Methods of determining health status of dogs based on one or more biomarkers and methods of treating risk of death identified by health status

Abstract

The present invention relates to a method for determining the risk of death and / or the probability of healthy lifespan of a dog; the method comprises determining a level of one or more biomarkers in one or more samples obtained from a dog, wherein the one or more biomarkers are selected from the group consisting of leukocyte count, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, average erythrocyte hemoglobin, serum glucose, average erythrocyte volume, serum globulin, serum calcium, platelet count, and / or erythrocyte count.

Description

Technical Field

[0001] The present invention relates to methods for determining the health status of a dog, in particular the risk of mortality and / or the probability of a longer healthy life span, based on biomarkers. Background Art

[0002] The ability to determine information about a dog's health is desirable to inform the dog's overall health and well-being. For example, the ability to determine information about a dog's health may be helpful in assessing whether a dog would benefit from a dietary or supplement-based intervention.

[0003] As is well known, chronological age is a major indicator of overall health status, wherein an increase in chronological age is associated with a decrease in health status. However, depending on genetics, nutrition, and lifestyle, the rate of individual aging may be slower or faster than chronological age. Therefore, chronological age may not always reflect the risk of reduced aging rate or health status of an individual. On the other hand, the biological age of an individual (based on, for example, clinical biochemistry and cell biology measurements) may be different from that of other individuals of the same chronological age. Methods for determining biological age may help to identify individuals at risk of age-related disorders earlier than expected based on the chronological age of the individual (see, for example, WO2019 / 165064).

[0004] However, additional methods are needed to determine information about a dog's health. Summary of the invention

[0005] The present invention relates to a method for quantifying the health status of a dog based on biomarkers, in particular blood biomarkers. The method is capable of determining the risk of death and / or the probability of healthy life span of a dog by assessing the phenotypic age of the dog.

[0006] Existing methods of using blood biomarkers to assess a dog's health state determine biological age based on a correlation between blood biomarkers and chronological age. Calculating an animal's biological age may include determining the difference in the level of one or more biomarkers compared to the expected level of the biomarkers given the chronological age. Thus, this method is based on using chronological age as a primary indicator of overall health.

[0007] In contrast, the present invention contemplates the direct predictive value of the biomarkers for the risk of death and / or the probability of healthy life span. For example, a given biomarker may not be directly correlated with chronological age, but may indicate a particular pathological condition, thereby indicating the probability of increased risk of death and / or decreased healthy life span. Thus, the present method can be described as identifying the phenotypic age (PhenoAge) of a dog. Thus, the biomarkers of the present invention are not necessarily correlated with chronological age, but are related to the difference between the phenotypic age and the chronological age of a dog.

[0008] The present invention provides a method for determining the risk of mortality and / or the probability of healthy life span in a dog; the method comprises determining the level of one or more biomarkers in one or more samples obtained from the dog.

[0009] In one aspect, the invention provides a method for determining the risk of mortality and / or probability of healthy life span in a dog; the method comprising determining the level 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 globulin, serum calcium, platelet count and / or red blood cell count.

[0010] The inventors have shown that each of these biomarkers individually correlates with phenotypic age and is therefore predictive of mortality risk and / or probability of healthy life span.

[0011] Determining the risk of death can refer to determining the likelihood that a dog will live longer or shorter than, for example, a comparable dog of the same chronological age, sex, and breed. Thus, the present method can determine the probability of a dog's healthy period or healthy lifespan compared to, for example, a comparable dog of the same chronological age, sex, and breed. Suitably, the risk of death can be equated to the probability of a dog's healthy lifespan; wherein a decrease in the risk of death is equated to an increased probability of a longer healthy lifespan for the dog, or an increase in the risk of death is equated to a decreased probability of a longer healthy lifespan for the dog. The risk of death can be expressed as the difference between the phenotypic age and the chronological age of the dog (phenotypic age is advanced). For example, an increase in phenotypic age compared to chronological age can indicate an increased risk of death for the dog; and a decrease in phenotypic age compared to chronological age can indicate a decreased risk of death for the dog.

[0012] In one embodiment, the method may include determining the white blood cell count in one or more samples obtained from the dog. The method may also include determining the level of one or more biomarkers in one or more samples 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.

[0013] In one embodiment, the method may further include determining the level of serum albumin in one or more samples obtained from the dog. The method may further include determining the level of one or more biomarkers in one or more samples selected from the group consisting of 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.

[0014] In one embodiment, the method may include determining the level of serum alkaline phosphatase in one or more samples obtained from the dog. The method may also include determining the level of one or more biomarkers in one or more samples selected from the group consisting of 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.

[0015] In one embodiment, the method may include determining the level of serum creatine kinase in one or more samples obtained from the dog. The method may also include determining the level of one or more biomarkers in one or more samples selected from the group consisting of 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.

[0016] In one embodiment, the method may include determining the level of hemoglobin in one or more samples obtained from the dog. The method may also include determining the level of one or more biomarkers in one or more samples selected from the group consisting of 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.

[0017] In one embodiment, the method may include determining the level of hematocrit in one or more samples obtained from the dog. The method may also include determining the level of one or more biomarkers in one or more samples selected from the group consisting of 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.

[0018] In one embodiment, the method may include determining the mean corpuscular hemoglobin level in one or more samples obtained from the dog. The method may also include determining the level of one or more biomarkers in one or more samples selected from the group consisting of 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.

[0019] In one embodiment, the method may include determining serum glucose levels in one or more samples obtained from the dog. The method may also include determining the levels of one or more biomarkers in one or more samples selected from the group consisting of white blood cells, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, mean corpuscular volume, serum globulin, serum calcium, platelet count, and / or red blood cell count.

[0020] In one embodiment, the method may include determining the mean corpuscular volume in one or more samples obtained from the dog. The method may also include determining the level of one or more biomarkers in one or more samples selected from the group consisting of white blood cells, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, serum globulin, serum calcium, platelet count, and / or red blood cell count.

[0021] In one embodiment, the method may include determining the level of serum globulin in one or more samples obtained from the dog. The method may also include determining the level of one or more biomarkers in one or more samples selected from the group consisting of 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.

[0022] In one embodiment, the method may include determining serum calcium levels in one or more samples obtained from the dog. The method may also include determining the levels of one or more biomarkers in one or more samples selected from the group consisting of white blood cells, serum albumin, serum alkaline phosphatase, serum creatine kinase, hemoglobin, hematocrit, mean corpuscular hemoglobin, serum glucose, mean corpuscular volume, serum globulins, platelet count, and / or red blood cell count.

[0023] In one embodiment, the method may include determining platelet count in one or more samples obtained from the dog. The method may also include determining the level of one or more biomarkers in one or more samples selected from the group consisting 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 and / or red blood cell count.

[0024] In one embodiment, the method may include determining the red blood cell count in one or more samples obtained from the dog. The method may also include determining the level of one or more biomarkers in one or more samples selected from the group consisting 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 and / or platelet count.

[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 determined that this combination of three biomarkers can significantly predict the risk of death and / or the probability of healthy life span. The predictive power can be further increased by combining one or more biomarkers in the 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.

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

[0027] In one embodiment, the present invention provides a method for determining the risk of mortality and / or the probability of healthy life span in a dog; the method comprising:

[0028] a. determining the levels 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 the dog; and

[0029] b. Determine the dog's phenotypic age (Phenoage) using formula (1):

[0030]

[0031] where xb is the sum of the values ​​of each biomarker, sex and breed multiplied by their corresponding coefficients according to formula (2):

[0032]

[0033] where sex is encoded as a numeric value, with 0 representing females and 1 representing males.

[0034] The breed is coded as a numeric value, where 0 represents a small breed and 1 represents a medium-sized breed.

[0035] and wherein phenotypic age is used to determine a dog's risk of mortality and / or probability of healthy life span.

[0036] Suitably, determining that the phenotypic age of the dog is greater than the chronological age of the dog indicates a higher risk of mortality. Suitably, determining that the phenotypic age of the dog is less than the chronological age of the dog indicates a reduced risk of mortality. Suitably, determining that the phenotypic age of the dog is greater than the chronological age of the dog indicates a reduced probability of a longer healthy lifespan. Suitably, determining that the phenotypic age of the dog is less than the chronological age of the dog indicates an increased probability of a longer healthy lifespan.

