embodiment, the user optionally inputs into the device one or more values of the biomarkers defined herein, along with chronological age, breed, and gender. The device then processes this information and provides an assessment of the risk of death and / or the probability of a healthy lifespan of the dog.

[0187]

[0210] The device may generally be a server on a network. However, any device may be used as long as it can process biomarker data and / or additional parameters or feature data using a processor, a central processing unit (CPU), etc. This device may be, for example, a smartphone, a tablet terminal, or a personal computer, and can output information indicating the risk of death and / or the probability of healthy life evaluated for a dog. The evaluated risk of death and / or the probability of healthy life may be presented in units of numerical scores or percentages, and may indicate, for example, the risk of death and / or the probability of healthy life evaluated in comparison with a control or reference population.

[0188]

[0211] Those skilled in the art will understand that all features of the present invention described herein can be freely combined without departing from the scope of the disclosed present invention.

[0189]

[0213] Next, the present invention will be further described by way of examples, which are meant to assist those skilled in the art in practicing the present invention and are not intended to limit the scope of the present invention in any way.

[0190]

[0214] Example 1 Determination of Blood Biomarkers Related to the Risk of Death in Dogs

[0191]

[0215] Predictive blood biomarkers were determined from a biomarker panel consisting of a standard clinical complete blood count (cbc) and a standard clinical blood chemistry analysis. Serum samples were collected after an overnight fast and measured by standard veterinary clinical practice.

[0192]

Table 1

[0193]

[0217] The inventors conducted a longitudinal study of dogs and repeatedly measured these parameters, as well as information on the dogs' status (alive or dead), gender, and breed. First, the inventors classified the breeds as small or medium (10 kg or less and over 10 kg, respectively) based on the average weight of adult dogs of this breed. Next, the data was organized using the R programming language. For each dog, the inventors recorded the biomarker as a time-dependent covariate using the time interval during which the left was open and the right was closed (i.e., (tstart, tstop]). The biomarker information corresponds to the start of this time interval, and the event (alive or dead) is recorded as the last tstop value. For this purpose, the inventors used the tmerge function of the survival package in R (v. 3.2 - 13). Next, the cox proportional hazards model was applied to the data of each of the 28 biomarkers, including gender and breed class (small or medium). Then, considering multiple comparisons (by false discovery rate (fdr)), the p-values of each parameter were corrected, and the features with a corrected fdr of less than 0.05 were selected (Figure 1).

[0194]

[0218] Using this method, the inventors identified 13 biomarkers that individually predict the survival probability in dogs. · White blood cell count (10^3 / μL) · Serum albumin (g / dL) · Serum alkaline phosphatase (U / L, ln-transformed) · Serum creatine kinase (IU / L, ln-transformed) · Hemoglobin (g / dL) · Hematocrit (%) · Mean corpuscular hemoglobin (pg) · Serum sodium (mmol / L) · Mean corpuscular volume (fL) · Serum globulin (g / dL) · Serum calcium (mg / dL) · Serum platelet count (10^3 / μL) · Red blood cell count (10^3 / μL)

[0195]

[0232] Example 2 Multi-parameter model for predicting the risk of death

[0196]

[0233] Next, the inventors constructed the best model that simultaneously considers multiple parameters as being more likely to cover the various organ dysfunctions that occur with age. However, selecting some features that can be correlated with each other is subject to bias. To avoid this problem, a penalized regression method using the glmnet package (v4.1-3) was used. The data was fitted to the LASSO of the penalized cox proportional hazards model, and 20-fold cross-validation was used to compare different values of the penalty parameter λ. This approach results in the selection of the top 10 most predictive blood biomarkers for survival in the order of importance shown below. · White blood cell count (10^3 / μL) · Serum albumin (g / dL) · Serum alkaline phosphatase (U / L, ln-transformed) · Serum creatine kinase (IU / L, ln-transformed) · Hemoglobin (g / dL) · Hematocrit (%) · Mean corpuscular hemoglobin (pg) · Serum glucose (mg / dL) · Mean corpuscular volume (fL) · Serum globulin (g / dL)

[0197]

[0244] The inventors also found that the first three biomarkers in this list are the most predictive, and that the performance can be improved by incorporating each of the next seven biomarkers.

