Diagnostic method

Abstract

The present application relates to personalized nutrition methods, in particular to methods of determining whether a human subject will benefit from taking a urolithin supplement and methods of determining a therapeutic dose of a urolithin supplement for a human subject. In particular methods comprising determining the level of urolithin or urolithin conjugate in a biological fluid such as a dried whole blood spot sample, dried plasma spot, m spot sample or urine sample. The present application also relates to systems for displaying whether a human subject will benefit from taking a urolithin supplement and for displaying a therapeutic dose of a urolithin supplement for a human subject. The present application also relates to computer implementations of the methods of the present application.

Description

Technical Field

[0001] This application relates to personalized nutrition methods, specifically to methods for determining whether a human subject will benefit from taking a urolithin supplement and for determining a therapeutic dose of the urolithin supplement for the human subject. In particular, it includes methods for determining the level of urolithin or urolithin conjugates in a biological fluid such as a dried whole blood spot sample, or a dried plasma spot or serum spot sample, or a urine sample. This application also relates to a system for indicating whether a human subject will benefit from taking a urolithin supplement and for indicating a therapeutic dose of the urolithin supplement for the human subject. This application also relates to a computer implementation of the methods of the invention. Background Technology

[0002] Urolithin has been proposed for the treatment of a variety of diseases associated with insufficient mitochondrial activity, including obesity, decreased metabolic rate, metabolic syndrome, diabetes, cardiovascular disease, hyperlipidemia, neurodegenerative diseases, cognitive impairment, mood disorders, stress, and anxiety disorders; for weight management, or to improve muscle function or mental performance, see WO2012 / 088519 (Amazentis SA). The use of urolithin for the treatment of various oncological diseases is described in WO2007 / 127263 (University of California Board of Trustees).

[0003] International Patent Publication WO2014 / 004902 (derived from application PCT / US2013 / 48310) discloses a method for increasing autophagy in cells, particularly mitochondrial autophagy, the method comprising contacting cells with an effective amount of urolithin or a pharmaceutically acceptable salt thereof, thereby increasing autophagy in cells, particularly mitochondrial autophagy. This medication may be used in subjects with a condition selected from the following diseases or conditions: metabolic stress, cardiovascular disease, endothelial dysfunction, sarcopenia, muscle degeneration, Duchenne muscular dystrophy, alcoholic liver disease, non-alcoholic fatty liver disease, drug-induced liver or muscle injury, alpha-1 antitrypsin deficiency, ischemia / reperfusion injury, inflammation, skin aging, inflammatory bowel disease, Crohn's disease, obesity, metabolic syndrome, type II diabetes, hyperlipidemia, osteoarthritis, neurodegenerative diseases, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, age-related macular degeneration, mitochondrial diseases (including, for example, growth retardation, loss of muscle coordination, muscle weakness, vision problems, hearing problems, heart disease, liver disease, kidney disease, gastrointestinal diseases, respiratory disorders, neurological problems, autonomic dysfunction, learning disabilities, and dementia caused by mitochondrial diseases), muscle diseases, cancer, cognitive impairment, stress, and mood disorders.

[0004] Specifically, urolithiasis has been proposed for the treatment of muscle-related pathological conditions. Muscle-related pathological conditions include muscular diseases and neuromuscular diseases. Examples of such conditions include Duchenne muscular dystrophy, acute sarcopenia such as muscle atrophy and / or cachexia associated with burns, bed rest, limb immobilization, or major chest, abdominal, neck, and / or orthopedic surgery, amyotrophic lateral sclerosis (ALS), and multiple sclerosis. Age-related muscle loss is a particularly common condition. Cachexia caused by prolonged inactivity or other diseases (such as cancer) is another condition typically characterized by poor muscle function.

[0005] Effective muscle function and physical fitness are important for both healthy and ill individuals, especially older adults, to achieve a high quality of life at all ages. Athletes are particularly interested in improving muscle function. For example, increased muscle contraction strength, increased muscle contraction amplitude, or a shortened muscle reaction time between stimulation and contraction are all beneficial to individuals, especially athletes. For older adults with age-related muscle degeneration diseases (including muscle loss / atrophy) or individuals with cachexia-associated muscular dystrophy, improving muscle function and physical fitness is important for fundamental aspects of daily functioning, such as the speed and distance they can walk independently.

[0006] Urolithin is a naturally occurring gut metabolite produced by the host gut microbiota upon exposure to dietary precursors (such as ellagitannins), which are found in high levels in certain fruits and nuts, such as pomegranates, berries, and walnuts. The production of urolithin in the gut exhibits significant inter-individual variability, which is associated with differences in the colonic microbiota [García-Villalba et al. (2013), J. Agric. Food Chem., 2013, 61, 8797–8806]. Some studies suggest that consuming these polyphenol-rich fruits is beneficial to health, but the results are inconsistent, likely due to significant inter-individual differences in gut microbiota leading to distinct metabolite profiles that greatly influence their bioavailability. This presents another major challenge in designing optimal dietary interventions and limits the interpretation of studies investigating the health benefits of consuming polyphenol-rich fruits. [For example, see Tomás-Barberán FA et al. (2014), *Agricultural and Food Chemistry* 62(28):6535-8 and González-Sarrías et al. (2017), *Molecular Nutrition and Food Research* 61(5). doi:10.1002 / mnfr.201600830]. Therefore, there is a need to develop assays to determine which individuals will benefit from urolithiasis and the required dosage. This application addresses this need. Summary of the Invention

[0007] One embodiment of the present invention provides a method for determining whether a human subject will benefit from taking a urolithin supplement, comprising the steps of: analyzing a biofluid sample collected from the subject to determine whether the biofluid contains urolithin or urolithin conjugates, and then determining whether the subject will benefit from ingesting a urolithin supplement based on the level of urolithin or urolithin conjugates in the biofluid, for example, wherein the biofluid sample is selected from urine or blood, and in the case of blood, is analyzed in the form of dried blood spots.

[0008] In one embodiment of the invention, the urolithin conjugate is urolithin A glucuronide, and the biofluid is dried blood spot, wherein if the level of urolithin A glucuronide is below 100 ng / ml, the subject will benefit from the dose of the urolithin supplement. In another embodiment, if the level of urolithin A glucuronide is below 50 ng / ml, the subject will benefit from the dose of the urolithin supplement. In yet another embodiment, if the level of urolithin A glucuronide is below 25 ng / ml, the subject will benefit from the dose of the urolithin supplement.

[0009] In another embodiment of the invention, the urolithin conjugate is urolithin A glucuronide, and the biofluid is urine, wherein if the level of urolithin A glucuronide is below 50,000 ng / ml, the subject will benefit from the dose of the urolithin supplement. In another embodiment, if the level of urolithin A glucuronide is below 25,000 ng / ml, the subject will benefit from the dose of the urolithin supplement. In yet another embodiment, if the level of urolithin A glucuronide is below 10,000 ng / ml, the subject will benefit from the dose of the urolithin supplement.

