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

Abstract

The present invention provides a method of assessing mortality risk and / or healthy life expectancy probability in a cat, the method comprising the step of measuring the value of one or more biomarkers in one or more samples obtained from the cat, wherein the one or more biomarkers are selected from white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, red blood cell count, serum sodium, serum cholesterol, serum potassium, serum alkaline phosphatase, and / or serum GGT.

Description

[Technical field]

[0001] The present invention relates to a method for assessing the health status, in particular the mortality risk and / or the longer healthy lifespan, of cats based on biomarkers. [Background technology]

[0002]

[0002] The ability to determine information regarding a cat's health is desirable to inform the health and well-being of the cat. For example, being able to assess information regarding a cat's health status can be useful in assessing whether a cat would benefit from a dietary or supplement-based intervention.

[0003]

[0003] Chronological age is known to be a major indicator of health status, and increasing chronological age is associated with declining health. However, genetics, nutrition, and lifestyle habits may cause an individual to age slower or faster than their chronological age. Thus, chronological age may not necessarily reflect an individual's rate of aging or risk of declining health. On the other hand, an individual's biological age (e.g., based on clinical biochemistry and cell biology measures) may differ compared to other individuals of the same chronological age. Methods for assessing biological age may help identify individuals who are at risk of age-related disorders earlier than expected based on their chronological age (see, e.g., WO 2019 / 165064).

[0004] However, additional methods for evaluating feline health information are needed. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2019 / 165064 Summary of the Invention

[0006] The present invention relates to a method for quantifying the health status of a cat based on biomarkers, in particular blood biomarkers. The method of the present invention allows the assessment of the phenotypic age of the cat to allow the calculation of the risk of mortality and / or the probability of a healthy lifespan for the cat.

[0007]

[0006] Existing methods for assessing cat health using blood biomarkers determine biological age based on correlation between blood biomarkers and chronological age. Calculating an animal's biological age may involve assessing the difference in the value of one or more biomarkers compared to the predicted value of the biomarker at a given chronological age. Such methods are therefore based on using chronological age as a primary indicator of overall health.

[0008]

[0007] In contrast, the present invention considers the direct predictive value of the described biomarkers in risk of mortality and / or probability of healthy lifespan. By way of example, a given biomarker may indicate a particular pathology without directly correlating with chronological age, and thus may indicate an increased risk of mortality and / or an increased probability of reduced healthy lifespan. Thus, the method of the present invention can be described as determining the phenotypic age (PhenoAge) of a cat. Thus, the biomarkers of the present invention do not necessarily correlate with chronological age, but rather relate to the difference between the phenotypic age and chronological age of a cat.

[0009]

[0008] The present invention provides a method for assessing mortality risk and / or healthy life expectancy probability in a cat, the method comprising the step of measuring the value of one or more biomarkers in one or more samples obtained from the cat.

[0010] In one aspect, the invention provides a method of assessing mortality risk and / or healthy life expectancy probability in a cat, the method comprising measuring the level of one or more biomarkers in one or more samples obtained from the cat, the one or more biomarkers being selected from white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, hematocrit, red blood cell count, serum albumin, and / or mean corpuscular hemoglobin concentration.

[0011]

[0010] The one or more biomarkers may be selected from white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, and / or red blood cell count.

[0012]

[0011] The inventors have shown that each of these biomarkers individually correlates with phenotypic age and is therefore predictive of mortality risk and / or healthy life expectancy.

[0013]

[0012] Assessing the risk of death can refer to assessing the likelihood that the cat will live for a longer or shorter period of time, for example, compared to a comparable cat of the same chronological age. Thus, the method of the present invention can assess the health span or healthy lifespan probability of the cat, for example, compared to a comparable cat of the same chronological age. Advantageously, the risk of death can be equivalent to the healthy lifespan probability of the cat, and a decrease in the risk of death is equivalent to an increase in the long-term healthy lifespan probability of the cat, or an increase in the risk of death is equivalent to a decrease in the long-term healthy lifespan probability of the cat. The risk of death can be expressed as the difference between the phenoage and chronological age of the cat (phenoage progression). For example, an increase in phenoage compared to chronological age can indicate an increase in the risk of death of the cat, whereas a decrease in phenoage compared to chronological age can indicate a decrease in the risk of death of the cat.

[0014] In one embodiment, the method of the invention may include measuring the white blood cell count in one or more samples obtained from the cat. The method of the invention may further include measuring the value of one or more biomarkers selected from hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, serum sodium, serum cholesterol, serum potassium, serum alkaline phosphatase, serum GGT, and / or red blood cell count in the one or more samples.

[0015] In one embodiment, the method of the invention may include measuring the level of hemoglobin in one or more samples obtained from the cat. The method of the invention may further include measuring the level of one or more biomarkers selected from white blood cell count, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, serum sodium, serum cholesterol, serum potassium, serum alkaline phosphatase, serum GGT, and / or red blood cell count in the one or more samples.

[0016] In one embodiment, the method of the invention may include measuring the level of serum urea nitrogen in one or more samples obtained from the cat. The method of the invention may further include measuring the level of one or more biomarkers selected from white blood cell count, hemoglobin, serum AST, serum chloride, serum total bilirubin, serum globulin, serum sodium, serum cholesterol, serum potassium, serum alkaline phosphatase, serum GGT, and / or red blood cell count in the one or more samples.

[0017] In one embodiment, the method of the invention may include measuring the level of serum AST in one or more samples obtained from the cat. The method of the invention may further include measuring the level of one or more biomarkers selected from white blood cell count, hemoglobin, serum urea nitrogen, serum chloride, serum total bilirubin, serum globulin, serum sodium, serum cholesterol, serum potassium, serum alkaline phosphatase, serum GGT, and / or red blood cell count in the one or more samples.

[0018] In one embodiment, the method of the invention may include measuring the level of serum chloride in one or more samples obtained from the cat. The method of the invention may further include measuring the level of one or more biomarkers selected from white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum total bilirubin, serum globulin, serum sodium, serum cholesterol, serum potassium, serum alkaline phosphatase, serum GGT, and / or red blood cell count in the one or more samples.

[0019] In one embodiment, the method of the present invention may include measuring the level of serum total bilirubin in one or more samples obtained from the cat. The method of the present invention may further include measuring the level of one or more biomarkers selected from white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum globulin, serum sodium, serum cholesterol, serum potassium, serum alkaline phosphatase, serum GGT, and / or red blood cell count in one or more samples.

[0020] In one embodiment, the method of the invention may include measuring the level of serum globulin in one or more samples obtained from the cat. The method of the invention may further include measuring the level of one or more biomarkers selected from white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum sodium, serum cholesterol, serum potassium, serum alkaline phosphatase, serum GGT, and / or red blood cell count in the one or more samples.

[0021] In one embodiment, the method of the invention may include measuring the level of serum sodium in one or more samples obtained from the cat. The method of the invention may further include measuring the level of one or more biomarkers selected from white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, serum cholesterol, serum potassium, serum alkaline phosphatase, serum GGT, and / or red blood cell count in the one or more samples.

[0022] In one embodiment, the method of the invention may include measuring the level of serum cholesterol in one or more samples obtained from the cat. The method of the invention may further include measuring the level of one or more biomarkers selected from white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, serum cholesterol, serum potassium, serum alkaline phosphatase, serum GGT, and / or red blood cell count in the one or more samples.

[0023] In one embodiment, the method of the invention may include measuring the level of serum potassium in one or more samples obtained from the cat. The method of the invention may further include measuring the level of one or more biomarkers selected from white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, serum cholesterol, serum alkaline phosphatase, serum GGT, and / or red blood cell count in the one or more samples.

[0024] In one embodiment, the method of the invention may include measuring the level of serum alkaline phosphatase in one or more samples obtained from the cat. The method of the invention may further include measuring the level of one or more biomarkers selected from white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, serum cholesterol, serum potassium, serum GGT, and / or red blood cell count in one or more samples.

[0025] In one embodiment, the method of the invention may include measuring the level of serum GGT in one or more samples obtained from the cat. The method of the invention may further include measuring the level of one or more biomarkers selected from white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, serum cholesterol, serum potassium, serum alkaline phosphatase, and / or red blood cell count in the one or more samples.

[0026] In one embodiment, the method of the invention may include measuring red blood cell count in one or more samples obtained from the cat. The method of the invention may further include measuring the value of one or more biomarkers selected from white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, serum cholesterol, serum potassium, serum alkaline phosphatase, serum GGT, and / or red blood cell count in the one or more samples.

[0027]

[0026] The method of the present invention may include the step of measuring each of the values ​​of white blood cell count, serum hemoglobin, serum urea nitrogen, and serum AST.