[0198]

[0245] To extract the phenotypic age of an animal, the inventors calculated two different Gompertz functions for their training set, namely a function that models survival as a function of the selected biomarker, age, breed class (small or medium dog), and sex (Model 1), and a second function that considers only age, breed class, and sex (Model 2). These models were fitted using the flexsurv package (v 2.1). The phenotypic age was defined as the time variable ("age") at which the survival probability of the animal given by Model 2 is equal to the survival probability at their chronological age given by Model 1. This yielded a mathematical function that relates the blood biomarker to the phenoage, given by the following equation.

Equation

[0199]

[0246] In the equation, xb is the sum of the values of the biomarker, sex, and breed, each multiplied by their respective coefficients. Sex and breed are coded as numerical values with female as 0, male as 1, small breed as 0, and medium breed as 1. The coefficients are given by the two Gompertz functions trained on the inventors' training set.

[0200]

[0247] As an example, the coefficients, as well as the γ and γ breed values were measured from the inventors' training set for the complete list of biomarkers and are shown in Table 2.

Equation

[0201]

Table 2

[0202]

[0249] Furthermore, starting from the top of the list and systematically removing one biomarker at a time to reduce the set of ten biomarkers, the inventors observed a decrease in the strength of the survival prediction (p-value). The decrease was most pronounced for the first parameter, confirming that it had the largest contribution, but the inventors also observed the change in the quality of prediction by reducing each of the sets, showing that each parameter contributed to the overall prediction (Figure 2).

[0203]

[0250] Example 3 Application of PhenoAge for Predicting the Risk of Death

[0204]

[0251] Subsequently, when the obtained PhenoAge was applied to a test set consisting only of dogs that were not used during the training of the algorithm, it was demonstrated that the difference between PhenoAge and chronological age (PhenoAge progression) was associated with a significant increase in the risk of death. The magnitude of the effect was calculated to be a hazard ratio of 1.75 when PhenoAge increased by one year compared to chronological age (Figure 3).

[0205]

[0252] Furthermore, the survival of dogs stratified by whether the median of the PhenoAge progression was high or low showed a statistically significant survival difference between the upper 50% and the lower 50% (Figure 4).

[0206]

[0253] PhenoAge progression (difference from chronological age) is altered in middle age by calorie restriction diet (75% of the baseline maintenance energy requirement (MER)). This change is earlier in females than in males (Figure 5).

[0207]

[0254] Stratification of dogs below 7 years and above 7 years reveals a significant difference in comparison between older dogs and younger dogs. This difference is larger in females than in males (Figure 6).

[0208]

[0255] Example 4 Protein-Restricted Diet Reduces PhenoAge Progression

[0209]

[0256] Dog

[0210]

[0257] Thirty dogs with a body condition score (BCS) of 7 or more were included in this weight loss trial. The baseline maintenance energy requirement (MER) for each dog was determined as the amount of food that stabilized the dog's body weight (change < 5% over 3 weeks). Subsequently, the baseline body fat % of each dog was measured by DEXA. Based on the baseline body weight, MER, age, gender, and body fat %, the dogs were randomly assigned to two groups of 15 dogs each.

[0211]

[0258] Test diet

[0212]

[0259] The two diets had equivalent metabolic energy (ME), but differed in protein, carbohydrate, fat, and fiber.

[0213]

Table 3

[0214]

[0260] Feeding instructions

[0215]

[0261] Both groups of dogs were fed 75% of their baseline MER for the first 4 months of the trial and 60% of their baseline MER for the last 2 months of the trial.

[0216]

[0262] Collection and analysis of blood samples

[0217]

[0263] Serum samples were collected at baseline, 2 months, 4 months, and 6 months of the trial. At the end of the trial, complete blood count (CBC) and blood chemistry panel analysis were performed on these serum samples.