[0010] In another embodiment of the invention, a method is provided for determining a therapeutic dose of a urolithiasis supplement for use in human subjects, comprising:

[0011] a) Determining the levels of urolithin or urolithin conjugates in biological fluids; and

[0012] b) Determine the therapeutic dose of urolithin supplements based on the level of urolithin or urolithin conjugates in the biofluid sample.

[0013] The biofluid samples were selected from urine or blood, and in the case of blood, they were analyzed as dried blood spots.

[0014] In one embodiment, when determining the therapeutic dose, the urolithin conjugate is urolithin A glucuronide, and the biofluid is dried blood spots, and:

[0015] a) If the level of urolithin A glucuronide is below 5 ng / ml, the therapeutic dose of urolithin supplement is 1000 mg or 1500 mg per day, for example, 1000 mg per day.

[0016] b) If the level of urolithin A glucuronide is between 5 ng / ml and 50 ng / ml, the therapeutic dose of urolithin supplement is 500 mg daily; and

[0017] c) If the level of urolithin A glucuronide is between 50 ng / ml and 100 ng / ml, the therapeutic dose of urolithin supplement is 250 mg / day.

[0018] In another embodiment, when determining the therapeutic dose, the urolithin conjugate is urolithin A glucuronide, and the biofluid is urine.

[0019] a) If the level of urolithin A glucuronide is below 10,000 ng / ml, the therapeutic dose of urolithin supplement is 1500 mg or 1000 mg per day, for example, 1000 mg per day.

[0020] b) If the level of urolithin A glucuronide is between 10,000 ng / ml and 25,000 ng / ml, the therapeutic dose of urolithin supplement is 500 mg daily; and

[0021] c) If the level of urolithin A glucuronide is between 25,000 ng / ml and 50,000 ng / ml, the therapeutic dose of urolithin supplement is 250 mg / day.

[0022] In another embodiment of the invention, a method is provided for determining whether a human subject will benefit from taking a urolithiasis supplement, comprising:

[0023] a) Determine the level of urolithin or urolithin conjugates in a first biofluid sample collected prior to the administration of the test dose of urolithin or urolithin precursor.

[0024] b) Administer a test dose of urolithin or a urolithin precursor, such as pomegranate juice;

[0025] c) Determine the level of urolithin or urolithin conjugates in the second biological fluid sample; and

[0026] d) Determine whether the subject will benefit from taking urolithin supplements based on the increase in urolithin or urolithin conjugate levels in the biological fluid sample;

[0027] The biofluid samples were selected from urine or blood, and in the case of blood, were analyzed as dried blood spots; and

[0028] For example,

[0029] (i) wherein urolithin is administered, the urolithin conjugate is urolithin A glucuronide, and the biofluid is a dried drug spot, and wherein if the level of urolithin glucuronide is higher than 5 ng / ml and lower than 100 ng / ml, the subject will benefit from the dose of the urolithin supplement; or

[0030] (ii) wherein a urolithin precursor is administered, the urolithin conjugate is urolithin A glucuronide, and the biofluid is a dried drug spot, and wherein if the level of urolithin glucuronide is less than 100 ng / ml, the subject will benefit from the dose of the urolithin supplement; or

[0031] (iii) wherein urolithin is administered, the urolithin conjugate is urolithin A glucuronide, and the biofluid is urine, and wherein the subject will benefit from the dose of urolithin supplement if the level of urolithin glucuronide is higher than 5,000 ng / ml and lower than 50,000 ng / ml; or

[0032] (iv) wherein a urolithin precursor is administered, the urolithin conjugate is urolithin A glucuronide, and the biofluid is urine, and wherein if the level of urolithin glucuronide is below 50,000 ng / ml, the subject will benefit from the dose of the urolithin supplement.

[0033] Biological samples can be collected at any time after administration of urolithin or its precursors when urolithin or urolithin conjugates are detectable in the biological sample. For example, samples can be collected at 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours after administration of urolithin or its precursors. For example, 6-8 hours, 6-10 hours, 6-12 hours, 6-18 hours, 6-24 hours, 6-30 hours, 6-36 hours, 6-42 hours, or 6-48 hours. For example, 12-18 hours, 12-24 hours, 12-30 hours, 12-36 hours, 12-42 hours, or 12-48 hours. For example, 18-24 hours, 18-30 hours, 18-36 hours, 18-42 hours, or 18-48 hours. For example, 24-30 hours, 24-36 hours, 24-42 hours, or 24-48 hours. For example, 36-42 hours or 36-48 hours. In one embodiment, the biofluid sample is collected within the following timeframes;

[0034] (a) Between approximately 18 and 30 hours after administration of urolithin or its precursor; or

[0035] (b) Approximately 24 hours after administration of urolithin or urolithin precursor;

[0036] (c) Between approximately 6 and 8 hours after administration of urolithin or urolithin precursor.

[0037] In one embodiment, the present invention provides a method for determining whether a human subject will benefit from taking a urolithin supplement, comprising the steps of: analyzing a dried blood spot sample collected from the subject to determine whether the dried blood spot contains urolithin or urolithin conjugates, and then determining whether the subject will benefit from ingesting a urolithin supplement based on the level of urolithin or urolithin conjugates in the dried blood spot. In other embodiments of the invention, the method further comprises administering urolithin to the subject.

[0038] In one embodiment, the dried blood spot is a dried whole blood sample.

[0039] In another embodiment, the dried blood spot is a dried plasma sample.

[0040] In another embodiment, the dried blood spot is a dried serum sample.

[0041] In one embodiment, the level of urolithin, such as urolithin A, is determined. In another embodiment, the level of urolithin conjugates, such as urolithin A glucuronide, is determined. In another embodiment, the level of sulfated urolithin, such as sulfated urolithin A, is determined. In yet another embodiment, the level of urolithin A glucuronide is determined.

[0042] In one embodiment, the human subject requires drug treatment. In another embodiment, the human subject is in good general condition but will benefit from taking urolithiasis.

[0043] Dried blood spot samples are reconstructed in aqueous fluids such as water or buffer solutions, or in suitable solvents such as methanol. Assays capable of detecting and determining levels of urolithin or urolithin conjugates in aqueous dried blood spot samples are well known in the art.