[0028]

[0027] The methods of the present invention may include a step of measuring the value of one or more additional biomarkers selected from hematocrit, red blood cell count, serum albumin, and / or mean corpuscular hemoglobin concentration in one or more samples obtained from the cat.

[0029]

[0028] In one embodiment, the method of the present invention includes measuring the values ​​of white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, hematocrit, red blood cell count, serum albumin, and mean corpuscular hemoglobin concentration.

[0030] In one embodiment, the present invention provides a method for assessing mortality risk and / or healthy life expectancy probability in a cat, the method comprising: a. measuring the levels of the following biomarkers in one or more samples obtained from the cat: white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, red blood cell count, serum sodium, serum cholesterol, serum potassium, serum alkaline phosphatase, and serum GGT; b. assessing the phenotypic age of the cat using formula (1):

number

[0031]

[0032] where xb is the sum of the values ​​of each biomarker multiplied by their respective coefficients according to equation (2).

number

[0032]

[0033] Phenotypic age is used to assess the cat's risk of mortality and / or healthy life expectancy.

[0033]

[0034] Preferably, assessing a cat's phenoage to be above its chronological age indicates a higher risk of mortality. Preferably, assessing a cat's phenoage to be below its chronological age indicates a reduced risk of mortality. Preferably, assessing a cat's phenoage to be above its chronological age indicates a reduced probability of long-term healthy lifespan. Preferably, assessing a cat's phenoage to be below its chronological age indicates an increased probability of long-term healthy lifespan.

[0034]

[0035] In one embodiment, one or more of the samples is from blood, for example a plasma sample.

[0035]

[0036] The present invention further relates to a method of selecting a lifestyle or diet plan for a cat, the method comprising: a. carrying out the method described in the first aspect of the present invention; b. selecting an appropriate lifestyle or diet plan based on the mortality risk and / or healthy life expectancy probability assessed in step a.

[0036]

[0039] The present invention further provides a method of assessing the effectiveness of a lifestyle or diet plan for reducing a risk of mortality in a cat, the method comprising the steps of: a. carrying out the method described in the first aspect of the invention; b. applying the lifestyle or diet plan changes to the cat; c. carrying out the method described in the first aspect of the invention after a period of applying the lifestyle or diet plan changes; and d. determining whether there has been a change in the risk of mortality of the cat between steps a and c.

[0037]

[0040] The invention further provides a dietary intervention for use in reducing the risk of mortality and / or increasing the probability of a healthy life span in a cat, the dietary intervention being administered to a cat whose risk of mortality has been assessed by the methods of the invention.

[0038]

[0041] The present invention also provides the use of dietary intervention to reduce an assessed risk of mortality in a cat and / or to increase a cat's healthy life expectancy, wherein the dietary intervention is administered to a cat whose risk of mortality and / or healthy life expectancy has been assessed by the method of the present invention.

[0039]

[0042] The present invention further provides a computer readable medium comprising instructions that, when executed, cause one or more processors to perform the methods of the present invention.

[0040]

[0043] The present invention also provides a computer system for assessing a cat's risk of mortality and / or healthy life probability, the computer system being programmed to perform one or more of the following steps: assessing the cat's risk of mortality and / or healthy life probability given values ​​of one or more biomarkers in a sample from the cat, where the one or more biomarkers are selected from white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, hematocrit, red blood cell count, serum albumin, and / or mean corpuscular hemoglobin concentration; and generating a report of the cat's risk of mortality and / or healthy life probability.

[0041]

[0044] The invention further provides a computer program product, the computer program product comprising computer executable instructions for causing a programmable computer to assess the mortality risk and / or healthy life expectancy probability of the cat given values ​​of one or more biomarkers from the cat, the one or more biomarkers being selected from white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, hematocrit, red blood cell count, serum albumin, and / or mean corpuscular hemoglobin concentration.

[0042]

[0045] Advantageously, embodiments of the invention in which the values ​​of multiple biomarkers are measured / assessed allow for the assessment of mortality risk and / or healthy life expectancy probability based on markers of multiple organ systems and functions. Thus, the use of multiple biomarkers advantageously encompasses a range of possible organ dysfunctions.

[0043]

[0046] By assessing the mortality risk or survival probability of cats, it is possible to examine several aspects of the well-being of animals.First, the measurement / assessment can predict whether this animal is more likely to need dietary or supplement-based intervention.Second, the measurement / assessment can be used to examine the effectiveness of dietary or supplement-based intervention on aging.It also provides a specific set of biomarkers that can be examined to evaluate the mortality risk or survival probability of animals and are routinely examined in veterinary clinics or clinics. [Brief description of the drawings]

[0044] [Figure 1] Figure 1 shows the identification of blood biomarkers predictive of risk of death. For each of the 28 biomarkers evaluated, including gender, a Cox proportional hazards model was fitted, and the p-values ​​of each parameter were adjusted to account for multiple comparisons (by false discovery rate (fdr)). Parameters with adjusted fdr less than 0.05 are shown. [Diagram 2] FIG. 1 shows demonstration of biomarkers contributing to the predictive ability of multi-parameter models for assessing phenoage. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045]

[0049] Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples, it being understood that a person skilled in the art can combine all features of the invention disclosed herein without departing from the scope of the invention disclosed.

[0046]

[0050] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0047]

[0051] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," or "containing," "contains," are inclusive or open-ended, and do not exclude additional, unspecified members, elements, or method steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0048]

[0052] Numeric ranges are inclusive of the numbers defining the range.

[0049]

[0053] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Any publications cited herein should not be construed as an admission that they constitute prior art to the claims appended hereto.

[0050]

[0054] The methods and systems disclosed herein can be used by veterinarians, medical professionals, laboratory technicians, pet care providers, and the like.

[0051]

[0055] subject

[0052]

[0056] The method of the present invention is directed to a feline subject. Thus, the subject of the present invention is a cat. Preferably, the cat is a domestic cat. More preferably, the cat is a domestic shorthair cat.

[0053]

[0057] sex

[0054]

[0058] Suitably, the sex of the cat may be categorised as male or female.

[0055]

[0059] Chronological age

[0056]

[0060] Chronological age may be defined as the amount of time that has elapsed since a subject's birth to a given date. Chronological age may be expressed in years, months, days, etc.

[0057]

[0061] Suitably, the method of the present invention can be applied to cats of any chronological age. In certain embodiments, the cat can be at least about 2 years old. Suitably, the cat can 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.

[0058]

[0062] sample

[0059]

[0063] The present invention involves measuring the level of one or more biomarkers in one or more samples obtained from a subject.

[0060]

[0064] Preferably, the sample is from blood. The sample may contain a blood fraction or may be whole blood. The sample preferably comprises plasma or serum. Techniques for obtaining samples from a subject are well known in the art.

[0061]

[0065] An appropriate sample may be selected based on the biomarker being measured. By way of example, if the biomarker is to be measured using a complete blood count (CBC), e.g., as part of a standard clinical complete blood count (CBC), then a whole blood sample should be used.

[0062]

[0066] The method of the invention may be performed on one or more samples obtained from a subject. For example, the method of the invention may be performed using a first sample obtained at a given time point and a second sample obtained after a time interval after the first sample was obtained. The method of the invention may be performed more than once on samples obtained from the same cat over a period of time. For example, samples may be repeatedly obtained once a month, once a year, or once every two years. Suitably, samples may be obtained about once a year (e.g., during an annual veterinary check-up). This may be useful, for example, in assessing the effect of a particular treatment, such as a change in dietary intervention or exercise regimen, or the effect of lifestyle changes.

[0063]

[0067] In one embodiment, the value of one or more biomarkers may be measured before a lifestyle change (e.g., a dietary product intervention or a change in exercise regimen). In another embodiment, the value of one or more biomarkers may be measured, for example, before and after a dietary product intervention or a change in exercise regimen. The value of the biomarkers may also be measured at predetermined time points, for example, during the dietary product intervention or during the change in exercise regimen. These predetermined time points may be periodic, for example, during the dietary product intervention or during the change in exercise regimen, for example, every day or every three days, or may vary depending on the subject being examined.

[0064]

[0068] Measuring the level of one or more biomarkers in a sample

[0065]

[0069] The biomarkers used in the present invention can be measured using standard methods in the art and are typically measured as part of a standard blood test to assess the disease state of an animal. For example, biomarkers are commonly measured as part of a standard clinical complete blood count (CBC) and standard clinical blood chemistry analysis.