[0218]

[0264] Results

[0219]

[0265] As shown in FIG. 7, compared to the baseline, all dogs except one showed a decrease in phenoage progression defined as the difference between the phenoage of dogs after a 6-month caloric restriction period and their chronological age. The mean difference in phenoage progression between baseline and post-intervention was 0.7 years and was significant in the paired t-test (p = 0.00093). The age of the dogs in the study ranged from 3 to 11 years old. The inventors did not detect a correlation between the increase in phenoage progression and the age of the dogs at the start of the study, indicating that the benefits extend across a wide range of the dogs' life stages. Furthermore, although the two diets had very different protein amounts, the effects on phenoage were equivalent between the two diets, suggesting that a reduction in calorie intake led to a younger phenoage regardless of the macronutrient ratio in dogs.

[0220]

[0266] All publications mentioned in the above specification are hereby incorporated by reference into this specification. Various modifications and variations of the disclosed methods, compositions, and uses of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. While the present invention has been disclosed in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications in the manner of the disclosed invention will be obvious to those skilled in the art and are intended to be within the scope of the following claims.

Claims

1. 1. A method for assessing a dog's risk of mortality and / or healthy life expectancy probability, the method comprising the step of measuring the value of one or more biomarkers in one or more samples obtained from the dog, the one or more biomarkers being selected from white blood cell count, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum globulins, serum calcium, platelet count, and / or red blood cell count.

2. The method of claim 1 , wherein the biomarker is white blood cell count.

3. The method of claim 1 or 2, wherein the biomarker is serum albumin.

4. The method of any one of claims 1 to 3, wherein the biomarker is serum alkaline phosphatase.

5. The method according to any one of claims 1 to 4, wherein the method comprises the steps of measuring each of white blood cell count, serum albumin, and serum alkaline phosphatase.

6. 2. The method of claim 1, wherein the method comprises measuring each of white blood cell count, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, and serum globulin.

7. The method of any one of claims 1 to 6, wherein the one or more samples is a blood sample.

8. 8. The method of any one of claims 1 to 7, wherein the method further comprises combining the value of the one or more biomarkers with one or more of the dog's chronological age, breed, and / or sex.

9. The method according to any one of claims 1 to 8, wherein the dog is classified as small, medium, large or giant size, and / or as robust or athletic.

10. 1. A method for assessing a mortality risk and / or healthy life expectancy probability of a dog, said method comprising: a. measuring the levels of the following biomarkers in one or more samples obtained from the dog: white blood cell count, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, and serum globulin; b. assessing the phenotypic age of the dog using formula (1); [0010] (wherein xb is the sum of the values ​​obtained by multiplying each of the values ​​of the biomarker, sex, and breed by each coefficient according to formula (2), [0025] Sex was coded as a number, with female being 0 and male being 1. Breed is coded as a number between 0 for small breeds and 1 for medium breeds). c) using said phenotypic age to assess the risk of mortality and / or healthy life expectancy of said dog.

11. 11. The method of claim 10, wherein assessing the dog's phenoage as exceeding its chronological age indicates a higher risk of mortality and / or a reduced probability of healthy life expectancy.

12. 11. The method of claim 10, wherein assessing the dog's phenoage to be less than its chronological age indicates a lower risk of mortality and / or an increased probability of healthy life expectancy.

13. A method according to any one of claims 1 to 12, wherein the method is performed on one or more samples obtained before and after a time interval to determine whether there has been a change in the mortality risk and / or healthy life expectancy probability of the dog during the time interval.

14. A method of calculating the rate of aging of a dog, the method comprising carrying out a method according to any one of claims 10 to 12, wherein (i) assessing the phenoage of the dog to be greater than its chronological age indicates a rapid rate of aging, or (ii) assessing the phenoage of the dog to be less than its chronological age indicates a slow rate of aging.

15. 1. A method for selecting a lifestyle or diet plan for a dog, the method comprising: a. carrying out the method according to any one of claims 1 to 13; b. selecting an appropriate lifestyle or diet plan based on the mortality risk and / or healthy life expectancy probability assessed in step a.