[0044] In one embodiment, when the urolithin glucoside level is below about 10 ng / ml, the subject will benefit from the dose of the urolithin supplement. In one embodiment, when the urolithin glucoside level is below about 20 ng / ml, the subject will benefit from the dose of the urolithin supplement. In one embodiment, when the urolithin glucoside level is below about 30 ng / ml, the subject will benefit from the dose of the urolithin supplement. In one embodiment, when the urolithin glucoside level is below about 40 ng / ml, the subject will benefit from the dose of the urolithin supplement. In one embodiment, when the urolithin glucoside level is below about 50 ng / ml, the subject will benefit from the dose of the urolithin supplement. In one embodiment, when the urolithin glucoside level is below about 60 ng / ml, the subject will benefit from the dose of the urolithin supplement. In one embodiment, when the urolithin glucoside level is below about 70 ng / ml, the subject will benefit from the dose of the urolithin supplement. In one embodiment, when the urolithin glucoside level is below about 80 ng / ml, the subject will benefit from the dose of the urolithin supplement. In another embodiment, when the urolithin glucoside level is below about 90 ng / ml, the subject will benefit from the dose of the urolithin supplement. In another embodiment, when the urolithin glucoside level is below about 100 ng / ml, the subject will benefit from the dose of the urolithin supplement. In another embodiment, when the urolithin glucoside level is below about 110 ng / ml, the subject will benefit from the dose of the urolithin supplement. In another embodiment, when the urolithin glucoside level is below about 120 ng / ml, the subject will benefit from the dose of the urolithin supplement. In another embodiment, when the urolithin glucoside level is below about 130 ng / ml, the subject will benefit from the dose of the urolithin supplement. In another embodiment, when the level of urolithin glucoside is below about 140 ng / ml, the subject will benefit from the dose of the urolithin supplement. In another embodiment, when the level of urolithin glucoside is below about 150 ng / ml, the subject will benefit from the dose of the urolithin supplement.

[0045] In addition to providing information about whether human subjects will benefit from taking urolithin supplements, the level of urolithin or urolithin conjugates can also provide information about the appropriate therapeutic dose for human subjects. Therefore, according to another aspect of the invention, a method for determining a therapeutic dose of urolithin supplement for human subjects is provided, comprising:

[0046] a) Determine the level of urolithin or urolithin conjugates in dried blood spot samples; and

[0047] b) Determine the therapeutic dose of urolithin supplements based on the level of urolithin or urolithin conjugates in the dried blood spot sample.

[0048] In another embodiment, the present invention provides a method for administering urolithin to a human subject, comprising:

[0049] a) Determine the level of urolithin or urolithin conjugates in dried blood spot samples;

[0050] b) Determine the therapeutic dose of urolithin supplements based on the level of urolithin or urolithin conjugates in the dried blood spot sample; and

[0051] c) Administer urolithiasis to the subject.

[0052] In one embodiment, the level of urolithin or urolithin conjugates in a dried blood spot sample can be used according to a continuous scale, for example, for an amount of urolithin or urolithin conjugates between 10 ng / ml and 100 ng / day, the indicated therapeutic dose is between 100 mg / day and 1500 mg / day, for example, 100 mg / day to 1000 mg / day. In another embodiment, the therapeutic dose will be selected from the range of amounts of urolithin or urolithin conjugates indicating a therapeutic dose per unit dose of urolithin. For example:

[0053] a) If the level of urolithin glucoside is below 10 ng / ml, the therapeutic dose of urolithin supplement is 1000 mg per day;

[0054] b) If the urolithin glucoside level is between 10 ng / ml and 50 ng / ml, the therapeutic dose of urolithin supplement is 500 mg daily; and

[0055] c) If the level of urolithin glucoside is between 50 ng / ml and 100 ng / ml, the therapeutic dose of urolithin supplement is 250 mg / day.

[0056] For example:

[0057] a) If the level of urolithin glucoside is below 5 ng / ml, the therapeutic dose of urolithin supplement is 1000 mg per day;

[0058] b) If the urolithin glucoside level is between 5 ng / ml and 50 ng / ml, the therapeutic dose of urolithin supplement is 500 mg daily; and

[0059] c) If the level of urolithin glucoside is between 50 ng / ml and 100 ng / ml, the therapeutic dose of urolithin supplement is 250 mg / day.

[0060] In addition to determining the amount of urolithin or urolithin conjugates in dried blood samples during separation, the present invention also provides a method in which a human subject ingests a urolithin source. Such a urolithin source may include unit dosage forms containing urolithin or urolithin precursors. Therefore, in another embodiment of the invention, a method is provided for determining whether a human subject will benefit from taking a urolithin supplement, comprising:

[0061] a) Determine the level of urolithin or urolithin conjugates in the first dried blood spot sample collected before the administration of the test dose of urolithin;

[0062] b) Administer a test dose of urolithiasis;

[0063] c) Determine the level of urolithin or urolithin conjugates in the second dried blood spot sample; and

[0064] d) Determine whether the subject will benefit from taking urolithin supplements based on the increased levels of urolithin or urolithin conjugates in dried blood spot samples.

[0065] In another embodiment of the invention, a method for administering urolithin to a human subject is provided, comprising:

[0066] a) Determine the level of urolithin or urolithin conjugates in the first dried blood spot sample collected before the administration of the test dose of urolithin;

[0067] b) Administer a test dose of urolithiasis;

[0068] c) Determine the level of urolithin or urolithin conjugates in the second dried blood spot sample;

[0069] d) Determine whether the subject will benefit from urolithin supplementation based on increased levels of urolithin or urolithin conjugates in dried blood spot samples; and

[0070] e) Administer urolithiasis to the subject.

[0071] To avoid ambiguity, for any method of the present invention including the administration of urolithin or a urolithin precursor, the method may be performed without collecting a first biological sample, such as urine or blood, and after the administration of urolithin or the urolithin precursor, only the biological sample is collected for urolithin / urolithin conjugate determination. The benefit of administering a urolithin supplement or a dose of said supplement can then be determined based on the level of urolithin or urolithin conjugate in the sample collected after the administration of urolithin or the urolithin precursor.

[0072] Following urolithin intake, the levels of urolithin and / or urolithin conjugates in dried blood samples will depend on the uptake and metabolic efficiency of urolithin or its precursors in human subjects, as well as the amount of urolithin ingested. In one embodiment, if the level of urolithin glucoside is above 10 ng / ml and below 150 ng / ml, the subject will benefit from the dose of urolithin supplementation. In another embodiment, if the level of urolithin glucoside is below 150 ng / ml, the subject will benefit from the dose of urolithin supplementation. In another embodiment, if the level of urolithin glucoside is above 5 ng / ml and below 100 ng / ml, the subject will benefit from the dose of urolithin supplementation. In another embodiment, if the level of urolithin glucoside is below 100 ng / ml, the subject will benefit from the dose of urolithin supplementation. In yet another embodiment, the level of urolithin glucoside is associated with therapeutic doses of 250 mg, 500 mg, 1000 mg, or 1500 mg as defined above.

[0073] The levels of urolithin and / or urolithin conjugates can be determined by methods known to those skilled in the art, such as high performance liquid chromatography coupled with mass spectrometry (HPLC / MS).