[0037] The present invention also relates to a method for selecting a lifestyle or dietary regimen for a dog, the method comprising:

[0038] a. Execute the method as described in the first aspect of the present invention; and

[0039] b. Selecting an appropriate lifestyle or dietary regimen based on the mortality risk and / or probability of healthy life span determined in step a.

[0040] The present invention also provides a method for determining the effectiveness of a lifestyle or dietary regimen in reducing the risk of death and / or increasing the probability of a dog's healthy life span, the method comprising: a. performing the method as described in the first aspect of the present invention; b. changing the dog's lifestyle or dietary regimen; c. performing the method as described in the first aspect of the present invention after a period of time of changing the lifestyle or dietary regimen; and d. determining whether there is a change in the dog's risk of death and / or the probability of a healthy life span between step a and step c.

[0041] The present invention also provides a dietary intervention for reducing the risk of mortality and / or increasing the probability of a healthy life span in a dog, wherein the dietary intervention is administered to a dog, wherein the risk of mortality is determined by the method of the present invention.

[0042] The invention also provides for the use of a dietary intervention to reduce the determined risk of mortality and / or increase the probability of healthy life span in a dog, wherein the dietary intervention is administered to a dog, wherein the risk of mortality and / or the probability of healthy life span is determined by the method of the invention.

[0043] The present invention also provides a computer readable medium comprising instructions which, when executed, cause one or more processors to perform the method of the present invention.

[0044] The present invention also provides a computer system for determining the risk of death and / or the probability of healthy life expectancy of a dog, the computer system being programmed to perform one or more of the following steps: determining the risk of death and / or the probability of healthy life expectancy of a dog given the level 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 globulin, serum calcium, platelet count and / or red blood cell count; and generating a report on the risk of death and / or the probability of healthy life expectancy of the dog.

[0045] The present invention also provides a computer program product comprising computer-implementable instructions for causing a programmable computer to determine the risk of mortality and / or probability of healthy life span for a dog given the level 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 globulin, serum calcium, platelet count and / or red blood cell count.

[0046] Advantageously, embodiments of the invention in which the levels of multiple biomarkers are determined enable the determination of mortality risk and / or probability of healthy life span based on markers of multiple organ systems and functions. Thus, the use of multiple biomarkers advantageously covers a range of potential organ dysfunctions.

[0047] Assessing a dog's risk of death or probability of survival allows one to test several aspects of the animal's well-being. First, it can predict whether the animal is more likely to require a dietary or supplement-based intervention. Second, it can be used to test the efficacy of dietary or supplement-based interventions on aging. It also provides a specific set of biomarkers that can be tested to assess an animal's risk of death or probability of survival and that can be routinely tested in a veterinary clinic or office. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 — Identification of blood biomarkers that predict mortality risk. Cox proportional hazards models were fit for each of the 28 biomarkers evaluated, including sex and breed category (small or medium). P values ​​for each parameter were adjusted to account for multiple comparisons (by false discovery rate (FDR)). Parameters shown are those with adjusted FDR less than 0.05.

[0049] Figure 2— Demonstration that biomarkers contribute to the predictive power of a multiparameter model for determining phenotypic age.

[0050] Figure 3 —The discrepancy between phenotypic age and chronological age (earlier phenotypic age) is associated with a significantly increased risk of mortality.

[0051] Figure 4 —Differences in survival of dogs stratified in advance according to high or low median phenotypic age.

[0052] Figure 5 - Advancement of phenotypic age (difference from chronological age) in midlife by calorie-restricted diets. This change occurs earlier in females than in males.

[0053] Figure 6 — Stratification of dogs aged 7 years or younger and 7 years or older revealed significant differences in the advancement of chronological age in older dogs compared to younger dogs.

[0054] Figure 7 —Dogs fed two calorie-restricted diets for 6 months showed a significant reduction in phenotypic age. DETAILED DESCRIPTION

[0055] Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples. The skilled person will appreciate that they can combine all features of the present invention disclosed herein without departing from the scope of the invention disclosed.

[0056] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0057] As used herein, the terms "comprising" and "consisting of" are synonymous with "including" or "containing" and are inclusive or open-ended and do not exclude additional unrecited members, elements, or method steps. The terms "comprising" and "consisting of" also include the term "composed of.

[0058] Numeric ranges are inclusive of the numbers defining the range.

[0059] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present patent application. Nothing herein should be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0060] The methods and systems disclosed herein can be used by veterinarians, healthcare professionals, laboratory technicians, pet care providers, and the like.

[0061] Subjects

[0062] The present method is directed to canine subjects. Therefore, the subject of the present invention is a dog.

[0063] Suitably, the dog may have a body condition score (BCS) of at least 7 (eg using the American Animal Hospital Association (AAHA) - 9 point scale; AAHA Guidelines; Nutritional Assessment Body and Muscle Condition Scores).

[0064] variety

[0065] The method may make use of information about the breed of the dog. For example, the dog may be classified as a toy, small, medium, large or giant breed. Suitably, the dog breeds may be classified based on the weight of the dog. Suitably, the dog breeds may be classified based on the average weight of a dog of a given breed.

[0066] Dogs can be classified based on their genetic information obtained through DNA sequencing or SNP detection. For example, dogs can be classified based on the breed branch to which they belong.

[0067] Additionally or alternatively, the dog may be a mixed breed. Mixed breed dogs may be classified based on the weight of the dog. For example, mixed breed dogs may be classified as small, medium, large, or giant.

[0068] Additionally or alternatively, mixed breed dogs can be classified based on genetic information, such as DNA sequence, SNP, haplotype or haplogroup. For example, mixed breed dogs can be classified based on the closest breed or branch through genetic analysis.

[0069] Additionally or alternatively, genetic information known to be associated with breed identity, such as DNA sequences, SNPs, haplotypes, or haploblocks, can be used to classify mixed-breed or pure-bred dogs.

[0070] Additionally or alternatively, genetic information known to correlate with longevity, such as DNA sequences, SNPs, haplotypes or haploblocks, can be used to classify mixed or purebred dogs.

[0071] Additionally or alternatively, mixed or purebred dogs may be classified as robust or athletic based on morphometric measurements (e.g., as disclosed in US8091509, entitled “Method for improving dog food” by Perez-Camargo et al. or US2017 / 0042194, entitled “Methods using morphometric measurements of a smalldog to improve food for the small dog” by Bouthegourd et al., both of which are incorporated herein by reference in their entirety).

[0072] Preferably, the dog can be classified as a small or medium breed. Preferably, the classification is determined by the average weight of an adult dog of the breed. Preferably, a breed with an average weight below 10 kg is classified as a small breed and / or a breed with an average weight above 10 kg is classified as a medium breed.

[0073] gender

[0074] Suitably, the gender of a dog may be classified as male or female.

[0075] Chronological age

[0076] Chronological age can be defined as the amount of time that has passed from the subject's birth to a given date. Chronological age can be expressed in years, months, days, etc.

[0077] Suitably, the present methods may be applied to dogs of any chronological age. In certain embodiments, the dog may be at least about 2 years old. Suitably, 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.

[0078] Suitably, the dog may be at least about 7 years old.

[0079] sample

[0080] The present invention includes the step of determining the level of one or more biomarkers in one or more samples obtained from an individual.

[0081] Preferably, the sample is derived from blood. The sample may comprise blood components or may be whole blood. The sample preferably comprises plasma and / or serum. Techniques for collecting samples from individuals are well known in the art.

[0082] The appropriate sample can be selected based on the biomarker to be determined. For example, if a complete blood count (CBC) is to be used to determine a biomarker, such as as part of a standard clinical complete blood count (CBC), a whole blood sample should be used.

[0083] The method can be performed on one or more samples obtained from a subject. For example, the method can 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 is obtained. The method can be performed more than once on samples obtained from the same dog over a period of time. For example, samples can be obtained repeatedly once a month, once a year, or once every two years. Suitably, samples can be obtained approximately once a year (e.g., during an annual veterinary health checkup). This may help determine the effect of a particular treatment or lifestyle change, such as a dietary intervention or a change in an exercise regimen.