[0066]

[0070] A complete blood count provides information about blood cells and their characteristics, such as red blood cells, white blood cells, and platelets. An exemplary complete blood count may include an automated process that uses flow cytometry or a Coulter counter to measure the number of cells in the blood. In addition to measuring cell counts, such automated systems may also be capable of measuring other blood biomarker readings depending on their complexity. Such systems can simultaneously measure blood cell counts, as well as red blood cell volume, hemoglobin value, mean corpuscular hemoglobin value, and hematocrit. For example, IDEXX Laboratories offers a hematology analyzer that can measure white blood cell count (WBC), red blood cell count (RBC), platelet count (PLT), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) (IDEXX Laboratories Inc., ProCyte Dx Hematology Analyzer).

[0067]

[0071] The values ​​of other biomarkers not related to blood cells, such as serum protein / enzyme and / or molecular biomarkers, can be measured using chemical tests, particularly using automated chemistry analyzer systems. These methods may utilize colorimetric-based approaches for quantification. For example, IDEXX Laboratories offers an automated chemistry analyzer that can quantify serum albumin, serum alkaline phosphatase, serum creatine kinase, serum glucose, serum globulins, and serum calcium (IDEXX Laboratories Inc., Catalyst One Chemistry Analyzer).

[0068]

[0072] Thus, methods for measuring the levels of biomarkers used in the present invention may include assays that result in a spectrophotometric change (e.g., a chemical or antibody binding change that results in a detectable signal at a particular wavelength). Such tests can be highly automated and efficient and form the basis of many routine veterinary health tests.

[0069]

[0073] Suitably, biomarker levels are assessed after an overnight fast and may be measured according to standard veterinary clinical practice.

[0070]

[0074] The value of each biomarker in a sample can be measured or evaluated by any suitable method known in the art. For example, mass spectrometry (MS), antibody detection methods such as enzyme-linked immunosorbent assay (ELISA), non-antibody protein scaffolds (e.g., fibronectin scaffolds), radioimmunoassays (RIA), or aptamers can be used. Other spectroscopic, chromatographic, labeling techniques, or quantitative chemical methods can also be used.

[0071]

[0075] Antibodies suitable for use in the above methods are known in the art and / or can be generated using known techniques. Suitable testing methods for detecting antibody levels include, but are not limited to, immunoassays, such as enzyme-linked immunosorbent assays, radioimmunoassays, Western blots, and immunoprecipitations.

[0072]

[0076] Biomarkers

[0073]

[0077] white blood cell count

[0074]

[0078] White blood cells, also written as leukocytes, are a type of cell found in blood. They have various immune-related functions depending on the subtype: monocytes, lymphocytes, neutrophils, basophils, and eosinophils. White blood cells contain a nucleus and have a variable cell shape depending on the subtype. The white blood cell count is the number of cells of this type per volume of blood.

[0075]

[0079] Methods for counting white blood cell count, typically expressed as thousands of cells per microliter (10^3 / μL), are known in the art. White blood cell counts can be performed manually on blood smears using staining and microscopic techniques, but can also be performed as part of an automated complete blood count (CBC). IDEXX Laboratories offers an automated hematology analyzer capable of counting white blood cells.

[0076]

[0080] Advantageously, an increased white blood cell count may be associated with a negative effect on reducing the risk of death. Accordingly, an increased white blood cell count may be associated with an increased risk of death.

[0077]

[0081] hemoglobin

[0078]

[0082] Hemoglobin is a transport protein in red blood cells. It consists of a tetramer of two α-chains and two β-chains. Each peptide chain is bound to a heme group, which consists of a porphyrin ring with an iron ion attached. The heme group can reversibly bind oxygen, allowing hemoglobin to function as an oxygen transporter protein.

[0079]

[0083] Methods for measuring hemoglobin levels, typically expressed as grams per deciliter (g / dL), are well known in the art. The International Committee for Standardization in Haematology has described a standardized method that utilizes spectrophotometric measurement of cyanide hemoglobin (Br J Haematol. 1967 Apr; 13: 71-5), and this approach can be used in commercially available automated chemistry analyzers, such as those provided by IDEXX Laboratories.

[0080]

[0084] Advantageously, an increase in hemoglobin level may be associated with a positive effect on a reduced risk of mortality. Thus, an increase in hemoglobin may be associated with a reduced risk of mortality.

[0081]

[0085] Serum urea nitrogen

[0082]

[0086] Urea is a waste product resulting from the breakdown of nitrogenous compounds and is the main means of nitrogen excretion from the body. It is produced in the liver from ammonia, a toxic by-product of protein metabolism. Urea circulates in the blood until it is removed by the kidneys and excreted in the urine. Changes in the value of serum urea nitrogen, also known as blood urea nitrogen, is a measure of the amount of urea in the blood and can indicate certain conditions and diseases. An overall increase in serum urea nitrogen can indicate kidney disease or starvation, while a decrease in the value can indicate chronic or severe liver disease or a low-protein diet.

[0083]

[0087] Methods for measuring serum urea nitrogen, usually expressed in milligrams per deciliter (mg / dL, natural log transformed), are well known in the art. A test commonly referred to as the blood urea nitrogen (BUN) test is a common test performed on serum as part of a chemistry panel. Generally, serum urea nitrogen can be measured by colorimetry or enzymatic conductivity, for example as described in EP 0163367 and U.S. Patent Application Publication No. 2006 / 0046275. The automated chemistry analyzer available from IDEXX is a commercially available system that can provide serum urea nitrogen readings.

[0084]

[0088] Advantageously, an increase in serum urea nitrogen may be associated with a negative effect on reducing the risk of mortality. Thus, an increase in serum urea nitrogen may be associated with an increase in the risk of mortality.

[0085]

[0089] serum AST

[0086]

[0090] AST, aspartate aminotransferase, is an enzyme involved in amino acid metabolism. AST is found primarily in the liver and muscles. Serum AST is a measure of the amount of this enzyme present in the blood. High levels of AST in the blood can indicate liver or muscle damage.

[0087]

[0091] Methods for measuring serum AST, commonly expressed in units per liter (U / L, natural logarithm transformed), are well known in the art. AST serum values ​​can be measured using colorimetric or electrochemical methods, such as those described in EP 0523227 and WO 2017 / 117231. The automated chemistry analyzer available from IDEXX (IDEXX Laboratories Inc., Catalyst One Chemistry Analyzer) is a commercially available system that can provide serum AST readings.

[0088]

[0092] Advantageously, increased serum AST may be associated with a negative effect on reducing the risk of mortality. Thus, increased serum AST may be associated with an increased risk of mortality.

[0089]

[0093] Serum Chloride

[0090]

[0094] Serum chloride is a measure of the concentration of chloride in the blood. Chloride is one of the most abundant electrolytes found in blood and is important for regulating the water content of blood. Changes in blood chloride levels can indicate fluctuations in total body water, which can be caused by conditions such as vomiting or diarrhea.

[0091]

[0095] Methods for measuring serum chloride are usually expressed in millimoles per liter (mmol / L) and are well known in the art. For example, colorimetry, coulometry, mercurymetry, and chloride-specific ion electrode-based methods (Walker HK, Hall WD, Hurst JW, editors. Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd edition. Boston: Butterworths; 1990. Chapter 197). Commercially available automated analyzers can measure serum chloride levels, as described in Adin et al. (2020; Journal of veterinary internal medicine, 34(1), 53-64.), or using analyzers such as those commercially available from IDEXX laboratories.

[0092]

[0096] Advantageously, an increase in serum chloride level may be associated with a positive effect on a decreased risk of death. Thus, an increase in serum chloride level may be associated with a decreased risk of death.

[0093]

[0097] Serum total bilirubin

[0094]

[0098] Serum total bilirubin is a measure of the amount of bilirubin in blood. Bilirubin is a compound produced during the breakdown of heme from hemoglobin in old or damaged red blood cells, or in the breakdown of other heme-containing proteins, such as myoglobin, in other tissues. Bilirubin is processed by the liver, and the products are excreted via bile and urine. Increased serum total bilirubin levels may indicate impaired liver or bile duct function, or may indicate increased levels of red blood cells being destroyed. Increased serum bilirubin levels may be observed based on the yellowing of the skin, whites of the eyes, and mucous membranes, known as jaundice.

[0095]

[0099] Methods for measuring serum total bilirubin, usually expressed in milligrams per deciliter (mg / dL), are well known in the art. Bilirubin levels can be measured from blood samples by spectrophotometric assays, enzymatic assays, or, most commonly, diazo dye colorimetric assays (as described in Westwood A. (1991) The analysis of bilirubin in serum. Ann Clin Biochem 28;119-130). Automated analyzers are also commercially available from IDEXX for diagnostic use.

[0096]

[0100] Advantageously, an increase in serum total bilirubin may be associated with a negative effect on reducing the risk of mortality. Accordingly, an increase in serum total bilirubin may be associated with an increase in the risk of mortality.