[0074] Prior to ingestion of urolithin supplements, the levels of urolithin or urolithin conjugates in dried blood spots will depend primarily on whether the subject possesses the gut microbiome required to metabolize urolithin precursors into urolithin. However, this will also depend on the diet of the human subject. In another embodiment, urolithin precursors may be administered to the human subject prior to collection of dried blood spot samples. Therefore, according to another embodiment of the invention, a method for determining whether a human subject will benefit from taking urolithin supplements is provided, comprising:

[0075] a) Determine the level of urolithin or urolithin conjugates in the first dried blood spot sample collected before the application of a urolithin precursor (e.g., pomegranate juice);

[0076] b) Administer urolithiasis precursor;

[0077] c) Determine the level of urolithin or urolithin conjugates in the second dried blood spot sample; and

[0078] d) Determine whether the subject will benefit from taking urolithin supplements based on the increase in urolithin or urolithin conjugate levels in the second dried blood spot sample compared to the first dried blood spot sample.

[0079] Therefore, according to another embodiment of the present invention, a method for administering urolithin to a human subject is provided, comprising:

[0080] a) Determine the level of urolithin or urolithin conjugates in the first dried blood spot sample collected before the application of a urolithin precursor (e.g., pomegranate juice);

[0081] b) Administer urolithiasis precursor;

[0082] c) Determine the level of urolithin or urolithin conjugates in the second dried blood spot sample;

[0083] d) Determine whether the subject will benefit from urolithin supplementation based on the increased levels of urolithin or urolithin conjugates in the second dried blood spot sample compared to the first dried blood spot sample; and

[0084] e) Administer urolithiasis to the subject.

[0085] Following ingestion of the urolithin precursor, the levels of urolithin and / or urolithin conjugates in dried blood spot samples will depend on the metabolic efficiency of the urolithin precursor in human subjects and the amount of urolithin precursor ingested. In one embodiment, if the level of urolithin glucoside is above 10 ng / ml and below 150 ng / ml, the subject will benefit from the dose of the urolithin supplement. In another embodiment, if the level of urolithin glucoside is below 150 ng / ml, the subject will benefit from the dose of the urolithin supplement. In another embodiment, if the level of urolithin glucoside is above 5 ng / ml and below 100 ng / ml, the subject will benefit from the dose of the urolithin supplement. In another embodiment, if the level of urolithin glucoside is below 100 ng / ml, the subject will benefit from the dose of the urolithin supplement. In yet another embodiment, the level of urolithin glucoside is associated with therapeutic doses of 250 mg, 500 mg, 1000 mg, or 1500 mg as defined above.

[0086] In any embodiment of the invention, the urolithin test dose may be any dose of urolithin that produces a measurable amount of urolithin or urolithin conjugates in dried blood spots or urine samples. For example, the test dose may be selected from 50 mg, 100 mg, 250 mg, 500 mg, or 1000 mg, such as 250 mg, 500 mg, or 1000 mg. In one embodiment, the test dose is selected from 250 mg, 500 mg, or 1000 mg.

[0087] A dried blood spot sample can be collected at any time after ingestion of urolithin or a urolithin precursor when urolithin or urolithin conjugates are detectable in dried blood. For example, at any time between approximately 2 hours and approximately 72 hours after ingestion of urolithin or a urolithin precursor, such as between approximately 2 hours and approximately 60 hours after ingestion of a urolithin supplement, such as between approximately 2 hours and approximately 48 hours, such as between approximately 4 hours and approximately 48 hours, such as between approximately 4 hours and approximately 36 hours, such as between approximately 4 hours and approximately 24 hours. In one embodiment, at approximately 4 hours, approximately 6 hours, approximately 8 hours, approximately 10 hours, approximately 12 hours, or approximately 24 hours after ingestion of urolithin or a urolithin supplement, such as approximately 6 hours, approximately 8 hours, or approximately 24 hours.

[0088] Surprisingly, we observed a correlation between the subjects' ability to metabolize urolithin precursors (e.g., pomegranate juice) to form urolithin A and the health status of their microbiome. Therefore, the levels of urolithin or urolithin conjugates in dried blood or urine samples are biomarkers of a healthy gut microbiome. The health status of the subjects' microbiome is measured by the diversity and richness of the gut microbiota.

[0089] As described above, the level of urolithin or urolithin conjugates in dried blood spots will primarily depend on whether the subject possesses the gut microbiota required to metabolize urolithin precursors into urolithin. Therefore, the level of urolithin or urolithin conjugates in dried blood is a biomarker of a healthy gut microbiome. Accordingly, according to another aspect of the invention, a method for determining the health status of an individual's gut microbiome is provided, comprising the steps of: analyzing dried blood spot samples collected from a subject to determine whether the dried blood spots contain urolithin or urolithin conjugates as an indicator of the individual's gut microbiome health status.

[0090] According to another embodiment of the present invention, a method for determining the health status of a subject's gut microbiome is provided, comprising the following steps: analyzing a biofluid sample collected from the subject to determine whether the biofluid contains urolithin or urolithin conjugates as an indicator of the subject's gut microbiome health status;

[0091] The biofluid samples were selected from urine or blood, and in the case of blood, they were analyzed as dried blood spots.

[0092] According to another aspect of the present invention, a method for determining the gut microbiome health status of a subject is provided, comprising the steps of: analyzing a biological fluid sample collected from the subject, determining the level of urolithin or urolithin conjugates in the biological fluid, and optionally collecting a fecal sample for microbiome analysis as an indicator of the subject's gut health status.

[0093] The biofluid samples were selected from urine or blood, and in the case of blood, they were analyzed as dried blood spots.

[0094] According to another aspect of the present invention, a method for determining the health status of an individual's gut microbiome is provided, comprising the steps of: analyzing dried blood spot samples collected from a subject, and determining the level of urolithin or urolithin conjugates in the dried blood spots as an indicator of the health status of the individual's gut microbiome.

[0095] According to another aspect of the present invention, a method for determining the health status of a subject's gut microbiome is provided, comprising the steps of: analyzing dried blood spot samples collected from the subject, and determining the level of urolithin or urolithin conjugates in the dried blood spots as an indicator of the subject's gut microbiome health status.

[0096] According to another aspect of the present invention, a method for determining the health status of a subject's gut microbiome is provided, comprising the steps of: analyzing dried blood spot samples collected from the subject, determining the level of urolithin or urolithin conjugates in the dried blood spots, and optionally collecting fecal samples for microbiome analysis as an indicator of the subject's gut microbiome health status.

[0097] According to another aspect of the present invention, a method for determining the health status of a subject's gut microbiome is provided, comprising the steps of: analyzing a urine sample collected from the subject to determine whether the urine contains urolithin or urolithin conjugates as an indicator of the subject's gut microbiome health status.

[0098] According to another aspect of the present invention, a method for determining the gut microbiome health status of a subject is provided, comprising the steps of: analyzing a urine sample collected from the subject to determine the level of urolithin or urolithin conjugates in the urine, and optionally collecting a fecal sample for microbiome analysis as an indicator of the subject's gut health status.