16. 16. The method of claim 15, wherein the lifestyle or dietary plan is a dietary intervention.

17. 17. The method according to claim 15 or 16, wherein the dietary intervention is a calorie restricted diet, an elderly diet or a low protein diet, preferably a calorie restricted diet.

18. 1. A method for assessing the effectiveness of a lifestyle or dietary regimen for improving a dog's risk of mortality and / or the probability of a healthy life span in a dog, the method comprising: a. assessing a first mortality risk and / or healthy life expectancy probability of said dog according to a method according to any one of claims 1 to 13; b. administering a lifestyle or dietary regimen to said dog; c) assessing a second mortality risk and / or healthy life expectancy probability of said dog after a period of applying said lifestyle or diet plan to said dog according to the method of any one of claims 1 to 13; d. assessing whether there is a difference between said first risk of mortality and / or healthy life expectancy and said second risk of mortality and / or healthy life expectancy of said dog after a period of following said lifestyle or dietary plan.

19. 1. A method for assessing the effectiveness of a lifestyle or dietary regimen for reducing an assessed risk of mortality and / or increasing the probability of a healthy life span in a dog, the method comprising: a. applying to said dog the lifestyle or diet plan selected according to the method of any one of claims 15 to 17; b. carrying out the method according to any one of claims 1 to 13 after a period of applying said lifestyle or dietary plan to said dog; c) determining whether there has been a change in the dog's risk of mortality and / or healthy life expectancy probability after said period of following said lifestyle or dietary plan.

20. 1. A method for assessing the effectiveness of a lifestyle or dietary regimen for reducing risk of mortality and / or increasing healthy life expectancy in a dog, the method comprising: a. selecting a lifestyle or diet plan for the dog according to the method of any one of claims 15 to 17; b. administering said lifestyle or dietary plan to said dog; c) assessing a second mortality risk and / or healthy life expectancy probability of said dog after a period of applying said lifestyle or diet plan to said dog according to the method of any one of claims 1 to 13; d. determining whether there has been a change in the dog's risk of death and / or healthy life expectancy probability between steps a and c.

21. 21. The method of any one of claims 18 to 20, further comprising modifying the lifestyle or dietary plan to reduce the dog's risk of mortality and / or increase its chances of healthy lifespan.

22. 1. A method for assessing the likelihood that a dog will benefit from a lifestyle or dietary regimen, said method comprising: a) assessing the risk of mortality and / or healthy life expectancy of the dog according to the method of any one of claims 1 to 13; b) identifying a dog as likely to respond to a lifestyle or diet plan if the dog has an increased risk of mortality and / or a decreased probability of healthy life expectancy compared to chronological age.

23. 1. A method for selecting a dog as suitable for receiving a lifestyle or dietary plan, the method comprising: a) assessing the risk of mortality and / or healthy life expectancy of the dog according to the method of any one of claims 1 to 13; b) selecting the dog as suitable for receiving a lifestyle or diet plan if the dog has an increased risk of mortality and / or a decreased probability of healthy life expectancy compared to chronological age.

24. 1. A method for reducing the risk of mortality and / or increasing the probability of healthy life span in a dog, said method comprising: a) assessing a dog's risk of mortality and / or healthy life expectancy probability, comprising measuring the value of one or more biomarkers in one or more samples obtained from the dog, wherein the one or more biomarkers are selected from white blood cell count, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum globulins, serum calcium, platelet count, and / or red blood cell count; b. Selecting an appropriate lifestyle or diet plan based on the mortality risk and / or healthy life expectancy probability assessed in step a; c) administering said lifestyle or dietary plan to said dog.

25. The method according to claim 24, wherein step a comprises the method according to any one of claims 1 to 13.

26. A method for reducing the risk of mortality and / or increasing the probability of a healthy lifespan in a dog, comprising a step of administering dietary intervention to a dog assessed as needing dietary intervention by the method of any one of claims 1 to 13.