[0084] In one embodiment, the level of one or more biomarkers can be determined before a lifestyle change (e.g., a dietary product intervention or a change in exercise regimen). In another embodiment, the level of one or more biomarkers can be determined before and after, for example, a dietary product intervention or a change in exercise regimen. Biomarker levels can also be determined at predetermined times during, for example, the entire dietary product intervention or the change in exercise regimen. These predetermined times can be periodic, for example, every day or every three days, or can depend on the subject being tested, for example.

[0085] Determine the level of one or more biomarkers in a sample

[0086] The biomarkers used in the present invention can be determined using standard methods in the art and are typically measured as part of a standard blood test to determine the disease state of an animal. For example, biomarkers are typically determined as part of a standard clinical complete blood count (CBC) and standard clinical blood chemistry analysis.

[0087] Complete blood cell count provides information about blood cells and their properties; For example, red blood cells, white blood cells and platelets. Exemplary complete blood cell counts may include an automated process using flow cytometry or a Coulter counter to determine the number of cells in the blood. In addition to determining cell counts, such automated systems may also be able to determine other blood biomarker readings depending on their complexity. Such systems can simultaneously measure blood cell counts and red blood cell volume, hemoglobin levels, mean corpuscular hemoglobin levels and hematocrit. For example, IDEXX Laboratories provides a hematology analyzer (ProCyte Dx Hematology Analyzer of IDEXX Laboratories Inc.) that can determine 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).

[0088] The levels of other biomarkers not related to blood cells, such as serum protein / enzyme and / or molecular biomarker levels, can be measured using chemical tests, particularly using automated chemical analyzer systems. These methods can be quantified using colorimetric-based methods. For example, IDEXX Laboratories provides an automated chemical analyzer (Catalyst One Chemistry Analyzer from IDEXX Laboratories Inc.) that can quantify serum albumin, serum alkaline phosphatase, serum creatine kinase, serum glucose, serum globulin, and serum calcium.

[0089] Thus, methods for determining the levels of biomarkers used in the present invention may include assays that result in spectrophotometric changes (e.g., chemical or antibody-related changes that result in a detectable signal at certain wavelengths). Such tests can be highly automated and efficient, and are the basis of many routine veterinary health examinations.

[0090] Suitably, biomarker levels may be determined after an overnight fast and measured using standard veterinary clinical practice.

[0091] The levels of individual biomarker species in a sample can be measured or determined by any suitable method known in the art. For example, mass spectrometry (MS), antibody detection methods (e.g., enzyme-linked immunosorbent assay (ELISA)), non-antibody protein scaffolds (e.g., fibronectin scaffolds), radioimmunoassays (RIA), or nucleic acid ligands can be used. Other spectrophotometric methods, chromatography, labeling techniques, or quantitative chemical methods can also be used.

[0092] Suitable antibodies for use in the above methods are known in the art and / or can be generated using known techniques. Suitable assays for detecting antibody levels include, but are not limited to, immunoassays such as enzyme-linked immunosorbent assays, radioimmunoassays, Western blotting, and immunoprecipitation.

[0093] Biomarkers

[0094] White blood cell count

[0095] Leukocytes, also known as white blood cells, are a type of cell found in the blood. They have various immune-related functions, depending on their subtype: monocytes, lymphocytes, neutrophils, basophils, and eosinophils. Leukocytes contain a nucleus and have variable cell shapes, also depending on the subtype. The white blood cell count is the number of such cells per volume of blood.

[0096] Methods for measuring white blood cell counts are known in the art and are typically expressed as thousands of cells per microliter (10^3 / uL). White blood cell count measurements can be performed manually on blood smears using staining and microscopy techniques, but can also be performed as part of an automated complete blood count (CBC). IDEXX Laboratories offers an automated hematology analyzer capable of performing white blood cell count measurements.

[0097] Suitably, an increase in white blood cell count may be associated with a negative impact on reducing the risk of death. Thus, an increase in white blood cell count may be associated with an increased risk of death.

[0098] Serum albumin

[0099] Serum albumin is a globular protein found in the blood. It is a 65 kDa protein that includes three homologous domains. Albumin regulates osmotic pressure, prevents fluid loss from the blood to the tissues, and serves as a transport protein for fatty acids, bilirubin, heme, heavy metals, hormones, and certain drugs. Albumin is present in high levels in the blood, accounting for 25-50% by weight of total plasma protein, and is produced by the liver. Abnormally high or low levels of albumin in the blood may indicate liver or kidney disease.

[0100] Methods for measuring serum albumin levels are well known in the art, and serum albumin levels are usually expressed in grams per deciliter (g / dL); and include zone electrophoresis, dye binding assays involving bromocresol green (BCG) or bromocresol purple (BCP), and ELISA methods. For example, Eagle Biosciences provides an ELISA-based canine serum albumin assay (Eagle Biosciences Inc., product code SKU: CAE49-K01). Stokol et al. describe an automated system for using a BCG binding assay in a clinical setting to determine serum albumin levels in dogs (Vet Clin Pathol. 2001; 30(4): 170-176). In addition, IDEXX Laboratories provides an automated chemistry analyzer that can measure serum albumin levels as part of a combined blood test.

[0101] Suitably, an increase in serum albumin levels may be associated with a positive effect on reducing the risk of death. Thus, an increase in serum albumin levels may be associated with a reduced risk of death.

[0102] Serum alkaline phosphatase

[0103] Alkaline phosphatase (ALP) is an enzyme that plays an important role in liver metabolism and bone development. It is an 86 kDa homodimeric protein. High levels of this protein in the blood may indicate liver damage or bone disease.

[0104] Methods for measuring serum alkaline phosphatase levels are well known in the art, and serum alkaline phosphatase levels are usually expressed in units / liter (U / L); and the most common method consists of quantifying enzyme activity via a colorimetric assay. Automated chemistry analyzers capable of measuring serum alkaline phosphatase levels are available from IDEXX Laboratories.

[0105] Suitably, an increase in serum alkaline phosphatase levels may be associated with a negative impact on reducing the risk of death. Thus, an increase in serum alkaline phosphatase levels may be associated with an increased risk of death.

[0106] Serum creatine kinase

[0107] Creatine kinase (CK) is an enzyme found primarily in muscle. The enzyme converts creatine and ATP into creatine phosphate for rapid energy generation during muscle contraction. The presence of high levels of this enzyme in the blood can indicate muscle damage.

[0108] Methods for determining creatine kinase levels in blood are well known in the art, with creatine kinase levels typically expressed in international units per liter (IU / L); and examples of these methods use enzyme assays to quantify creatine kinase levels. The automated chemistry analyzer provided by IDEXX Laboratories is an example of a commercially available tool for measuring serum creatine kinase levels.

[0109] Suitably, an increase in creatine kinase levels may be associated with a negative impact on reducing the risk of death. Thus, an increase in creatine kinase levels may be associated with an increased risk of death.

[0110] Hemoglobin

[0111] Hemoglobin is a transport protein in red blood cells. It is composed of a tetramer of two alpha chains and two beta chains. Each peptide chain is bound to a heme group, which is composed of a porphyrin ring bound to an iron ion. This group can reversibly bind oxygen, allowing hemoglobin to be used as an oxygen transport carrier protein.

[0112] Methods for determining hemoglobin levels are well known in the art, and hemoglobin levels are usually expressed in grams per deciliter (g / dL). A standardized method for the spectrophotometric determination of hemoglobin cyanide has been described by the International Committee for Standardization in Hematology (Br J Haematol. 1967 Apr; 13: 71-5), and the method can be used in commercially available automated chemistry analyzers, such as those provided by IDEXX Laboratories.

[0113] Suitably, an increase in haemoglobin levels may be associated with a positive effect on reducing the risk of death. Thus, an increase in haemoglobin may be associated with a reduced risk of death.