[0097]

[0101] Serum globulins

[0098]

[0102] Serum globulin is a measure of the concentration of globular proteins in the blood. Globular proteins are secreted primarily by the liver and to a lesser extent by immune cells. Albumin is the most abundant serum globulin. The remaining serum globulins can be separated into fractions based on their behavior in electrophoretic separation methods. Immunoglobulins are an important part of the immune system and are secreted by immune cells. Examples of other serum globulins are immune system proteins such as complement, hormones, and carrier proteins such as ferritin. Changes in total serum globulin protein values ​​can indicate certain conditions or diseases. A generalized increase in serum globulins can indicate infection and an inflammatory immune response, while a decrease in values ​​can indicate bleeding, gastrointestinal disease, or severe malnutrition.

[0099]

[0103] Methods for measuring serum globulin levels, typically expressed in grams per deciliter (g / dL), are well known in the art, for example chemical and physical methods are described by Tothova et al. (Veterinarni Medicina, 2016, 61:475-496), and automated chemistry analyzers available from IDEXX can also measure serum globulin levels.

[0100]

[0104] Advantageously, an increase in serum globulin levels may be associated with a negative effect on reducing the risk of mortality. Thus, an increase in serum globulin levels may be associated with an increase in the risk of mortality.

[0101]

[0105] Hematocrit

[0102]

[0106] Hematocrit is the percentage by volume of red blood cells in blood. Hematocrit values ​​that deviate from normal values ​​may indicate a disease or condition in which the percentage of red blood cells in the blood is higher or lower than normal. High hematocrit may indicate conditions such as dehydration, while low hematocrit may indicate anemia due to bleeding, hemolysis, or decreased red blood cell production.

[0103]

[0107] Methods for measuring hematocrit, typically expressed as a percentage (%) of blood volume, are well known in the art. Measurements can be performed manually using the packed cell volume by centrifuging blood in a microhematocrit tube. Alternatively, hematocrit can be calculated from the mean corpuscular volume and red blood cell count, both of which can be measured directly by modern hematology analyzers in a standard complete blood count (CBC).

[0104]

[0108] Advantageously, an increase in hematocrit may be associated with a positive effect on a decreased risk of mortality. Thus, an increase in hematocrit may be associated with a decreased risk of mortality.

[0105]

[0109] red blood cell count

[0106]

[0110] Red blood cells, also known as red blood corpuscles, are the most abundant cells present in blood. Red blood cells do not contain a nucleus, but instead consist mainly of hemoglobin contained within the cell membrane, which maximizes the oxygen-carrying capacity of red blood cells. Red blood cell counts above or below normal are indicative of a disorder or disease. A low red blood cell count may indicate hemolysis, blood loss, or decreased red blood cell production, which may result from multiple causes. 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 due to increased red blood cell production.

[0107]

[0111] Methods for counting red blood cells, typically expressed as thousands of cells per microliter (10^3 / μL), are well known in the art. Red blood cell counts can be performed manually on blood smears by microscopic examination, but are commonly performed as part of an automated complete blood count (CBC).

[0108]

[0112] Advantageously, an increase in red blood cell count may be associated with a positive effect on a reduced risk of mortality. Thus, an increase in red blood cell count may be associated with a reduced risk of mortality.

[0109]

[0113] Serum albumin

[0110]

[0114] Serum albumin is a globular protein found in blood. It is a 65 kDa protein consisting of three homologous domains. Albumin plays an important role in blood, regulating oncotic pressure, preventing loss of fluid from blood to tissues, and acting as a transport protein for fatty acids, bilirubin, heme, heavy metals, hormones, and certain drugs. Albumin is very abundant in 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 blood may indicate liver or kidney disease.

[0111]

[0115] Methods for measuring serum albumin levels, typically expressed in grams per deciliter (g / dL), are well known in the art and include zone electrophoresis, dye-binding assays including bromocresol green (BCG) or bromocresol purple (BCP), and ELISA. For example, Eagle Biosciences offers an ELISA-based assay for feline serum albumin (Eagle Biosciences Inc., product code SKU: CAE49-K01). Stokol et al. describe the use of an automated system that uses a BCG binding assay to measure feline serum albumin levels in a clinical setting (Vet Clin Pathol. 2001;30(4):170-176). Additionally, IDEXX Laboratories offers an automated chemistry analyzer that can measure serum albumin levels as part of a combined blood test.

[0112]

[0116] Advantageously, increased serum albumin levels may be associated with a positive effect on reduced risk of mortality. Thus, increased serum albumin levels may be associated with reduced risk of mortality.

[0113]

[0117] Mean corpuscular hemoglobin concentration

[0114]

[0118] Mean corpuscular hemoglobin concentration (MCHC) is the average amount of hemoglobin per volume of packed red blood cells. MCHC values ​​outside the normal range may indicate certain disorders, such as hypochromic anemia.

[0115]

[0119] Methods for measuring MCHC, typically expressed in grams per deciliter (g / dL), are well known in the art. Methods generally involve calculation from observed values ​​of hemoglobin and hematocrit, which can be measured during a complete blood count (CBC). When a CBC is performed using a hematology analyzer such as those described above, MCHC is calculated using the hemoglobin concentration, red blood cell count (RBC), and mean hemoglobin volume (MCV).

[0116]

[0120] Advantageously, an increase in MCHC may be associated with a positive effect on reducing the risk of death. Thus, an increase in MCHC may be associated with a reduction in the risk of death.

[0117]

[0121] Serum sodium

[0118]

[0122] Serum sodium is a measure of the concentration of sodium in the blood. Sodium is the most abundant cation in blood and functions to maintain fluid distribution and osmotic pressure in the blood. Changes in serum sodium levels can indicate kidney disease or can be the result of severe diarrhea or vomiting.

[0119]

[0123] Methods for measuring serum sodium levels, usually expressed in millimoles per liter (mmol / L), are well known in the art. Most modern tests use direct or indirect ion-selective electrode techniques. Automated analyzers capable of performing this measurement on blood samples are also commercially available, such as those available from IDEXX.

[0120]

[0124] Advantageously, increased serum sodium levels may be associated with a negative effect on reducing the risk of mortality. Thus, increased serum sodium levels may be associated with an increased risk of mortality.

[0121]

[0125] Serum cholesterol

[0122]

[0126] Serum cholesterol is a measure of the amount of cholesterol present in the blood. Cholesterol is an essential sterol that forms part of cell membranes, is involved in signal transduction, and is a metabolic precursor of several steroid hormones. Cholesterol in the blood is transported via lipoproteins, particles that contain amphipathic proteins and lipids. Changes in cholesterol levels in the blood can indicate several conditions or diseases. Low cholesterol can indicate liver disease, severe small intestinal disease, or a low-lipid diet.

[0123]

[0127] Methods for measuring serum cholesterol, typically expressed in milligrams per deciliter (mg / dL), are well known in the art. These include classical chemical methods such as the Abell-Kendall method, fluorescent-enzymatic assays commonly used in automated systems, and mass spectrometry-based methods, all of which are disclosed in Li et al. (2019; J Food Drug Anal.; 27(2):375-386). Commercially available automated analyzers capable of measuring serum cholesterol levels are available, such as those from IDEXX.

[0124]

[0128] Advantageously, increased serum cholesterol may be associated with a negative effect on reducing the risk of death. Thus, increased serum cholesterol may be associated with an increased risk of death.

[0125]

[0129] Serum potassium

[0126]

[0130] Serum potassium is a measure of the concentration of potassium in the blood. Potassium is an important electrolyte found primarily within cells, and the potassium gradient across the extracellular membrane is involved in regulating fluid balance and generating action potentials essential for nerve and muscle function. Although a low percentage of potassium is found in the extracellular fluid, serum concentrations may indicate certain diseases and conditions. Low serum potassium may indicate chronic kidney disease, diarrhea and vomiting, or a potassium-deficient diet, which can result in muscle weakness. High serum potassium may indicate kidney disease or hormonal disorders.

[0127]

[0131] Methods for measuring serum potassium levels, usually expressed in millimoles per liter (mmol / L), are well known in the art. Serum potassium levels can be measured using colorimetric methods or direct or indirect ion-selective electrode methods. Commercially available automated analyzers capable of measuring serum potassium levels are available from IDEXX.

[0128]

[0132] Advantageously, increased serum potassium levels may be associated with a negative effect on reducing the risk of mortality. Thus, increased serum potassium levels may be associated with an increased risk of mortality.

[0129]

[0133] Serum alkaline phosphatase

[0130]

[0134] Alkaline phosphatase (ALP) is an enzyme that plays an important role in liver metabolism and skeletal development and is a homodimeric protein of 86 kDa. High levels of this protein in the blood can indicate liver damage or bone disease.