[0099] In one embodiment, a method for determining the health status of a subject’s microbiome includes administering a urolithin precursor, such as pomegranate juice, before measuring the level of urolithin or urolithin conjugates in a biological sample.

[0100] The biofluid samples were selected from urine or blood, and in the case of blood, they were analyzed as dried blood spots.

[0101] In another embodiment, if the subject is a low- or high-yield producer of urolithin precursors to urolithin (e.g., urolithin A), then the subject has a rich microbiome.

[0102] In another embodiment, after administration of a urolithin precursor (e.g., pomegranate juice), low-yielders were subjects whose urolithin A glucuronide levels were measured in dried blood spot samples from 5 ng / ml to 100 ng / ml, while high-yielders were subjects whose urolithin A glucuronide levels were greater than 100 ng / ml.

[0103] In another embodiment, after administration of a urolithin precursor (e.g., pomegranate juice), low-yielders were subjects whose urolithin A glucuronide levels in urine were between 5,000 g / ml and 25,000 ng / ml, while high-yielders were subjects whose urolithin A glucuronide levels in urine were greater than 50,000 ng / ml, for example, greater than 25,000 ng / ml.

[0104] It is believed that the higher the production of urolithin A or urolithin A glucuronide, the richer the subject's microbiome.

[0105] In one embodiment, urolithin or urolithin conjugates are measured as an indicator of microbiome health status approximately 6 to 8 hours after administration of the urolithin precursor or approximately 24 hours after administration of the urolithin precursor.

[0106] In another aspect of the invention, a method is provided for determining a therapeutic dose of a urolithiasis supplement for use in human subjects, comprising:

[0107] a) Determine the level of urolithin or urolithin conjugates in dried blood spot samples; and

[0108] b) Determine the therapeutic dose of urolithin supplements based on the level of urolithin or urolithin conjugates in the dried blood spot sample.

[0109] In another embodiment, a method for estimating the concentration of urolithin or urolithin conjugates in blood, plasma, or serum is provided, comprising:

[0110] (a) Calculate the level of urolithin or urolithin conjugates in dried blood spots;

[0111] (b) Using the amount of blood used to prepare dried blood spots, the level is converted into a concentration of urolithin.

[0112] (c) Multiply the concentration by a factor of about 3.5 to about 4.5, for example about 3.7 to about 4.3, for example about 3.5 to about 4.0, for example about 3.8 to about 4.2, for example about 3.7 to 3.9.

[0113] Another embodiment of the present invention provides a method for determining whether a human subject will benefit from taking a urolithin supplement, comprising the steps of: analyzing a urine sample collected from the subject to determine whether the urine contains urolithin or urolithin conjugates, and then determining whether the subject will benefit from taking a urolithin supplement based on the level of urolithin or urolithin conjugates in the urine.

[0114] In another embodiment, the present invention provides a method for administering urolithin to a human subject, comprising the steps of: analyzing a urine sample taken from the subject to determine whether the urine contains urolithin or urolithin conjugates, then determining whether the subject will benefit from ingesting a urolithin supplement based on the level of urolithin or urolithin conjugates in the urine, and administering urolithin to the subject.

[0115] In one embodiment, the method of the present invention is used to determine the level of urolithin. In another embodiment, the method of the present invention is used to determine the level of urolithin conjugates. In another embodiment, the method of the present invention is used to determine the level of sulfated urolithin. In yet another embodiment, the method of the present invention is used to determine the level of urolithin glucuronide, for example, urolithin A or urolithin A conjugates, such as urolithin A glucuronide.

[0116] In one embodiment, the human subject requires drug treatment. In another embodiment, the human subject is in good general condition but will benefit from taking urolithiasis.

[0117] Tests capable of determining the level of urolithin or urolithin conjugates in urine samples are well known in the art.

[0118] In another embodiment, when the urolithin glucoside level is below about 10,000 ng / ml, the subject will benefit from the dose of the urolithin supplement. In another embodiment, when the urolithin glucoside level is below about 20,000 ng / ml, the subject will benefit from the dose of the urolithin supplement. In another embodiment, when the urolithin glucoside level is below about 30,000 ng / ml, the subject will benefit from the dose of the urolithin supplement. In another embodiment, when the urolithin glucoside level is below about 40,000 ng / ml, the subject will benefit from the dose of the urolithin supplement. In another embodiment, when the urolithin glucoside level is below about 50,000 ng / ml, the subject will benefit from the dose of the urolithin supplement. In another embodiment, when the urolithin glucoside level is below about 60,000 ng / ml, the subject will benefit from the dose of the urolithin supplement.

[0119] In addition to providing information about whether human subjects will benefit from taking urolithin supplements, the level of urolithin or urolithin conjugates can also provide information about the appropriate therapeutic dose for human subjects. Therefore, according to another aspect of the invention, a method for determining a therapeutic dose of urolithin supplement for human subjects is provided, comprising:

[0120] a) Determining the level of urolithin or urolithin conjugates in urine samples; and

[0121] b) Determine the therapeutic dose of urolithin supplements based on the level of urolithin or urolithin conjugates in the urine sample.

[0122] In another aspect of the invention, a method for administering a urolithin supplement to a human subject is provided, comprising:

[0123] a) Determine the level of urolithin or urolithin conjugates in urine samples;

[0124] b) Determine the therapeutic dose of urolithin supplements based on the level of urolithin or urolithin conjugates in the urine sample; and

[0125] c) Administer therapeutic doses of urolithiasis to human subjects.

[0126] In one embodiment, the level of urolithin or urolithin conjugates in the urine sample may be used according to a continuous scale, for example, for amounts of urolithin or urolithin conjugates between 10,000 ng / ml and 50,000 ng / day, the indicated therapeutic dose is between 100 mg / day and 1500 mg / day, or between 100 mg / day and 1000 mg / day. In another embodiment, the therapeutic dose will be selected from the range of amounts of urolithin or urolithin conjugates indicating a therapeutic dose per unit dose of urolithin. For example,

[0127] a) If the level of urolithin glucuronide is below about 10,000 ng / ml, the therapeutic dose of urolithin supplement is 1500 mg or 1000 mg per day, for example, 100 mg per day;

[0128] b) If the urolithin glucoside level is between approximately 10,000 ng / ml and approximately 25,000 ng / ml, the therapeutic dose of urolithin supplement is 500 mg daily; and

[0129] c) If the level of urolithin glucoside is between approximately 25,000 ng / ml and approximately 50,000 ng / ml, the therapeutic dose of urolithin supplement is 250 mg / day.

[0130] In addition to determining the amount of urolithin or urolithin conjugates in a urine sample during separation, the present invention also provides a method in which a human subject ingests a urolithin source. Such a urolithin source may include unit dosage forms containing urolithin or urolithin precursors. Therefore, in another embodiment of the invention, a method is provided for determining whether a human subject will benefit from taking a urolithin supplement, comprising:

[0131] a) Optionally, determine the level of urolithin or urolithin conjugates in a urine sample collected prior to the administration of the test dose of urolithin or urolithin precursor.