[0114] Hematocrit

[0115] Hematocrit is the volume percentage of red blood cells in the blood. Hematocrit levels above normal can indicate a disease or condition that causes a higher or lower than normal proportion of red blood cells in the blood. For example, a high hematocrit can indicate a condition such as dehydration; while a low hematocrit can indicate anemia, hemolysis, or decreased red blood cell production.

[0116] Methods for determining hematocrit are well known in the art and hematocrit is usually expressed as a percentage (%) of the blood volume. It can be measured manually by centrifuging blood in a microhematocrit tube, using the packed cell volume. Alternatively, hematocrit can be calculated from the mean corpuscular volume and the red blood cell count, both of which can be measured directly in a standard complete blood count (CBC) by modern hematology analyzers.

[0117] Suitably, an increase in hematocrit level may be associated with a positive effect on reducing the risk of death. Thus, an increase in hematocrit may be associated with a reduced risk of death.

[0118] Mean corpuscular hemoglobin

[0119] Mean corpuscular hemoglobin (MCH) is the average mass of hemoglobin per red blood cell. MCH values ​​outside the normal range may indicate certain diseases, such as macrocytic or hypochromic anemia.

[0120] Methods of measuring MCH are well known in the art and MCH is typically expressed in picograms (pg); and generally involves calculation from observed values ​​of hemoglobin levels and red blood cell counts, which may be measured during a complete blood count (CBC) using a hematology analyzer as described above.

[0121] Suitably, an increase in MCH may be associated with a positive effect on reducing the risk of death.Thus, an increase in MCH may be associated with a reduced risk of death.

[0122] Serum glucose

[0123] Serum glucose is a measure of the amount of glucose present in the blood. Glucose levels in the blood are controlled by hormones such as insulin to keep glucose levels within a normal range. When glucose levels in the blood are outside of normal levels, it can indicate a disease, such as diabetes.

[0124] Methods for measuring serum glucose levels are well known in the art, and serum glucose levels are usually expressed in milligrams per deciliter (mg / dL). Most glucose determinations are photometric, and there are many commercial devices available. For example, Acon Pharmaceuticals offers a veterinary glucose monitoring system (Acon Pharmaceuticals Inc.'s GK), and Carradini et al. described the use of a continuous glucose monitoring device in dogs (FreeStyle Libre from Abbott Laboratories).

[0125] Suitably, an increase in serum glucose levels may be associated with a negative impact on reducing the risk of death. Thus, an increase in serum glucose levels may be associated with an increased risk of death.

[0126] Mean corpuscular volume

[0127] The mean corpuscular volume, or mean corpuscular volume (MCV), is a measure of the average volume of red blood cells in the blood. MCV is a diagnostic criterion that can classify possible anemia as microcytic, normocytic, or macrocytic, and can help identify underlying diseases or conditions. A high MCV can indicate conditions such as vitamin B12 deficiency, while a low MCV can indicate conditions such as iron deficiency.

[0128] Methods for determining mean corpuscular volume, which is typically expressed in femtoliters (fL), are well known in the art. Although modern hematology analyzers (e.g., ProCyte DxHematology Analyzer from IDEXX Laboratories Inc.) are able to measure MCV directly as part of a complete blood count (CBC), it can also be calculated from other measurements of hematocrit and red blood cell count.

[0129] Suitably, 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 reduced risk of death.

[0130] Serum globulin

[0131] Serum globulin is a measure of the concentration of globular proteins in the blood. Globular proteins are mainly secreted by the liver, and a small portion is secreted by immune cells. The most abundant of serum globulins is albumin. The remaining serum globulins can be separated into various components based on their behavior in electrophoretic separation methods. Immunoglobulins are important components 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 level of serum globulins can indicate certain conditions or diseases. An overall increase in serum globulins can indicate infection and inflammatory immune responses, while a decrease in levels can indicate bleeding, gastrointestinal disease, or severe malnutrition.

[0132] Methods for determining serum globulin levels are well known in the art, and serum globulin levels are usually expressed in grams per deciliter (g / dL). For example, chemical and physical methods are described in Tothova et al. (Veterinarni Medicina, 2016, 61: 475-496), and automated chemical analyzers from Idexx are also capable of measuring serum globulin levels.

[0133] Suitably, an increase in serum globulin levels may be associated with a negative impact on reducing the risk of death. Thus, an increase in serum globulin levels may be associated with an increased risk of death.

[0134] Serum calcium

[0135] Serum calcium is a measure of the total concentration of calcium in the blood. Calcium in the blood may be ionized, complexed, or bound to proteins. Calcium is necessary for a wide range of intracellular and extracellular functions in the body, including muscle contraction and blood coagulation, and is a major component of bone. Excessively high calcium levels may be the result of certain cancers or bone disorders, while low calcium levels may be the result of kidney disease, pancreatitis, or decreased serum albumin.

[0136] Methods for determining serum calcium levels are well known in the art and are usually expressed in milligrams per deciliter (mg / dL). A colorimetric method for determining calcium in serum was described by F. Gran (Acta Physiologica Scandinavica; 1960, 49: 192-197), and modern automated chemistry analyzers are capable of measuring calcium levels in serum.

[0137] Suitably, an increase in serum calcium levels may be associated with a positive effect on reducing the risk of death.Thus, an increase in serum calcium levels may be associated with a reduced risk of death.

[0138] Platelet count

[0139] Platelets, also known as thrombocytes, are small cells that make up blood. They are small cells without a nucleus and are produced by the cytoplasm of bone marrow cells called megakaryocytes. Platelets help the clotting process to stop bleeding at sites of damaged blood vessels. Platelet count levels above or below the normal range can indicate a condition or disease. In particular, a decrease in platelet count may be the result of certain infections, cancer, immune system disorders, or pancreatitis.

[0140] Methods for measuring platelet counts are well known in the art, and platelet counts are typically expressed as thousands of cells per microliter (10^3 / uL). Platelet counts can be performed manually on blood smears using staining and microscopy methods, but are typically performed as part of an automated complete blood count (CBC).

[0141] Suitably, an increase in platelet count may be associated with a negative impact on reducing the risk of death. Thus, an increase in platelet count may be associated with an increased risk of death.

[0142] Red blood cell count

[0143] Red blood cells, also known as erythrocytes, are the most abundant cells present in the blood. These cells do not contain a nucleus, but are composed primarily of hemoglobin contained within the cell membrane to maximize their oxygen-carrying potential. Red blood cell counts that are higher or lower than normal indicate a condition or disease. A low red blood cell count may indicate hemolysis, blood loss, or decreased red blood cell production, which may be caused by a variety of reasons. A high red blood cell count may indicate a relative increase in red blood cells per volume of blood compared to normal due to dehydration or increased red blood cell production.

[0144] Methods for measuring red blood cell counts are well known in the art and are typically expressed in thousands of cells per microliter (10^3 / uL). Red blood cell count measurements can be performed manually on a blood smear using microscopy, but are typically performed as part of an automated complete blood count (CBC).

[0145] Suitably, 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 reduced risk of death.

[0146] Combination of biomarkers

[0147] Although a single biomarker may have predictive value in the methods of the invention, the quality and / or predictive power of the method may be improved by combining values ​​from multiple biomarkers.

[0148] Thus, the method may involve determining the level of at least two of the biomarkers described herein. For example, the method may include determining the level of two or more biomarkers in one or more samples 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, platelet count and / or red blood cell count.

[0149] As used herein, the term "one or more biomarkers" may include at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or at least thirteen biomarkers.

[0150] As used herein, the term "one or more biomarkers" may include one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen biomarkers.

[0151] Suitably, the method may comprise determining the level of white blood cell count, serum albumin and serum alkaline phosphatase in one or more samples. Advantageously, it has been determined that this combination of three biomarkers can significantly predict the risk of death and / or the probability of healthy life span. The predictive power may be further increased by combining one or more of the 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.

[0152] Suitably, the method may comprise determining the level of each 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.