[0131]

[0135] Methods for measuring serum alkaline phosphatase levels, typically expressed as units per liter (U / L, natural logarithmic transformation), are well known in the art and most often consist of quantification of enzyme activity by colorimetric assay. Automated chemistry analyzers capable of measuring serum alkaline phosphatase levels are available from IDEXX Laboratories.

[0132]

[0136] Advantageously, an increase in serum alkaline phosphatase levels may be associated with a positive effect on reducing the risk of death. Thus, a decrease in serum alkaline phosphatase levels may be associated with an increase in the risk of death.

[0133]

[0137] Serum GGT

[0134]

[0138] Gamma-glutamyltransferase (GGT) is an enzyme involved in glutathione metabolism and found primarily in the liver. Serum GGT is a measure of the amount of GGT in the blood. Increased levels of GGT in the blood may indicate liver disease, such as cholestasis.

[0135]

[0139] Methods for measuring serum GGT levels, usually expressed as grams per deciliter (g / dL, natural log transformed), are well known in the art. Colorimetric enzyme assays can be used to measure GGT levels, and commercially available automated analyzers, such as those available from IDEXX, are available for this purpose.

[0136]

[0140] Advantageously, an increase in serum GGT levels may be associated with a negative effect on reducing the risk of death. Thus, an increase in serum GGT levels may be associated with an increase in the risk of death.

[0137]

[0141] Biomarker combinations

[0138]

[0142] In the methods of the invention, while individual biomarkers may have predictive value, the quality and / or predictive power of the methods of the invention may be improved by combining values ​​obtained from multiple biomarkers.

[0139]

[0143] Thus, the methods of the invention may comprise measuring the value of at least two biomarkers from the biomarkers defined herein. For example, the methods of the invention may comprise measuring the value of two or more biomarkers selected from white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, hematocrit, red blood cell count, serum albumin, mean corpuscular hemoglobin concentration, serum sodium, serum cholesterol, serum potassium, serum alkaline phosphatase, and / or serum GGT in one or more samples.

[0140]

[0144] The term "one or more biomarkers" as used herein can include at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 biomarkers.

[0141]

[0145] As used herein, the term "one or more biomarkers" can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 biomarkers, as described herein.

[0142]

[0146] Advantageously, the method of the present invention may include measuring the values ​​of white blood cell count, serum hemoglobin, serum urea nitrogen, and serum AST in one or more samples.Advantageously, it is concluded that this combination of four biomarkers provides significant prediction of mortality risk and / or healthy life expectancy probability.Prediction ability can be further improved by incorporating one or more additional biomarkers selected from serum chloride, serum total bilirubin, serum globulin, hematocrit, red blood cell count, serum albumin, mean red blood cell hemoglobin concentration, serum sodium, serum cholesterol, serum potassium, serum alkaline phosphatase, and serum GGT.

[0143]

[0147] Suitably, the method of the invention may comprise the step of measuring in one or more samples the values ​​of each of white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, red blood cell count, serum sodium, serum cholesterol, serum potassium, serum alkaline phosphatase and serum GGT.

[0144]

[0148] Comparison with a standard or control

[0145]

[0149] The method of the present invention may further comprise comparing the value of each biomarker in the test sample with one or more reference or control values. The reference value may be associated with a predetermined risk of death and / or a healthy life expectancy probability. In some embodiments, the reference value is a previously obtained value for a subject or group of subjects with a known mortality outcome. The reference value may be based on an average value, e.g., the mean or median value, from a group of subjects with a known chronological age, breed, sex, and / or mortality outcome. Preferably, the reference value may be based on an average value, e.g., the mean or median value, from a group of subjects with a known chronological age, breed, sex, and mortality outcome.

[0146]

[0150] Combining biomarker values ​​with additional measurements and / or features

[0147]

[0151] Suitably, the methods of the invention further comprise combining the value of one or more biomarkers with one or more of the cat's chronological age and / or sex. Combining this information provides an improved model of the cat's risk of mortality and / or healthy life expectancy.

[0148]

[0152] In a preferred embodiment, the values ​​of one or more biomarkers defined herein are measured in a sample from the cat and those values ​​are combined with the cat's chronological age to assess the cat's risk of mortality and / or healthy life expectancy probability.

[0149]

[0153] Preferably, the mortality risk and / or healthy life expectancy probability is expressed as phenotypic age and is given by the following formula:

number

[0150]

[0154] (where xb is the sum of the values ​​of each biomarker multiplied by their respective coefficients according to formula (2).)

number

[0151] Phenotypic age is used to assess the cat's risk of mortality and / or healthy life expectancy.

[0152]

[0157] The coefficient value of each parameter typically depends on the units of measure of all variables in the model. Thus, as will be appreciated by those skilled in the art, the exact value of each coefficient value will vary depending, for example, on the number and nature of the different parameters used in the model, as well as the nature of the training data provided (e.g., the breeds of cats in the training data set). Thus, customary statistical methods can be applied to the training data set to arrive at coefficient values ​​for use in the above formula. Such methods include, for example, the calculation of two Gompertz functions for the training set (e.g., when the status of the cats (alive or dead) is known), one that models survival as a function of the selected biomarkers, chronological age, and sex (Model 1), and a second function that only considers chronological age and sex (Model 2). These models can be fitted using the flexsurv package (v 2.1) in the R software environment.

[0153]

[0158] Suitably, a negative coefficient for a given biomarker means that a higher value of the biomarker has a positive effect on reducing the risk of mortality and / or a lower value of the biomarker has a negative effect on reducing the risk of mortality. Suitably, a positive coefficient for a given biomarker means that a higher value of the biomarker has a negative effect on reducing the risk of mortality and / or a lower value of the biomarker has a positive effect on reducing the risk of mortality.

[0154]

[0159] Phenotypic age can be defined as the time variable ("chronological age") at which the survival probability of animals given by Model 2 is equal to the survival probability at their chronological age given by Model 1.

[0155]

[0160] The phenotypic age (ie, phenoage) of a cat may be expressed in years, months, days, etc.

[0156]

[0161] Preferably, the mortality risk and / or healthy life expectancy probability is expressed as the difference between the cat's phenoage and its chronological age, which may also be referred to as the cat's phenoage progression.

[0157]

[0162] For example, an increase in phenoage compared to chronological age may indicate an increase in the risk of death of a cat. For example, a decrease in phenoage compared to chronological age may indicate a decrease in the risk of death of a cat. By way of illustration, the inventors have concluded that the difference between phenoage and chronological age (phenoage progression) is associated with a significant increase in risk of death, and the effect size is calculated to be a hazard ratio of 1.36 for one year increase in phenoage compared to chronological age (see Example 3). In other words, the inventors have concluded that one year increase in phenoage compared to chronological age is associated with approximately 35% higher risk of death at any given time in life.

[0158]

[0163] Subject stratification

[0159]

[0164] The mortality risk and / or healthy life expectancy probability assessed by the method of the present invention can also be compared with one or more predefined thresholds. Such thresholds can be used to stratify subjects into categories that indicate the assessed mortality risk, for example, assessed mortality risk and / or healthy life expectancy probability is low, medium, or high. The degree of deviation from the threshold is useful for identifying which subjects will benefit most from a particular intervention. In this way, dietary intervention and lifestyle modification can be optimized. The assessed mortality risk and / or healthy life expectancy probability can be presented in terms of a numerical score or percentage, for example, it can indicate the assessed mortality risk and / or healthy life expectancy probability compared to a control or reference population.

[0160]

[0165] How to select / monitor a subject's lifestyle or diet plan

[0161]

[0166] In a further aspect, the present invention provides a method for selecting a lifestyle or diet plan for a subject. The lifestyle modification may be any modification described herein, such as a dietary intervention and / or a modification of an exercise regimen. The lifestyle modification may be the administration of a therapeutic modality.

[0162]

[0167] The lifestyle or diet plan may be applied to the cat for any suitable period of time. After such period, the cat's mortality risk and / or healthy life expectancy probability may be evaluated again using the method of the present invention to evaluate the effectiveness of the lifestyle or diet plan to reduce the cat's mortality risk and / or increase the cat's healthy life expectancy probability. By way of example, the lifestyle or diet plan may be applied for at least 2 weeks, at least 4 weeks, at least 8 weeks, at least 16 weeks, at least 32 weeks, or at least 64 weeks. The lifestyle or diet plan may be applied for at least 3 months, at least 6 months, at least 12 months, at least 24 months, at least 36 months, at least 48 months, or at least 60 months.

[0163]

[0168] The modification is preferably a dietary intervention as described herein. The term "dietary intervention" refers to an external factor applied to a subject that causes the subject to change their diet. More preferably, the dietary intervention includes at least providing a dietary product or a meal plan or a dietary supplement.