[0132] b) Administer a test dose of urolithin or urolithin precursor;

[0133] c) Determine the level of urolithin or urolithin conjugates in urine samples collected after administration of the test dose, for example, approximately 24 hours after administration of the test dose; and

[0134] d) Determine whether the subject will benefit from taking urolithin supplements based on the level or elevated level of urolithin or urolithin conjugates in the urine sample.

[0135] In another embodiment of the invention, a method for administering urolithin to a human is provided, comprising:

[0136] a) Optionally, determine the level of urolithin or urolithin conjugates in a urine sample collected prior to the administration of the test dose of urolithin or urolithin precursor.

[0137] b) Administer a test dose of urolithin or urolithin precursor;

[0138] c) Determine the level of urolithin or urolithin conjugates in urine samples collected after administration of the test dose, for example, approximately 24 hours after administration of the test dose;

[0139] d) Determine whether the subject will benefit from urolithin supplementation based on the level or elevated level of urolithin or urolithin conjugates in the urine sample; and

[0140] e) Administer urolithin or urolithin precursor to the subject.

[0141] Following ingestion of urolithin or its precursors, the levels of urolithin and / or urolithin conjugates in urine will depend on the uptake and metabolic efficiency of urolithin or its precursors in human subjects. In one embodiment, if the level of urolithin glucoside is above 10,000 ng / ml and below 50,000 ng / ml, the subject will benefit from the dose of the urolithin supplement. In another embodiment, if the level of urolithin glucoside is below 50,000 ng / ml, the subject will benefit from the dose of the urolithin supplement. In yet another embodiment, the level of urolithin glucoside is associated with therapeutic doses of 250 mg, 500 mg, 1000 mg, or 1500 mg as defined above.

[0142] The levels of urolithin and / or urolithin conjugates can be determined by methods known to those skilled in the art, such as high performance liquid chromatography coupled with mass spectrometry (HPLC / MS).

[0143] Prior to ingesting a urolithin supplement, the level of urolithin or urolithin conjugates in urine will depend primarily on whether the subject possesses the gut microbiota required to metabolize urolithin precursors into urolithin. However, this will also depend on the diet of the human subject. In another embodiment, a urolithin precursor may be administered to the human subject prior to urine collection. Therefore, according to another embodiment of the invention, a method for determining whether a human subject will benefit from taking a urolithin supplement is provided, comprising:

[0144] a) Determine the level of urolithin or urolithin conjugates in the first urine sample collected before the administration of a urolithin precursor (e.g., pomegranate juice);

[0145] b) Administer urolithiasis precursor;

[0146] c) Determine the level of urolithin or urolithin conjugates in the second urine sample; and

[0147] d) Determine whether the subject will benefit from taking urolithin supplements based on the increased levels of urolithin or urolithin conjugates in the second urine sample compared to the first urine sample.

[0148] In another embodiment of the invention, a method for administering urolithin to a human subject is provided, comprising:

[0149] a) Determine the level of urolithin or urolithin conjugates in the first urine sample collected before the administration of a urolithin precursor (e.g., pomegranate juice);

[0150] b) Administer urolithiasis precursor;

[0151] c) Determine the level of urolithin or urolithin conjugates in the second urine sample;

[0152] d) Determine whether the subject will benefit from taking urolithin supplements based on the increased levels of urolithin or urolithin conjugates in the second urine sample compared to the first urine sample.

[0153] e) Administer urolithiasis to the subject.

[0154] Following ingestion of the urolithin precursor, the levels of urolithin and / or urolithin conjugates in the urine sample will depend on the metabolic efficiency of the urolithin precursor in human subjects. In one embodiment, if the level of urolithin glucoside is above 5,000 ng / ml and below 50,000 ng / ml, the subject will benefit from the dose of the urolithin supplement. In another embodiment, if the level of urolithin glucoside is below 50,000 ng / ml, the subject will benefit from the dose of the urolithin supplement. In yet another embodiment, the level of urolithin glucoside is associated with therapeutic doses of 250 mg, 500 mg, 1000 mg, or 1500 mg as defined above.

[0155] Following administration of urolithin or a urolithin precursor, the level of urolithin or urolithin conjugates in urine can also be used to determine the therapeutic dose of urolithin for the subject. In one embodiment, the level of urolithin or urolithin conjugates in the urine sample can be used according to a continuous scale, for example, for amounts of urolithin or urolithin conjugates between 5,000 ng / ml and 50,000 ng / day, the indicated therapeutic dose is between 100 mg / day and 1500 mg / day, for example, 100 mg / day to 1000 mg / day. In another embodiment, the therapeutic dose will be selected from the range of amounts of urolithin or urolithin conjugates indicating a therapeutic dose per unit dose of urolithin. For example,

[0156] a) If the level of urolithin glucoside is below approximately 10,000 ng / ml, the therapeutic dose of urolithin supplement is 1000 mg per day;

[0157] b) If the urolithin glucoside level is between approximately 10,000 ng / ml and approximately 25,000 ng / ml, the therapeutic dose of urolithin supplement is 500 mg daily; and

[0158] c) If the level of urolithin glucoside is between approximately 25,000 ng / ml and approximately 50,000 ng / ml, the therapeutic dose of urolithin supplement is 250 mg / day.

[0159] The test dose of urolithin can be any dose of urolithin that produces a measurable amount of urolithin or urolithin conjugates in the urine. For example, the test dose can be selected from 50 mg, 100 mg, 250 mg, 500 mg, or 1000 mg, such as 250 mg, 500 mg, or 1000 mg, such as 250 mg or 500 mg.

[0160] Urine samples can be collected at any time after ingestion of urolithin or its precursor, when urolithin or urolithin conjugates are detectable in the urine. For example, at any time between approximately 2 hours and approximately 72 hours after ingestion of urolithin or its precursor, such as between approximately 2 hours and approximately 60 hours after ingestion of the urolithin precursor, such as between approximately 2 hours and approximately 48 hours, such as between approximately 4 hours and approximately 48 hours, such as between approximately 4 hours and approximately 36 hours, such as between approximately 4 hours and approximately 24 hours. In one embodiment, at approximately 4 hours, approximately 6 hours, approximately 8 hours, approximately 10 hours, approximately 12 hours, approximately 18 hours, or approximately 24 hours after ingestion of urolithin or its precursor, for example, approximately 24 hours after ingestion of urolithin or its precursor.

[0161] In another aspect of the invention, a method is provided for determining a therapeutic dose of a urolithiasis supplement for use in human subjects, comprising:

[0162] a) Determining the level of urolithin or urolithin conjugates in urine samples; and

[0163] b) Determine the therapeutic dose of urolithin supplements based on the level of urolithin or urolithin conjugates in the urine sample.