[0153] Compare with a reference or control

[0154] The method may also include the step of comparing the levels of various biomarkers in the test sample with one or more reference or control values. The reference value may be associated with a predefined risk of death and / or probability of healthy life span. In some embodiments, the reference value is a value previously obtained for a subject or subject group with a known death outcome. The reference value may be based on the average level, such as mean or median level, from a subject group with a known chronological age, breed, sex and / or death outcome. Preferably, the reference value may be based on the average level, such as mean or median level, from a subject group with a known chronological age, breed, sex and death outcome.

[0155] Combining biomarker levels with additional measures and / or characteristics

[0156] Suitably, the method further comprises combining the level of one or more biomarkers with one or more of the chronological age, breed and / or sex of the dog. By combining this information, an improved model for the probability of mortality risk and / or healthy life span of the dog is provided.

[0157] In a preferred embodiment, the levels of one or more biomarkers defined herein are determined in a sample from a dog and these levels are combined with the chronological age, breed and sex of the dog in order to determine the risk of mortality and / or probability of healthy life span for the dog.

[0158] Preferably, the risk of death and / or the probability of healthy life span is expressed as phenotypic age (Phenoage), which is given by the following formula:

[0159]

[0160] where xb is the sum of the values ​​of each biomarker, sex and breed multiplied by their corresponding coefficients according to formula (2):

[0161]

[0162] where sex is encoded as a numeric value, with 0 representing females and 1 representing males.

[0163] The breed is coded as a numeric value, where 0 represents a small breed and 1 represents a medium breed.

[0164] The coefficient value of each parameter usually depends on the measurement unit of all variables in the model. As the technician will understand, the exact value of each coefficient value will therefore depend on the quantity and property of the different parameters used in the model and the property of the training data provided. Therefore, conventional statistical methods can be applied to the training data set to obtain the coefficient value for the above formula. This method includes, for example, calculating two gompertz functions (for example, in the case of the state (survival or death) of a known dog) on ​​the training set, a function models the survival rate as a function of selected biomarkers, chronological age, breed category (small or medium-sized dog) and sex (model 1), and the second function only considers chronological age, breed category and sex (model 2). These models can be fitted using flexsurv package (v 2.1) in the R software environment.

[0165] Suitably, a negative coefficient for a given biomarker means that a higher level of the biomarker has a positive effect on reducing the risk of death, and / or a lower level of the biomarker has a negative effect on reducing the risk of death. Suitably, a positive coefficient for a given biomarker means that a higher level of the biomarker has a negative effect on reducing the risk of death, and / or a lower level of the biomarker has a positive effect on reducing the risk of death.

[0166] Phenotypic age can be defined as the time variable ("chronological age") at which the probability of survival of an animal given by Model 2 is equal to the probability of survival given by Model 1 at their chronological age.

[0167] The phenotypic age of a dog can be expressed in years, months, days, etc.

[0168] Preferably, the risk of death and / or the probability of healthy life span is expressed as the difference between the phenotypic age and the chronological age of the dog. This difference can be referred to as the phenotypic age advance of the dog.

[0169] For example, an increase in phenotypic age compared to chronological age may indicate an increased risk of death for a dog. For example, a decrease in phenotypic age compared to chronological age may indicate a decreased risk of death for a dog. As an illustration, the inventors determined that a difference between phenotypic age and chronological age (an advance in phenotypic age) was associated with a significant increase in risk of death, and the magnitude of the effect was calculated as: a hazard ratio of 1.75 for a 1-year increase in phenotypic age compared to chronological age (see Example 3). In other words, the inventors determined that a 1-year increase in phenotypic age compared to chronological age was associated with a 75% increase in risk of death at any given point in life.

[0170] Individual classification

[0171] The probability of the risk of death and / or healthy life span determined by the method of the present invention can also be compared with one or more predetermined threshold values. Using this threshold value, the subject can be stratified into each category indicating the probability of the risk of death and / or healthy life span determined (e.g., low, medium or high determination of the risk of death). The divergence degree with the threshold value can be used to determine which individuals will benefit most from certain interventions. In this way, dietary intervention and lifestyle changes can be optimized. The probability of the determined risk of death and / or healthy life span can be represented by a numerical score or percentage, for example, it can indicate the probability of the determined risk of death and / or healthy life span compared to a control or reference group.

[0172] Method for selecting / monitoring a subject's lifestyle or dietary regimen

[0173] In another aspect, the invention provides a method for selecting a lifestyle or dietary regimen for a subject. The change in lifestyle can be any change described herein, such as a change in dietary intervention and / or an exercise regimen. The change in lifestyle can be the administration of a therapeutic approach.

[0174] Lifestyle or dietary regimen can be applied to dogs in any suitable time period. After the period of time, the method can be used to determine the risk of death and / or the probability of healthy life span of the dog again, so as to determine the efficacy of the lifestyle or dietary regimen to reduce the risk of death and / or increase the probability of healthy life span of the dog. For example, the lifestyle or dietary regimen can 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 dietary regimen can 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.

[0175] Preferably, the change is a dietary intervention as described herein. The term "dietary intervention" refers to an external factor that is applied to a subject and causes a change in the subject's diet. More preferably, the dietary intervention comprises the administration of at least one dietary product or dietary regimen or nutritional supplement.

[0176] Dietary interventions can be meals, meal regimens, supplements, or supplement regimens.

[0177] The dietary intervention or dietary product described herein can be any suitable dietary regimen, such as a calorie-restricted diet, an elderly diet, a low-protein diet, a phosphorus diet, a low-protein 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 ω3-6 ratio diet, or a diet supplemented with carnitine, branched-chain amino acids or derivatives, nucleotides, nicotinamide precursors (such as nicotinamide mononucleotide (MNM) or nicotinamide riboside (NR)), or any combination of the foregoing.

[0178] Suitably, the dietary intervention or dietary product may be a calorie restricted diet, an elderly diet or a low protein diet. Suitably, the dietary intervention or dietary product may be a calorie restricted diet. Suitably, the dietary intervention or dietary product may be a low protein diet.

[0179] The dietary intervention may be determined based on the dog's baseline maintenance energy requirement (MER). Suitably, the MER may be the amount of food that stabilizes the dog's body weight (varies by less than 5% over three weeks).

[0180] For example, it is generally believed that young, growing dogs benefit from a high-energy / high-protein diet; however, older dogs may have lower energy requirements, so the diet may be adjusted appropriately. In particular, many manufacturers produce "senior" lines of dog foods that are lower in calories and higher in fiber, but with protein and fat levels appropriate for older dogs.

[0181] Suitably, the calorie restricted diet may comprise about 60%, about 65%, about 75% or about 80% of the dog's MER. Suitably, the calorie restricted diet may comprise about 60% or about 75% of the dog's MER.

[0182] Suitably, the low protein diet may comprise less than 20% protein (dry matter %).For example, the low protein diet may comprise less than 15% or less than 10% protein (dry matter %).

[0183] These diets are often recommended based on the dog's chronological age. For example, it may be recommended that a dog be switched to a senior diet around 7 or 8 years old. However, in the context of the present invention, determining that a dog has an increased risk of death and / or a decreased probability of healthy life span than would be expected given its chronological age may allow for a determination to switch a dog to a senior diet at an earlier age. In contrast, a dog with a decreased risk of death and / or an increased probability of healthy life span compared to its chronological age may be able to continue on a high energy / high protein diet for longer.

[0184] Dietary interventions may include foods, supplements and / or beverages that contain nutrients and / or bioactive agents that mimic the benefits of caloric restriction (CR) without limiting daily caloric intake. For example, foods, supplements and / or beverages may contain functional ingredients with benefits similar to CR. Suitably, foods, supplements and / or beverages may contain autophagy inducers. Suitably, foods, supplements and / or beverages may contain fruits and / or nuts (or their extracts). Suitable examples include, but are not limited to, pomegranates, strawberries, blackberries, camu camu, walnuts, chestnuts, pistachios, pecans. Suitably, foods, supplements and / or beverages may contain probiotics, with or without fruit extracts or nut extracts.

[0185] Modifying the subject's lifestyle also includes indicating that the subject needs to make a lifestyle change, such as prescribing more exercise. Similar to dietary intervention, determining that the dog has an increased risk of mortality and / or a decreased probability of healthy lifespan compared to what would be expected given its chronological age can allow for a determination to switch the dog to an appropriate exercise regimen.