[0164]

[0169] The dietary intervention may be a diet, a dietary plan, a dietary supplement, or a dietary supplement plan.

[0165]

[0170] The dietary intervention or dietary product described herein may be any suitable dietary plan, for example a calorie restricted diet, an elderly diet, a low protein diet, a phosphorus diet, a potassium supplemented diet, a polyunsaturated fatty acid (PUFA) supplemented diet, an antioxidant supplemented diet, a vitamin B supplemented diet, a liquid diet, a selenium supplemented diet, an omega 3-6 ratio diet, or a diet supplemented with carnitine, branched chain amino acids or derivatives, nucleotides, nicotinamide precursors such as nicotinamide mononucleotide (MNM) or nicotinamide riboside (NR), or any combination of the above.

[0166]

[0171] 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.

[0167]

[0172] Dietary intervention may also be determined based on the cat's baseline maintenance energy requirement (MER), which may suitably be the amount of food that stabilizes the cat's weight (less than 5% change over a 3 week period).

[0168]

[0173] As an example, it is generally understood that young, growing cats benefit from a high energy / high protein diet. However, older cats may have lower energy requirements, so the diet can be modified appropriately. In particular, many manufacturers produce a "senior" range of cat foods that are low in calories, high in fiber, but with amounts of protein and fat appropriate for older cats.

[0169]

[0174] Preferably, the calorie restricted diet comprises about 60%, about 65%, about 75%, or about 80% of the MER of the cat. Preferably, the calorie restricted diet comprises about 60% or about 75% of the MER of the cat.

[0170]

[0175] 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).

[0171]

[0176] These diets are generally recommended based on the cat's chronological age. For example, it may be recommended to switch cats to senior diets at around 7 or 8 years old. However, in the context of the present invention, the assessment of increased mortality risk and / or decreased healthy life expectancy compared to what would be expected given the cat's chronological age may allow the decision to switch cats' diets to senior diets at an earlier age. In contrast, cats that have decreased mortality risk and / or increased healthy life expectancy compared to their chronological age may be able to continue on high energy / high protein diets for a longer period of time.

[0172]

[0177] Dietary intervention may include foods, supplements, and / or beverages that contain nutrients and / or bioactives that mimic the benefits of calorie restriction (CR) without restricting daily calorie intake. For example, the foods, supplements, and / or beverages may contain functional ingredients with CR-like benefits. Suitably, the foods, supplements, and / or beverages may contain autophagy inducers. Suitably, the foods, supplements, and / or beverages may contain fruits and / or nuts. Suitably, examples include, but are not limited to, pomegranate, strawberry, blackberry, camu camu, walnut, chestnut, pistachio, and pecan. Suitably, the foods, supplements, and / or beverages may contain probiotics with or without fruit or nut extracts.

[0173]

[0178] Modifying the subject's lifestyle also includes instructing the subject to change their lifestyle, for example, by instructing them to exercise more. As with dietary interventions, an assessment of the cat's increased risk of death and / or decreased probability of healthy life span compared to that expected given its chronological age may allow for the decision to switch the cat to a more appropriate exercise regime.

[0174]

[0179] Modifying the subject's lifestyle also includes recommending a treatment modality or plan. The treatment modality or plan may be, for example, a modality useful for treating and / or preventing arthritis, dental disease, endocrine disorders, heart disease, diabetes, liver disease, kidney disease, prostate disorders, cancer, and behavioral or cognitive disorders. Preventive therapy may be administered to cats identified as at risk for such disorders due to increased mortality risk (phenoage) and / or based on certain biomarkers known to be associated with disease-related pathways. In other embodiments, cats assessed as at risk for certain conditions (due to increased mortality risk (phenoage) and / or based on certain biomarkers known to be associated with disease-related pathways) may be followed up more regularly so that diagnosis and treatment can be initiated as soon as possible.

[0175]

[0180] Thus, the present invention can also advantageously allow for the identification of cats that are expected to respond particularly well to a given intervention (e.g., a lifestyle or dietary plan), such that the intervention can be applied in a more targeted manner to those cats expected to respond.

[0176]

[0181] The present invention further provides a method for assessing the effectiveness of a lifestyle or dietary regimen for reducing a risk of mortality in a cat and / or increasing a cat's chance of healthy life span, the method comprising: a. assessing a first mortality risk and / or healthy life expectancy probability of a cat according to the method of the first aspect of the invention; b. administering the lifestyle or diet plan to the cat; c. assessing a second risk of mortality and / or a second healthy life span probability of the cat after a period of applying the lifestyle or dietary plan to the cat according to the method of the first aspect of the invention; d. Determining whether there has been a change between the first risk of mortality and / or the first healthy life span probability and the second risk of mortality and / or the second healthy life span probability of the cat after a period of following the lifestyle or dietary plan.

[0177]

[0186] The present invention also provides a method for assessing the effectiveness of a lifestyle or diet plan for reducing an assessed risk of mortality and / or increasing the probability of a healthy life span in a cat, the method comprising: a. applying to the cat a lifestyle or diet plan selected according to a method comprising carrying out a method according to the first aspect of the invention, and selecting an appropriate lifestyle or diet plan based on the assessed mortality risk and / or healthy life expectancy probability; b. assessing a second risk of mortality and / or a second healthy life span probability of the cat after a period of applying the lifestyle or dietary plan to the cat by carrying out the method of the first aspect of the invention; c. Determining whether there has been a change in the cat's risk of mortality and / or healthy life expectancy probability after a period of following the lifestyle or diet plan.

[0178]

[0190] The present invention further provides a method for assessing the effectiveness of a lifestyle or dietary regimen for reducing risk of mortality and / or increasing healthy life expectancy in a cat, the method comprising: a. selecting a lifestyle or diet plan for a cat according to a method comprising carrying out a method according to the first aspect of the invention, selecting an appropriate lifestyle or diet plan based on the assessed mortality risk; b. administering the lifestyle or diet plan to the cat; c. assessing a second risk of mortality and / or a second healthy life span probability of the cat after a period of applying the lifestyle or dietary plan to the cat according to the method of the first aspect of the invention; d. determining whether there has been a change in the cat's risk of death and / or healthy life expectancy probability between steps a and c.

[0179]

[0195] A decrease in the second (or subsequent) risk of mortality assessed for the cat compared to the first (or earlier) risk of mortality and / or the first (or earlier) healthy life expectancy probability assessed for the cat after the period of application of the lifestyle or diet plan indicates that the lifestyle or diet plan is effective in reducing the risk of mortality and / or increasing the healthy life expectancy probability of the cat.

[0180]

[0196] The mortality risk and / or healthy life expectancy probability of the cat may be evaluated before and after the lifestyle or diet plan is applied to the cat. The mortality risk and / or healthy life expectancy probability of the cat may also be evaluated at predetermined time points throughout the application of the lifestyle or diet plan. These predetermined time points may be periodic during the lifestyle or diet plan, such as daily or every 3 days, weekly, every 2 weeks, monthly, every 2 months, every 6 months, annually, or every 2 years. The predetermined time points may vary depending on the subject being examined. Advantageously, the lifestyle or diet plan may have been applied to the cat during the period before the first mortality risk is evaluated. However, the effectiveness of the lifestyle or diet plan to reduce the mortality risk may be followed up by evaluating the mortality risk at two or more predetermined time points during the application of the lifestyle or diet plan.

[0181]

[0197] The present invention further provides a method for assessing the effectiveness of a lifestyle or dietary regimen for reducing risk of mortality and / or increasing healthy life expectancy in a cat, the method comprising: a. assessing a first mortality risk and / or a first healthy life span probability of a cat according to the method of the first aspect of the invention; b. administering the lifestyle or diet plan to the cat; c. assessing a second risk of mortality and / or a second healthy life span probability of the cat after a period of applying the lifestyle or dietary plan to the cat according to the method of the first aspect of the invention; d. Determining whether there has been a change between the first risk of mortality and / or first healthy life span probability and the second risk of mortality and / or second healthy life span probability of the cat after a period of following the lifestyle or dietary plan.

[0182]

[0202] The present invention also provides a method for assessing the effectiveness of a lifestyle or diet plan for reducing an assessed risk of mortality and / or increasing the probability of a healthy life span in a cat, the method comprising: a. applying to the cat a lifestyle or diet plan selected according to a method comprising carrying out a method according to the first aspect of the invention and selecting an appropriate lifestyle or diet plan based on the assessed mortality risk; b. assessing a second risk of mortality and / or a second healthy life span probability of the cat after a period of applying the lifestyle or dietary plan to the cat by carrying out the method of the first aspect of the invention; c. Determining whether there has been a change in the cat's risk of mortality and / or healthy life expectancy probability after a period of following the lifestyle or diet plan.