[0164] In another aspect of the invention, a method for administering urolithin to a human subject is provided, comprising:

[0165] a) Determine the level of urolithin or urolithin conjugates in urine samples;

[0166] b) Determine the therapeutic dose of urolithin supplements based on the level of urolithin or urolithin conjugates in the urine sample; and

[0167] c) Administer the therapeutic dose to the subject.

[0168] The reagent kit of the present invention

[0169] One consideration when using the methods of the present invention is to make these methods available to subjects. One approach to this is to provide a kit containing all the materials required for a subject to collect and detect dried blood spots using the method. Therefore, in one embodiment of the invention, a kit is provided for determining whether a human subject will benefit from taking a urolithin supplement or for determining a therapeutic dose of a urolithin supplement for a human subject, and includes:

[0170] (a) One of several fluid collection devices used for collecting dried blood;

[0171] (b) Optionally, urolithin or urolithin precursor;

[0172] (c) Instructions for taking dried blood spot samples or dried blood samples; and

[0173] (d) A device for detecting dried blood spot samples or a device for obtaining test dried blood samples (e.g., a suitable mailing envelope or mailing container).

[0174] Fluid collection devices can be any solid absorbent carrier suitable for collecting whole blood, plasma, or serum samples, on which the blood, plasma, or serum sample can be dried. Such devices can be stored for subsequent urolithiasis assays.

[0175] In one embodiment, the filter collection device is a card. Such cards contain filter paper, such as cellulose filter paper, for example, Whatman 903 filter paper or Whatman 903 sample collection card (GE Healthcare, Little Halford, Buckinghamshire, UK) or Perkin Elmer (Boston, Massachusetts, USA) blood collection card.

[0176] Fluid acquisition cards also include cards capable of separating blood sample components, such as red blood cells from plasma for analysis within the plasma. For example, the ADX100 Blood Card (Advance Dx Inc., Chicago, Illinois, USA). Further information about the ADX Blood Card can be found in U.S. Patent 8,062,608.

[0177] In another embodiment, the filter collection device includes an absorbent rod capable of absorbing whole blood, plasma, or serum samples and drying them. The tip of such a rod is made of a hydrophilic porous material for sample collection, such as a tip made of a hydrophilic polymer material (e.g., polyethylene). Examples of such devices include: Mitra... TM Microsampler (Neoteryx, LLC., Torrance, California, USA). For more information, please see International Patent Application No.: WO 2013 / 067520.

[0178] In another embodiment, the blood collection device includes a sealable container. The container includes absorbent material and a desiccant. Once a whole blood, plasma, or serum sample has been applied to the absorbent material, the container is sealed for storage and transport. Examples of such devices include: hemaspot HF. TM (Spot on Sciences, San Francisco, California, USA). About hemaspot HF TM More information about the device can be found in U.S. Patent No. 9759640.

[0179] Blood samples are typically collected by pricking a finger with a lancet or similar sharp instrument (preferably a sterile lancet or similar sharp instrument). The blood sample consists of many drops of blood, for example, 3 to 4 drops, which may be dripped onto or absorbed into a fluid collection device. The blood sample may also be a sample of blood, such as venous blood obtained using a syringe or cannula.

[0180] As described above, the method of the present invention is applicable to whole blood dried spots, serum dried spots, or plasma dried spots. Therefore, before drying, whole blood samples can be separated into plasma or serum, for example, by centrifugation or coagulation.

[0181] It can collect a single dried blood spot sample or collect several dried blood spot samples at different times.

[0182] In another embodiment, a kit is provided for determining whether a human subject will benefit from taking a urolithin supplement or for determining a therapeutic dose of a urolithin supplement for a human subject, and includes:

[0183] (a) One of several fluid collection devices used to collect dried blood spots;

[0184] (b) Instructions for taking dried blood spot samples; and

[0185] (c) A device for detecting dried blood spot samples or a device for obtaining test dried blood spot samples (e.g., a suitable mailing envelope or mailing container).

[0186] In another embodiment, a kit is provided for determining whether a human subject will benefit from taking a urolithin supplement or for determining a therapeutic dose of a urolithin supplement for a human subject, and includes:

[0187] (d) One of several fluid collection devices used for collecting dried blood;

[0188] (e) Urolithin or urolithin precursors, such as urolithin A;

[0189] (f) Instructions for taking dried blood spot samples; and

[0190] (g) Apparatus for detecting dried blood spot samples or for obtaining test dried blood spot samples (e.g., a suitable mailing envelope or mailing container).

[0191] Another approach is to provide a kit containing all the materials required for a subject to collect and test urine for this method. Therefore, in one embodiment of the invention, a kit is provided for determining whether a human subject will benefit from taking a urolithin supplement or for determining a therapeutic dose of a urolithin supplement for a human subject, and includes:

[0192] (a) One of several containers used for collecting urine;

[0193] (b) Optionally, urolithin or a urolithin precursor; and

[0194] (c) Instructions for collecting urine samples.

[0195] In another embodiment of the invention, a kit is provided for determining whether a human subject will benefit from taking a urolithin supplement or for determining a therapeutic dose of a urolithin supplement for a human subject, and includes:

[0196] (a) One of several containers used for collecting urine;

[0197] (b) Optionally, urolithin or urolithin precursor;

[0198] (c) Instructions for urine sample collection; and

[0199] (d) A device for testing a urine sample or a device for transporting a urine sample to a testing site (e.g., a suitable mailing envelope or mailing container).

[0200] The container can be any container suitable for collecting urine samples, such as a BD container. Plus urine analysis tubes (Becton Dickinson, Franklin Lake, New Jersey, USA). The containers can be empty or filled with reagents, such as lyophilization preservatives (e.g., boric acid, sodium formate, and sodium borate), which maintain the integrity of the urine sample at room temperature.

[0201] A single urine sample can be collected, or several urine samples can be collected at different times.

[0202] In another embodiment, a kit is provided for determining whether a human subject will benefit from taking a urolithin supplement or for determining a therapeutic dose of a urolithin supplement for a human subject, and includes:

[0203] (a) One of several containers used for collecting urine;

[0204] (b) Urolithin, such as urolithin A or urolithin precursors, such as pomegranate juice;

[0205] (c) Instructions for urine sample collection; and

[0206] (d) A device for testing a urine sample or a device for transporting a urine sample to a testing site (e.g., a suitable mailing envelope or mailing container).

[0207] The system of the present invention

[0208] In another embodiment of the invention, an apparatus is provided for presenting relevant information about the method of the invention. The relevant information includes the determination status of urolithin or urolithin conjugate levels in dried blood spots or urine samples, the results of dried blood spot or urine sample testing, such as the level of urolithin or urolithin conjugates in dried blood spots or urine samples, or the therapeutic dose of urolithin, and / or conclusions drawn based on the level of urolithin or urolithin conjugates in the dried blood spot or urine samples, such as whether a subject will benefit from taking urolithin or urolithin supplements and / or the dose of urolithin or urolithin supplements.