27. A dietary intervention for use in reducing the risk of mortality and / or increasing healthy life expectancy in dogs, said dietary intervention being administered to dogs whose risk of mortality and / or healthy life expectancy have been assessed by a method according to any one of claims 1 to 13.

28. Use of dietary intervention to reduce the risk of mortality and / or increase the healthy life expectancy of a dog, said dietary intervention being administered to a dog whose risk of mortality and / or healthy life expectancy has been assessed by a method according to any one of claims 1 to 13.

29. 29. The method, dietary intervention for use or use according to any one of claims 23 to 28, wherein the dietary intervention is a calorie restricted diet, an ageing diet or a low protein diet, preferably a calorie restricted diet.

30. A computer readable medium comprising instructions which, when executed, cause one or more processors to perform the method of any one of claims 1-13.

31. 1. A computer system for assessing a mortality risk and / or a healthy life expectancy probability of a dog, the computer system comprising: assessing the risk of mortality and / or healthy life expectancy of the dog given the value of one or more biomarkers in a sample from the dog, wherein the one or more biomarkers are selected from white blood cell count, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum globulins, serum calcium, platelet count, and / or red blood cell count; and generating a report of the mortality risk and / or healthy life expectancy probability of the dog.

32. 1. A computer program product comprising computer executable instructions for causing a programmable computer to assess a dog's risk of mortality and / or healthy life expectancy probability given values ​​of one or more biomarkers from the dog, wherein the one or more biomarkers are selected from white blood cell count, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum globulins, serum calcium, platelet count, and / or red blood cell count.

33. The computer system or computer program product according to any one of claims 31 to 32, wherein the biomarker is white blood cell count.

34. 34. The computer system or computer program product of any one of claims 31 to 33, wherein the biomarker is serum albumin.

35. A computer system or computer program product according to any one of claims 31 to 34, wherein said biomarker is serum alkaline phosphatase.

36. 1. A method for addressing a risk of mortality in a dog that is at higher risk of mortality than a comparable dog of the same chronological age, sex, and breed, said method comprising: Identifying said dog as having a higher risk of mortality than said comparable dogs using the method of any one of claims 1 to 13; providing a dietary intervention to the dog based on the determination that the dog's risk of mortality is higher than that of comparable dogs.

37. 37. The method of claim 36, comprising, prior to administering the dietary intervention to the dog, selecting the dietary intervention based on the dog's risk of mortality.

38. 38. The method of claim 36 or 37, further comprising assessing the risk of mortality of the dog after administering the dietary intervention to the dog to assess the effectiveness of the dietary intervention, and then optionally (i) continuing to administer the dietary intervention to the dog based on the effectiveness of the dietary intervention meeting a threshold, or (ii) administering a different dietary intervention to the dog based on the effectiveness of a previously administered dietary intervention that does not meet the threshold.

39. The method according to any one of claims 36 to 38, wherein the dietary intervention is administered to the dog for at least two weeks.

40. 40. The method of any one of claims 36 to 39, wherein the dietary intervention is selected from the group consisting of a calorie restricted diet, an elderly diet, a low protein diet, a low phosphorus diet, a low protein diet, a potassium supplement, a polyunsaturated fatty acid (PUFA) supplemented diet, an antioxidant supplement, a B vitamin supplement, a liquid diet, a selenium supplement, an omega 3-6 ratio diet, carnitine, one or more branched chain amino acids or derivatives thereof, nucleotides, one or more nicotinamide precursors, and any combination thereof.

41. 41. The method of any one of claims 36 to 40, further comprising administering a lifestyle modification regimen to the dog based on the dog's risk of mortality, wherein the lifestyle modification regimen preferably comprises at least one of: (i) exercise therapy; or (ii) a therapeutic modality for treating and / or preventing a condition selected from the group consisting of arthritis, dental disease, endocrine disorders, cardiac disease, diabetes, liver disease, kidney disease, prostate disorders, cancer, behavioral disorders, cognitive disorders, and combinations thereof.

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