[0186] Changing the lifestyle of the experimenter also includes recommending a treatment mode or scheme. Treatment mode or scheme can be a mode for treating and / or preventing, for example, arthritis, dental disease, endocrine disorders, heart disease, diabetes, liver disease, nephropathy, prostate disorders, cancer, and behavioral or cognitive disorders. Suitably, preventive treatment can be applied to a dog identified as being in the risk of such a disease due to an increase in the risk of death (phenotype age) and / or a specific biomarker based on a known disease-related pathway. In other embodiments, a dog determined to be in the risk of certain morbidities (due to an increase in the risk of death (phenotype age) and / or a specific biomarker based on a known disease-related pathway) can be monitored more regularly so that diagnosis and treatment can be started as early as possible.

[0187] Thus, the present invention can advantageously enable identification of dogs that are expected to respond particularly well to a given intervention (e.g., lifestyle or dietary regimen). Thus, intervention can be applied in a more targeted manner to dogs that are expected to respond.

[0188] The present invention also provides a method for determining the efficacy of a lifestyle or dietary regimen in reducing the risk of mortality and / or increasing the probability of a healthy life span in a dog, the method comprising:

[0189] a. determining a dog's first mortality risk and / or probability of healthy life span according to the method of the first aspect of the present invention;

[0190] b. administering a lifestyle or dietary regimen to a dog;

[0191] c. determining the second mortality risk and / or health life expectancy of the dog according to the method of the first aspect of the present invention after the lifestyle or dietary regimen has been applied to the dog for a period of time;

[0192] d. Determining whether there is a change between the first risk of death and / or probability of healthy life span and the second risk of death and / or probability of healthy life span for the dog after a period of following the lifestyle or dietary regimen.

[0193] The present invention also provides a method for determining the efficacy of a lifestyle or dietary regimen in reducing the risk of mortality and / or increasing the probability of a healthy life span in a dog, the method comprising:

[0194] a. administering a lifestyle or dietary regimen to a dog, wherein the lifestyle or dietary regimen has been selected according to a method comprising performing a method according to the first aspect of the invention; and selecting an appropriate lifestyle or dietary regimen based on the determined risk of death and / or probability of healthy life span;

[0195] b. determining the probability of a second mortality risk and / or healthy life span of the dog by performing the method of the first aspect of the present invention after the lifestyle or dietary regimen has been applied to the dog for a period of time;

[0196] c. Determine whether there is a change in the dog's risk of mortality and / or probability of healthy life span after a period of following the lifestyle or dietary regimen.

[0197] The present invention also provides a method for determining the efficacy of a lifestyle or dietary regimen in reducing the risk of mortality and / or increasing the probability of a healthy life span in a dog, the method comprising:

[0198] a. selecting a lifestyle or dietary regimen for a dog according to a method comprising performing the method according to the first aspect of the present invention; and selecting an appropriate lifestyle or dietary regimen based on the determined risk of death and / or probability of healthy life expectancy;

[0199] b. administering a lifestyle or dietary regimen to a dog;

[0200] c. determining the second mortality risk and / or health life expectancy of the dog according to the method of the first aspect of the present invention after the lifestyle or dietary regimen has been applied to the dog for a period of time;

[0201] d. Determine whether the dog's risk of mortality has changed between step a and step c.

[0202] After a period of administration of the lifestyle or dietary regimen, a reduction in the second (or subsequent) risk of death determined for the dog compared to the first (or earlier) risk of death determined for the dog indicates that the lifestyle or dietary regimen is effective in reducing the dog's risk of death.

[0203] The probability of the risk of death and / or healthy life span of a dog can be determined before and after the lifestyle or dietary regimen is applied to the dog. The probability of the risk of death and / or healthy life span of a dog can also be determined at the scheduled time during the application of the whole lifestyle or dietary regimen. These scheduled times can be periodic during the whole lifestyle or dietary regimen, such as every day or every three days, every week, every two weeks, every month, every two months, every 6 months, every year or every two years. The scheduled time can depend on the subject being tested. Suitably, before determining the probability of the first risk of death and / or healthy life span, a lifestyle or dietary regimen may have been applied to a dog for a period of time; However, the effectiveness of the probability of the risk of death and / or healthy life span can still be monitored by determining the probability of the risk of death and / or healthy life span at two or more scheduled times during the application of the lifestyle or dietary regimen to reduce the risk of death and / or increase the probability of healthy life span.

[0204] Uses of dietary intervention

[0205] In one aspect, the invention provides a dietary intervention for reducing the risk of mortality and / or increasing the probability of healthy life span in a dog, wherein the dietary intervention is administered to a dog, wherein the risk of mortality and / or the probability of healthy life span is determined by the present method.

[0206] In another aspect, the invention provides for use of a dietary intervention in reducing predicted mortality risk and / or increasing probability of healthy life span in a dog, wherein the dietary intervention is administered to the dog, wherein the mortality risk and / or probability of healthy life span is determined by the present method.

[0207] As described herein, a dietary intervention can be a dietary product or a dietary regimen or a nutritional supplement.

[0208] Computer program product

[0209] The method can be performed using a computer. Therefore, the method can be performed on a computer.

[0210] The methods described herein can be implemented as computer programs running on general-purpose hardware such as one or more computer processors. In some embodiments, the functions described herein can be implemented by devices such as smart phones, tablet terminals or personal computers.

[0211] In one aspect, the present invention provides a computer program product comprising computer-implementable instructions for causing a programmable computer to determine the risk of mortality and / or probability of healthy life span for a dog as described herein.

[0212] In another aspect, the invention provides a computer program product comprising computer-implementable instructions for causing a device to determine a dog's risk of death and / or probability of healthy life span given the level of one or more biomarkers from a user, wherein the biomarker is selected from one or more biomarkers as defined herein. Preferably, the biomarker level is a fasting level. Additional parameters or characteristics of the dog may also be provided to the computer program product. As described herein, additional parameters or characteristics may include chronological age, breed, and gender.

[0213] In one embodiment, the user inputs the level of one or more of the biomarkers as defined herein into the device, optionally along with chronological age, breed and sex. The device then processes this information and provides a determination of the dog's risk of mortality and / or probability of healthy life span.

[0214] The device can generally be a server on a network. However, any device can be used as long as it can process biomarker data and / or additional parameters or characteristics using a processor, a central processing unit (CPU), etc. The device can be, for example, a smart phone, a tablet terminal, or a personal computer, and outputs information indicating the determined risk of death and / or the probability of healthy life span of the dog. The determined risk of death and / or the probability of healthy life span can be expressed in numerical scores or percentages, for example, it can indicate the determined risk of death and / or the probability of healthy life span compared to a control or reference population.

[0215] Those skilled in the art will understand that they can freely combine all features of the present invention described herein without departing from the scope of the invention disclosed herein.

[0216] Example

[0217] The present invention will now be further described by way of examples, which are intended to help those skilled in the art to practice the present invention but are not intended to limit the scope of the present invention in any way.

[0218] Example 1 - Determination of blood biomarkers associated with mortality risk in dogs

[0219] Predictive blood biomarkers were determined from a biomarker panel consisting of a standard clinical complete blood count (CBC) and standard clinical blood chemistry analysis. Serum samples were collected after an overnight fast and measured using standard veterinary clinical practice.

[0220] Table 1 - Clinical complete blood count (CBC) and clinical blood chemistry analysis

[0221]

[0222]

[0223] *Values ​​were log-transformed using the natural logarithm.

[0224] We conducted a longitudinal study of dogs with repeated measures of these parameters as well as on the status (alive or dead), sex, and breed of the dogs. We first classified the breeds as small or medium based on the mean weight of adult dogs of the breed (less than 10 kg or more than 10 kg, respectively). We then organized the data using the R programming language. For each dog, we recorded the biomarkers as time-dependent covariates using time intervals open on the left and closed on the right, i.e., (tstart, tstop), where the biomarker information corresponded to the start of the interval and the event (alive or dead) was recorded as the last tstop value. To do this, we used the tmerge function of the survival package in R (v.3.2-13). We then fitted a Cox proportional hazards model to the data for each of the 28 biomarkers, including sex and breed category (small or medium). We then adjusted the p-value for each parameter to account for multiple comparisons (by the false discovery rate (FDR)), and selected features with an adjusted FDR below 0.05 ( Figure 1 ).