[0183]

[0206] The present invention further provides a method for assessing the effectiveness of a lifestyle or dietary regimen for reducing risk of mortality and / or increasing healthy life expectancy in a cat, the method comprising: a. selecting a lifestyle or diet plan for a cat according to a method comprising carrying out a method according to the first aspect of the invention, selecting an appropriate lifestyle or diet plan based on the assessed mortality risk; b. administering the lifestyle or diet plan to the cat; c. assessing a second risk of mortality and / or a second healthy life span probability of the cat after a period of applying the lifestyle or dietary plan to the cat according to the method of the first aspect of the invention; d. determining whether there has been a change in the cat's risk of death between steps a and c.

[0184]

[0211] A reduction in the second (or subsequent) risk of death assessed for the cat compared to the first (or earlier) risk of death assessed for the cat after the period of application of the lifestyle or diet plan indicates that the lifestyle or diet plan is effective in reducing the cat's risk of death.

[0185]

[0212] The mortality risk and / or healthy life expectancy probability for the cat may be evaluated before and after the lifestyle or diet plan is applied to the cat. The mortality risk and / or healthy life expectancy probability for the cat may also be evaluated at predetermined time points during the application of the lifestyle or diet plan. These predetermined time points may be periodic during the lifestyle or diet plan, for example, daily or every 3 days, weekly, every 2 weeks, monthly, every 2 months, every 6 months, annually, or every 2 years. The predetermined time points may vary depending on the subject being examined. Suitably, the lifestyle or diet plan may have been applied to the cat during a period before the first mortality risk and / or first healthy life expectancy probability is evaluated. However, the effectiveness of the lifestyle or diet plan to reduce the mortality risk and / or increase the healthy life expectancy probability may be followed up by evaluating the mortality risk and / or healthy life expectancy probability at two or more predetermined time points during the application of the lifestyle or diet plan.

[0186]

[0213] Use of dietary interventions

[0187]

[0214] In one aspect, the invention provides a dietary intervention for use in reducing the risk of mortality and / or increasing healthy life expectancy in a cat, the dietary intervention being administered to a cat whose risk of mortality and / or healthy life expectancy has been assessed by the method of the invention.

[0188]

[0215] In another aspect, the invention provides the use of dietary intervention to reduce the predicted risk of mortality and / or increase healthy life expectancy in a cat, the dietary intervention being administered to a cat whose risk of mortality and / or healthy life expectancy has been assessed by the method of the invention.

[0189]

[0216] As described herein, the dietary intervention may be a dietary product or a dietary plan or a dietary supplement.

[0190]

[0217] Computer Program Products

[0191]

[0218] The method of the present invention may be carried out using a computer, thus the method may be carried out in silico.

[0192]

[0219] The methods described herein may be implemented as computer programs running on general-purpose hardware, such as one or more computer processors. In some embodiments, the functionality described herein may be implemented by devices such as smartphones, tablets, or personal computers.

[0193]

[0220] In one aspect, the present invention provides a computer program product comprising computer executable instructions for causing a programmable computer to assess the mortality risk and / or healthy life expectancy probability of a cat as described herein.

[0194]

[0221] In another aspect, the present invention provides a computer program product comprising computer executable instructions for causing a device to assess the risk of death and / or healthy life expectancy of a cat given the value of one or more biomarkers from a user, the biomarkers being selected from one or more biomarkers defined herein. Preferably, the values ​​of the biomarkers are fasting values. The computer program product may also be provided with additional parameters or characteristics for the cat. As described herein, the additional parameters or characteristics may include chronological age, breed, and sex.

[0195]

[0222] In one embodiment, the user inputs values ​​for one or more of the biomarkers defined herein, optionally along with chronological age, breed, and sex, into the device, which then processes this information and provides an assessment of the cat's risk of mortality and / or healthy life expectancy probability.

[0196]

[0223] The device may generally be a server on a network. However, any device may be used as long as it is capable of processing the biomarker data and / or additional parameter or feature data using a processor, or a central processing unit (CPU), or the like. The device may be, for example, a smartphone, a tablet, or a personal computer, and may output information indicating the assessed mortality risk and / or healthy life expectancy probability for the cat. The assessed mortality risk and / or healthy life expectancy probability may be presented in terms of a numerical score or percentage, for example, indicating the assessed mortality risk and / or healthy life expectancy probability compared to a control or reference population.

[0197]

[0224] Those skilled in the art will understand that they can freely combine all features of the invention described herein without departing from the scope of the invention disclosed.

[0198]

[0226] The present invention will now be further described by way of examples, which are meant to be provided to aid those skilled in the art in practicing the invention, and are not intended to limit the scope of the invention in any way.

[0199]

[0227] Example 1 Determination of blood biomarkers associated with mortality risk in cats

[0200]

[0228] 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 according to standard veterinary clinical practice.

[0201] [Table 1]

[0202]

[0230] We performed a longitudinal study of cats, repeatedly measuring these parameters, as well as information on the cat's status (alive or dead) and sex. The analysis was focused on domestic shorthair breeds. The data are then organized using the R programming language. For each cat, we recorded the biomarkers as time-dependent covariates using a time interval (i.e. (tstart, tstop]) with the left open and the right closed. The biomarker information corresponds to the start of the interval, and the event (alive or dead) is recorded as the last tstop value. For this purpose, we use the tmerge function of the survival package in R (v.3.2-13). We then fit a Cox proportional hazards model to this data separately, with each of the 28 biomarkers and sex as covariates. We then corrected the p-value of each parameter to account for multiple comparisons (by false discovery rate (fdr)) and selected features with a corrected fdr below 0.05 (Figure 1).

[0203]

[0231] By this method, we identified 11 biomarkers that were individually predictive of survival in cats. Hemoglobin (g / dL) ·White blood cell count (10^3 / μL) Hematocrit (%) Red blood cell count (10^3 / ul) Serum urea nitrogen (mg / dL, natural logarithm transformed) Serum AST (U / L, natural logarithm transformed) Serum chloride (mmol / L) Serum total bilirubin (mg / dL) Serum globulin (g / dL) Serum albumin (g / dL) · Mean corpuscular hemoglobin concentration (g / dL)

[0204]

[0243] Example 2. A multi-parameter model for predicting mortality risk

[0205]

[0244] Next, we constructed the best model considering multiple parameters simultaneously, as it is more likely to cover the various organ dysfunctions that occur with age. However, selecting several features that may be correlated with each other is subject to bias. To avoid this problem, we used a penalized regression method using the glmnet package (v4.1-3). We fitted the data to a LASSO penalized cox proportional hazards model and compared different values ​​of the penalty parameter λ using 20-fold cross-validation. This approach resulted in a selection of the top 13 most predictive blood biomarkers for survival, in the order of importance shown below. ·White blood cell count (10^3 / μL) Hemoglobin (g / dL) Serum urea nitrogen (mg / dL, natural logarithm transformed) Serum AST (U / L, natural logarithm transformed) Serum chloride (mmol / L) Serum total bilirubin (mg / dL) Serum globulin (g / dL) Serum sodium (mmol / L) Serum cholesterol (mg / dL) Serum potassium (mmol / L) Serum alkaline phosphatase (U / L) ·Serum GGT (g / dL) Red blood cell count (10^3 / ul)

[0206]

[0258] To extract the phenotypic age of the animals, we calculate two different Gompertz functions for our training set: one that models survival as a function of selected biomarkers and age (Model 1), and a second function that only considers age (Model 2). These models are fitted using the flexsurv package (v 2.1). Phenotypic age is defined as the time variable ("age") where the survival probability of animals given by Model 2 is equal to the survival probability at their chronological age given by Model 1. This results in a mathematical function relating blood biomarkers to phenoage, given by the following equation:

number

[0207]

[0259] where xb is the sum of the values ​​of each biomarker multiplied by their respective coefficients, given by two Gompertz functions trained on our training set.

[0208]

[0260] As an example, the coefficients, and γ and γ2 values, measured from our training set for the complete list of biomarkers, are shown in Table 1.

number

[0209] [Table 2]

[0210]

[0262] Furthermore, by reducing the set of 13 biomarkers by systematically removing one biomarker at a time, starting from the top of the list, we observed a decrease in the strength of survival prediction (p-value). Although the decrease was most pronounced for the first four parameters, confirming their maximal contribution, we observed a change in the quality of prediction by reducing each of the set, indicating that each parameter contributes to the overall prediction (Figure 2).

[0211]

[0263] Example 3 Application of PhenoAge to predict risk of mortality

[0212]

[0264] When the resulting phenoage was subsequently applied to a test set consisting of only cats that had not been used during training of the algorithm, it was demonstrated that the difference between phenoage and chronological age (advancement of phenoage) was associated with a significantly increased risk of death.