[0225] Using this approach, we identified 13 biomarkers that individually predicted a dog's probability of survival:

[0226] White blood cell count (10^3 / ul)

[0227] Serum albumin (g / dL)

[0228] Serum alkaline phosphatase (U / L, ln conversion)

[0229] Serum creatine kinase (IU / L, ln conversion)

[0230] Hemoglobin (g / dL)

[0231] Hematocrit (%)

[0232] Mean corpuscular hemoglobin (pg)

[0233] Serum sodium (mmol / L)

[0234] Mean corpuscular volume (fL)

[0235] Serum globulin (g / dL)

[0236] Serum calcium (mg / dL)

[0237] Serum platelet count (10^3 / uL)

[0238] Red blood cell count (10^3 / uL)

[0239] Example 2 - Multi-parameter model for predicting mortality risk

[0240] Next, we constructed the best model that considered multiple parameters simultaneously, as this is more likely to cover the various organ dysfunctions that occur with aging. However, selecting several features that may be correlated with each other will be biased. To avoid this problem, we used a penalized regression approach using the glmnet package (v4.1-3). We fit a LASSO penalized cox proportional hazards model on the data and used 20-fold cross-validation to compare different values ​​of the penalty parameter λ. This approach selected the 10 most predictive blood biomarkers for survival, ranked in order of importance as follows:

[0241] White blood cell count (10^3 / ul)

[0242] Serum albumin (g / dL)

[0243] Serum alkaline phosphatase (U / L, ln conversion)

[0244] Serum creatine kinase (IU / L, ln conversion)

[0245] Hemoglobin (g / dL)

[0246] Hematocrit (%)

[0247] Mean corpuscular hemoglobin (pg)

[0248] Serum glucose (mg / dL)

[0249] Mean corpuscular volume (fL)

[0250] Serum globulin (g / dL)

[0251] We also found that the first 3 biomarkers in this list were the most predictive and that performance could be improved by combining each of the next 7 biomarkers.

[0252] To extract the phenotypic age of the animals, we calculated two different gompertz functions on our training set, one that modeled survival as a function of the selected biomarkers, age, breed class (small or medium dog) and sex (model 1), while the second function only considered age, breed class and sex (model 2). These models were fitted using the flexsurv package (v 2.1). Phenotypic age was defined as the time variable (“age”) under which the probability of survival of the animals given by model 2 was equal to the probability of survival given by model 1 at their chronological age. This resulted in a mathematical function that relates blood biomarkers to phenotypic age and is given by the following formula:

[0253]

[0254] where xb is the sum of the values ​​for each biomarker, sex, and breed multiplied by their corresponding coefficients. Sex and breed are encoded as numeric values, with 0 for females and 1 for males for sex and 0 for small breeds and 1 for medium breeds for breeds. The coefficients are given by two gompertz functions trained on our training set.

[0255] As an example, the coefficients γ and γ 品种 Values ​​have been measured for the full list of biomarkers from our training set and are given in Table 2 .

[0256]

[0257] Table 2 - Coefficients and γ and γ 品种 The values ​​have been measured from the training set

[0258] coefficient γ 0.491790219 <![CDATA[β0]]> -6.036261473 Beta cell count 0.091862564 Beta hemoglobin -0.009131623 β mean corpuscular volume -0.007486146 Beta hematocrit -0.018418391 β-mean corpuscular hemoglobin -0.128195615 β serum glucose 0.009169677 Beta serum globulin 0.132755858 Serum creatine kinase beta 0.332818902 Beta serum albumin -0.744060565 Serum alkaline phosphatase β 0.262594338 Beta Varieties 1.138018960 Beta Gender 0.151826455 <![CDATA[γ 品种 ]]> 0.5668399 <![CDATA[β 02 ]]> -9.5204440 <![CDATA[β 品种2 ]]> 1.2299804 <![CDATA[β 性别2 ]]> 0.2678798

[0259] Furthermore, we observed a decrease in the strength of survival prediction (p-value) by reducing the set of 10 biomarkers by systematically removing one biomarker from the top of the list. The decrease was most pronounced for the first parameters, confirming their largest contribution, but we observed changes in the quality of predictions each time the set was reduced, indicating that each parameter contributes to the overall prediction ( Figure 2 ).

[0260] Example 3 - Using phenotypic age to predict mortality risk

[0261] It was subsequently demonstrated that when the resulting phenotypic age was applied to a test set consisting only of dogs not used during algorithm training, the difference between phenotypic age and chronological age (advancing phenotypic age) was associated with a significant increase in the risk of mortality. The effect size was calculated as: the hazard ratio for a 1-year increase in phenotypic age relative to chronological age was 1.75 ( Figure 3 ).

[0262] Furthermore, survival of dogs stratified in advance by high or low median phenotypic age showed a statistically significant difference between the upper and lower 50% ( Figure 4 ).

[0263] Phenotypic age (difference from chronological age) is altered in midlife by calorie-restricted diet (75% baseline maintenance energy requirement (MER)). This change occurs earlier in females than in males ( Figure 5 ).

[0264] Stratification of dogs aged 7 years and under and 7 years and older revealed significant differences in older dogs compared to younger dogs. This was more severe in females than in males ( Figure 6 ).

[0265] Example 4 - Low protein diet reduces phenotypic age advancement

[0266] dog

[0267] Thirty dogs with a body condition score (BCS) of 7 or greater were enrolled in this weight loss study. The baseline maintenance energy requirement (MER) for each dog was determined as the amount of food that stabilized the dog's weight (less than 5% change over three weeks). The baseline body fat % for each dog was then determined by DEXA. The dogs were randomized into 2 groups of 15 dogs each based on baseline weight, MER, age, sex, and body fat %.

[0268] Test diet

[0269] The two diets were comparable in metabolizable energy (ME) but differed in protein, carbohydrates, fat, and fiber.

[0270]

[0271]

[0272] Feeding Instructions

[0273] Dogs in both groups were fed 75% of their baseline MER during the first 4 months of the study and 60% of their baseline MER during the second 2 months of the study.

[0274] Blood sample collection and analysis

[0275] Serum samples were collected at baseline, 2 months, 4 months, and 6 months of the study. At the end of the study, a complete blood count (CBC) and blood chemistry panel were performed on these serum samples.

[0276] result

[0277] like Figure 7As shown, all but one dog showed a reduction in phenotypic age advance compared to baseline, with phenotypic age advance defined as the difference between the dog's phenotypic age and the dog's chronological age after 6 months of the weight-reducing diet. The mean difference in phenotypic age advance between baseline and post-intervention was 0.7 years, which was significant in a paired t-test (p=0.00093). The age of the dogs in the study ranged from 3 to 11 years. We did not find a correlation between phenotypic age advance and age of the dogs at the start of the study, suggesting that the benefits extend across a wide range of dog life stages. Furthermore, despite the two diets having completely different protein levels, the effects of both diets on phenotypic age were comparable, suggesting that a reduction in caloric intake results in a younger phenotypic age regardless of the dog's macronutrient ratio.

[0278] All publications mentioned in the above description are incorporated herein by reference. Various modifications and variations of the methods, compositions and uses disclosed in the present invention will be apparent to the skilled person without departing from the scope and spirit of the present invention. Although the present invention has been disclosed in conjunction with specific preferred embodiments, it should be understood that the present invention protected by the claims should not be unduly limited to such specific embodiments. In fact, various modifications to the modes disclosed for practicing the present invention that are apparent to the skilled person are intended to fall within the scope of the following claims.

Claims

1. A method for determining the risk of mortality and / or probability of healthy life span in a dog; the method comprising determining the level of one or more biomarkers in one or more samples obtained from the dog, wherein the one or more biomarkers are selected from the group consisting 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 / 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 according to any preceding claim, wherein the biomarker is serum alkaline phosphatase.