[0213]

[0265] The effect size was calculated to be a hazard ratio of 1.36 per year increase in chronological age compared with phenoage.

[0214]

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

Claims

1. 1. A method for assessing the risk of mortality and / or healthy life expectancy of a cat, the method comprising the step of measuring the value of one or more biomarkers in one or more samples obtained from the cat, the one or more biomarkers being selected from white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, hematocrit, red blood cell count, serum albumin, and / or mean corpuscular hemoglobin concentration.

2. A method for assessing the risk of mortality and / or healthy life expectancy of a cat, the method comprising the step of measuring the value of one or more biomarkers in one or more samples obtained from the cat, the one or more biomarkers being selected from white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, and / or red blood cell count.

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

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

5. The method of any one of claims 1 to 4, wherein the biomarker is serum urea nitrogen.

6. The method of any one of claims 1 to 5, wherein the biomarker is serum AST.

7. The method according to any one of claims 1 to 6, wherein the method comprises the steps of measuring each of the values ​​of white blood cell count, serum hemoglobin, serum urea nitrogen, and serum AST.

8. 8. The method of any one of claims 1 to 7, wherein the value of one or more further biomarkers selected from serum sodium, serum cholesterol, serum potassium, serum alkaline phosphatase, and / or serum GGT is measured in the one or more samples obtained from the cat.

9. 9. The method according to any one of claims 2 to 8, wherein the method comprises measuring each of the values ​​of white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, red blood cell count, serum sodium, serum cholesterol, serum potassium, serum alkaline phosphatase, and serum GGT.

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

11. The method of any one of claims 1 to 10, wherein the method further comprises combining the value of the one or more biomarkers with the chronological age of the cat.

12. 1. A method for assessing the mortality risk and / or healthy life expectancy probability of a cat, the method comprising: a. measuring the levels of the following biomarkers in one or more samples obtained from the cat: white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, red blood cell count, serum sodium, serum cholesterol, serum potassium, serum alkaline phosphatase, and serum GGT; b. assessing the phenotypic age of the cat using formula (1); [0010] (where xb is the sum of the values ​​of each biomarker multiplied by their respective coefficients according to equation (2). [0025] And, c. using the phenotypic age to assess the risk of mortality and / or healthy life expectancy of the cat; A method comprising:

13. 13. The method of claim 12, wherein assessing the phenoage of the cat as exceeding its chronological age indicates a higher risk of mortality and / or a reduced probability of healthy lifespan.

14. 13. The method of claim 12, wherein assessing the phenoage of the cat to be less than its chronological age indicates a lower risk of mortality and / or an increased probability of healthy lifespan.

15. A method according to any one of claims 1 to 14, wherein the method is performed on one or more samples obtained before and after a time interval to determine whether there has been a change in the mortality risk and / or healthy life expectancy probability of the cat during the time interval.

16. 16. A method of calculating the ageing rate of a cat, the method comprising carrying out a method according to any one of claims 12 to 15, wherein (i) assessing the phenoage of the cat to be greater than its chronological age indicates a rapid rate of ageing, or (ii) assessing the phenoage of the cat to be less than its chronological age indicates a slow rate of ageing.

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

18. 18. The method of claim 17, wherein the lifestyle or dietary plan is a dietary intervention.

19. 19. The method, dietary intervention for use or use according to claim 17 or 18, wherein the dietary intervention is a calorie restricted diet, an elderly diet or a low protein diet, preferably a calorie restricted diet.

20. 1. A method for assessing the effectiveness of a lifestyle or dietary regimen for reducing risk of mortality and / or increasing healthy life expectancy in a cat, the method comprising: assessing a first mortality risk and / or a first healthy lifespan probability of said cat according to a method of any one of claims 1 to 15; b. administering a lifestyle or dietary regimen to said cat; c) assessing a second risk of mortality and / or a second healthy life expectancy probability of said cat after a period of applying said lifestyle or diet plan to said cat according to a method of any one of claims 1 to 15; d. determining whether there is a difference between the first and second mortality risks and / or between the first and second healthy life span probabilities of the cat after a period of following the lifestyle or diet plan.

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

22. 1. A method for assessing the effectiveness of a lifestyle or diet plan for reducing risk of mortality and / or increasing the probability of healthy life span in a cat, the method comprising: a. selecting a lifestyle or diet plan for the cat according to the method of any one of claims 17 to 19; b. administering the lifestyle or diet plan to the cat; c) assessing a second risk of mortality and / or a second healthy life expectancy probability of said cat after a period of applying said lifestyle or diet plan to said cat according to a method of any one of claims 1 to 15; d. Determining whether there has been a change in the mortality risk and / or healthy life expectancy probability of the cat between steps a and c.

23. 23. The method of any one of claims 20 to 22, further comprising modifying the lifestyle or dietary plan to reduce the risk of mortality and / or increase the chances of a healthy lifespan of the cat.

24. 1. A method for assessing the likelihood that a cat will benefit from a lifestyle or dietary regimen, said method comprising: a. assessing the mortality risk and / or healthy life expectancy probability of the cat according to the method of any one of claims 1 to 15; b. Identifying the cat as likely to respond to a lifestyle or diet plan if the cat has an increased risk of mortality and / or a decreased probability of healthy life span compared to chronological age.

25. 1. A method of selecting a cat as suitable for receiving a lifestyle or diet plan, said method comprising: a. assessing the mortality risk and / or healthy life expectancy probability of said cat according to the method of any one of claims 1 to 15; b. Selecting the cat as suitable for receiving a lifestyle or diet plan if the cat has an increased risk of mortality and / or a decreased probability of healthy life expectancy compared to chronological age.

26. 1. A method for reducing the risk of mortality and / or increasing the probability of healthy life span in a cat, the method comprising: assessing the risk of mortality and / or healthy life expectancy of the cat, comprising measuring the value of one or more biomarkers in one or more samples obtained from the cat, wherein the one or more biomarkers are selected from white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, red blood cell count, serum sodium, serum cholesterol, serum potassium, serum alkaline phosphatase, serum GGT, and / or red blood cell count; b. Selecting an appropriate lifestyle or diet plan based on the mortality risk and / or healthy life expectancy probability assessed in step a; c) administering said lifestyle or dietary plan to said cat.

27. The method according to claim 26, wherein step a comprises the method according to any one of claims 1 to 15.

28. A method for reducing the risk of mortality and / or increasing the probability of a healthy lifespan in a cat, comprising a step of administering dietary intervention to a cat assessed as needing dietary intervention by the method of any one of claims 1 to 15.

29. A dietary intervention for use in reducing the risk of mortality and / or increasing healthy life expectancy in cats, said dietary intervention being administered to cats having a risk of mortality and / or healthy life expectancy assessed by a method according to any one of claims 1 to 15.

30. Use of dietary intervention to reduce the risk of mortality and / or increase healthy life expectancy in a cat, said dietary intervention being administered to a cat whose risk of mortality and / or healthy life expectancy has been assessed by a method according to any one of claims 1 to 15.

31. 31. The method, dietary intervention for use or use according to any one of claims 26 to 30, wherein said dietary intervention is a calorie restricted diet, an ageing diet or a low protein diet; preferably a calorie restricted diet.

32. A computer readable medium comprising instructions that, when executed, cause one or more processors to perform the method of any one of claims 1 to 15.

33. 1. A computer system for assessing a mortality risk of a cat, the computer system comprising: assessing the risk of mortality and / or healthy life expectancy of the cat given the value of one or more biomarkers in a sample from the cat, wherein the one or more biomarkers are selected from white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, red blood cell count, serum sodium, serum cholesterol, serum potassium, serum alkaline phosphatase, and / or serum GGT; and generating a report of the mortality risk and / or healthy life expectancy probability of the cat.

34. 1. A computer program product comprising computer executable instructions for causing a programmable computer to assess the risk of mortality and / or healthy life expectancy of a cat given values ​​of one or more biomarkers from the cat, wherein the one or more biomarkers are selected from white blood cell count, hemoglobin, serum urea nitrogen, serum AST, serum chloride, serum total bilirubin, serum globulin, red blood cell count, serum sodium, serum cholesterol, serum potassium, serum alkaline phosphatase, and / or serum GGT.

35. The computer system or computer program product according to any one of claims 33 to 34, wherein the biomarker is white blood cell count.

36. 36. A computer system or computer program product according to any one of claims 33 to 35, wherein the biomarker is hemoglobin.

37. 37. The computer system or computer program product of any one of claims 33 to 36, wherein the biomarker is serum urea nitrogen.

38. 38. The computer system or computer program product of any one of claims 33 to 37, wherein the biomarker is serum albumin.

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