5. A method according to any preceding claim, wherein the method comprises measuring each of white blood cell count, serum albumin and serum alkaline phosphatase.

6. 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. A method according to any preceding claim, wherein the one or more samples are blood samples.

8. The method of any preceding claim, wherein the method further comprises combining the levels of the one or more biomarkers with one or more of the chronological age, breed and / or sex of the dog.

9. A method according to any preceding claim, wherein the dog is classified as small, medium, large or giant and / or stocky or athletic.

10. A method for determining the risk of mortality and / or the probability of healthy life span in a dog; the method comprising: a. Determine the levels 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 globulins in one or more samples obtained from the dog; and b. Determine the phenotypic age (Phenoage) of the dog using formula (1): where xb is the sum of the values ​​of each biomarker, sex and breed multiplied by their corresponding coefficients according to formula (2): where sex is encoded as a numeric value, with 0 representing females and 1 representing males. The breed is coded as a numerical value, where 0 represents a small breed and 1 represents a medium breed; as well as c. Using the phenotypic age to determine the risk of mortality and / or probability of healthy life span for the dog.

11. The method of claim 10, wherein determining the phenotypic age of the dog to be greater than the chronological age of the dog indicates a higher risk of mortality and / or a decreased probability of healthy lifespan.

12. The method of claim 10, wherein determining that the phenotypic age of the dog is less than the chronological age of the dog indicates a lower risk of mortality and / or an increased probability of healthy lifespan.

13. A method according to any preceding claim, wherein the method is performed on one or more samples obtained before and after a time interval and determining whether the risk of mortality and / or probability of healthy life span of the dog has changed during the time interval.

14. A method for assessing the aging rate of a dog, the method comprising performing the method of any one of claims 10 to 12; wherein (i) determining the phenotypic age of the dog to be greater than the chronological age of the dog indicates a faster aging rate, or (ii) determining the phenotypic age of the dog to be less than the chronological age of the dog indicates a slower aging rate.

15. A method for selecting a lifestyle or dietary regimen for a dog, the method comprising: a. Execute the method as claimed in any one of claims 1 to 13; as well as b. Selecting a suitable lifestyle or dietary regimen based on the mortality risk and / or probability of healthy life expectancy determined in step a.

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

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. A method for determining the efficacy of a lifestyle or dietary regimen on improving mortality risk and / or probability of healthy life span in a dog, the method comprising: a. Determining the first mortality risk and / or the probability of healthy life span of the dog according to the method of any one of claims 1 to 13; b. administering a lifestyle or dietary regimen to the dog; c. after administering the lifestyle or dietary regimen to the dog for a period of time, Determining a second mortality risk and / or a probability of healthy life span of the dog according to the method of any one of claims 1 to 13; d. determining whether there is a difference between said first risk of mortality and / or probability of healthy life span and said second risk of mortality and / or probability of healthy life span for said dog after said period of following said lifestyle or dietary regimen.

19. A method for determining the efficacy of a lifestyle or dietary regimen in reducing the risk of mortality and / or the probability of healthy life span determined for a dog, the method comprising: a. administering a lifestyle or dietary regimen to the dog, wherein the lifestyle or dietary regimen has been selected according to the method of any one of claims 15 to 17; b. performing the method according to any one of claims 1 to 13 after the lifestyle or dietary regimen has been applied to the dog for a period of time; c. determining whether there is a change in the risk of mortality and / or probability of healthy life span of the dog after following the lifestyle or dietary regimen for the period of time.

20. A method for determining the efficacy of a lifestyle or dietary regimen in reducing the risk of mortality and / or increasing the probability of a healthy life span in a dog, the method comprising: a. The method according to any one of claims 15 to 17 selects a lifestyle or dietary regimen for the dog; b. administering the lifestyle or dietary regimen to the dog; c. after administering the lifestyle or dietary regimen to the dog for a period of time, Determining a second mortality risk and / or a probability of healthy life span of the dog according to the method of any one of claims 1 to 13; d. Determining whether the risk of mortality and / or probability of healthy life span of the dog has changed between step a and step c.

21. The method of any one of claims 18 to 20, comprising further modifying the lifestyle or dietary regimen so as to reduce the risk of mortality of the dog and / or increase the probability of a healthy life span of the dog.

22. A method of determining the likelihood that a dog will benefit from a lifestyle or dietary regimen; the method comprising: a) determining the risk of mortality and / or the probability of healthy life span of the dog according to the method of any one of claims 1 to 13; b) Identifying the dog as likely to respond to the lifestyle or dietary regimen if the dog has an increased risk of mortality and / or a decreased probability of healthy lifespan compared to the dog's chronological age.

23. A method for selecting a dog suitable for receiving a lifestyle or dietary regimen; the method comprising: a) determining the risk of mortality and / or the probability of healthy life span of the dog according to the method of any one of claims 1 to 13; b) The dog is selected as suitable for receiving the lifestyle or dietary regimen if the dog has an increased risk of mortality and / or a decreased probability of healthy lifespan compared to the dog's chronological age.

24. A method for reducing the risk of mortality and / or the probability of healthy life span in a dog; the method comprising: a. determining the risk of mortality and / or probability of healthy life span of the dog; the method comprising determining the level 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 globulin, serum calcium, platelet count and / or red blood cell count; b. selecting an appropriate lifestyle or dietary regimen based on the mortality risk and / or health life expectancy probability determined in step a; c. administering the selected lifestyle or dietary regimen to the dog.

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

26. A method for reducing the risk of mortality and / or increasing the probability of a healthy life span in a dog, the method comprising administering a dietary intervention to a dog identified as being in need of the dietary intervention by a method according to any one of claims 1 to 13.

27. A dietary intervention for reducing the risk of mortality and / or increasing the probability of a healthy life span in a dog, wherein the dietary intervention is administered to a dog, wherein the risk of mortality and / or the probability of a healthy life span is determined by a method according to any one of claims 1 to 13.

28. Use of a dietary intervention to reduce the risk of mortality and / or increase the probability of a healthy life span in a dog, wherein the dietary intervention is administered to a dog, wherein the risk of mortality and / or the probability of a healthy life span is determined by a method according to any one of claims 1 to 13.

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 elderly 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 to 13.

31. A computer system for determining the risk of mortality and / or probability of healthy life span for a dog, the computer system being programmed to perform one or more of the following steps: determining the risk of mortality and / or the probability of healthy lifespan for the dog given the level of one or more biomarkers in a sample from the dog, wherein the one or more biomarkers are selected from the group consisting 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 / or red blood cell count, and generating a report of the mortality risk and / or probability of healthy life span for the dog.

32. A computer program product comprising computer-implementable instructions for causing a programmable computer to determine the risk of mortality and / or probability of healthy life span for a dog given the level 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 globulin, serum calcium, platelet count and / or red blood cell count.

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

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

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

36. A method of treating a dog's risk of mortality that is greater than the risk of mortality of a comparable dog of the same chronological age, sex, and breed, the method comprising: using the method of any one of claims 1 to 13 to determine that the dog has a higher risk of death than the equivalent dog; as well as Based on the determination that the dog has a higher risk of mortality than the peer dogs, a dietary intervention is administered to the dog.

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

38. The method of claim 36 or claim 37, comprising, after administering the dietary intervention to the dog, additionally determining the risk of mortality for the dog to determine the efficacy of the dietary intervention; and then optionally (i) continuing to administer the dietary invention to the dog based on the efficacy of the dietary invention meeting a threshold, or (ii) administering a different dietary intervention to the dog based on the efficacy of the previously administered dietary intervention not meeting the threshold.

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

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) supplement, an antioxidant supplement, a vitamin B 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. The method of any one of claims 36 to 40, further comprising administering to the dog a lifestyle change based on the risk of death of the dog, the lifestyle change preferably comprising at least one of: (i) an exercise regimen, or (ii) a therapeutic modality that treats and / or prevents a condition selected from the group consisting of arthritis, dental disease, endocrine disorders, heart disease, diabetes, liver disease, kidney disease, prostate disorders, cancer, behavioral disorders, cognitive disorders, and combinations thereof.

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