[0209] One method of presenting relevant information is via a software program through a user interface, such as a mobile application, like a mobile application on a mobile phone or tablet device, referred to as an "app". In one embodiment, the user interface is an internet user interface.

[0210] Therefore, according to another embodiment of the present invention, a system is provided for determining whether a human subject will benefit from taking a urolithiasis supplement, and includes the following steps:

[0211] a) Analyze dried blood spot samples collected from subjects;

[0212] b) Determine the level of urolithin or urolithin conjugates in dried blood spot samples;

[0213] c) Determine whether the subject will benefit from urolithin supplementation based on the level of urolithin in dried blood spots; and

[0214] d) Present on the user interface whether the subject will benefit from the dosage of the urolithiasis supplement.

[0215] In another embodiment of the invention, a system is provided for delivering a therapeutic dose of a urolithiasis supplement for human subjects, and includes the following steps:

[0216] a) Analyze dried blood spot samples collected from subjects;

[0217] b) Determine the level of urolithin or urolithin conjugates in dried blood spot samples;

[0218] c) Determine the therapeutic dose of urolithin supplement for the subject based on the level of urolithin or urolithin conjugates in the dried blood spot sample; and

[0219] d) Present the therapeutic dose of urolithiasis supplement for the subject on the user interface.

[0220] In another embodiment of the invention, a system is provided for determining whether a human subject will benefit from taking a urolithiasis supplement, and includes the following steps:

[0221] a) Analyze urine samples collected from the subjects;

[0222] b) Determine the level of urolithin or urolithin conjugates in the urine sample;

[0223] c) Determine whether the subject will benefit from urinary lithiform supplementation based on the level of lithiform in the urine; and

[0224] d) Present on the user interface whether the subject will benefit from the dosage of the urolithiasis supplement.

[0225] In another embodiment of the invention, a system is provided for delivering a therapeutic dose of a urolithiasis supplement for human subjects, and includes the following steps:

[0226] a) Analyze urine samples collected from the subjects;

[0227] b) Determine the level of urolithin or urolithin conjugates in the urine sample;

[0228] c) Determine the therapeutic dose of urolithin supplement for the subject based on the level of urolithin or urolithin conjugates in the urine sample; and

[0229] d) Present the therapeutic dose of urolithiasis supplement for the subject on the user interface.

[0230] In another embodiment of the invention, a system is provided for determining whether a human subject will benefit from taking a urolithiasis supplement, and includes the following steps:

[0231] a) Determine the level of urolithin or urolithin conjugates in the first dried blood spot sample collected before the administration of the test dose of urolithin;

[0232] b) Administer a test dose of urolithiasis;

[0233] c) Determine the level of urolithin or urolithin conjugates in second dried blood spot samples collected at time intervals (e.g., approximately 6-8 hours or approximately 24 hours); and

[0234] d) Determine whether the subject will benefit from urolithin supplementation based on increased levels of urolithin or urolithin conjugates in dried blood spot samples; and

[0235] e) Present the therapeutic dose of urolithiasis supplement for the subject on the user interface.

[0236] In another embodiment of the invention, a system is provided for determining whether a human subject will benefit from taking a urolithiasis supplement, and includes the following steps:

[0237] a) Determine the level of urolithin or urolithin conjugates in the first dried blood spot sample collected before the application of a urolithin precursor (e.g., pomegranate juice);

[0238] b) Administer urolithiasis precursor;

[0239] c) Determine the level of urolithin or urolithin conjugates in the second dried blood spot sample; and

[0240] d) Determine whether the subject will benefit from urolithin supplementation based on the increased levels of urolithin or urolithin conjugates in the second dried blood spot sample compared to the first dried blood spot sample; and

[0241] e) Present the therapeutic dose of urolithiasis supplement for the subject on the user interface.

[0242] In another embodiment of the invention, a system is provided for determining whether a human subject will benefit from taking a urolithiasis supplement, and includes the following steps:

[0243] a) Determine the level of urolithin or urolithin conjugates in the first urine sample collected before the administration of the test dose of urolithin;

[0244] b) Administer a test dose of urolithiasis;

[0245] c) Determine the level of urolithin or urolithin conjugates in the second urine sample; and

[0246] d) Determine whether the subject will benefit from urolithin supplementation based on elevated levels of urolithin or urolithin conjugates in the urine sample; and

[0247] e) Present the therapeutic dose of urolithiasis supplement for the subject on the user interface.

[0248] In another embodiment of the invention, a system is provided for determining whether a human subject will benefit from taking a urolithiasis supplement, and includes the following steps:

[0249] a) Determine the level of urolithin or urolithin conjugates in the first urine sample collected before the administration of a urolithin precursor (e.g., pomegranate juice);

[0250] b) Administer urolithiasis precursor;

[0251] c) Determine the level of urolithin or urolithin conjugates in the second urine sample; and

[0252] d) Determine whether the subject will benefit from urolithin supplementation based on the increased levels of urolithin or urolithin conjugates in the second urine sample compared to the first urine sample; and

[0253] e) Present the benefits of taking urolithiasis on the user interface.

[0254] The urolithin or urolithin level used for use with the system of the present invention is the same as that disclosed in the method of the present invention described above.

[0255] The computer implementation method of the present invention

[0256] The method of the present invention can be implemented using a computer to facilitate sample collection and the transmission of results to subjects. For example, subjects can be provided with a computer program, such as an "app" on a mobile computer device (e.g., a mobile phone or tablet).

[0257] Such computer programs allow subjects to input data, such as:

[0258] (i) Record the biological samples that have been collected.

[0259] (ii) To facilitate the sending of such samples for testing, such as by making it easy to print address labels;

[0260] (iii) Optional input data, such as time and date, age, gender, weight, race, sample type, etc.

[0261] This type of data can be shared with remote computers to aid in sample processing and results reporting.

[0262] Such computer programs can transmit results to subjects, such as urolithin or urolithin conjugate levels, whether the subject will benefit from urolithin supplementation, and the required dose of urolithin supplementation.

[0263] In one embodiment of the present invention, a computer-implemented method is provided, comprising the following steps:

[0264] (a) Optionally receive data confirming the transport of biofluid samples from the subject;

[0265] (b) Receive data on the levels of urolithin or urolithin conjugates in biological fluids;

[0266] (c) Optionally receive subject data, such as time and date, age, sex, weight, race, sample type, etc.;

[0267] (d) Compare the level of urolithin or urolithin conjugate with data that correlates the level with a therapeutic dose of urolithin (e.g., urolithin A) to calculate the therapeutic dose;

[0268] (e) Optionally, the results may be communicated to the subjects.

[0269] In one embodiment of the present invention, a non-transitory computer-readable medium comprising computer-executable instructions, which, when executed by a processor of a computing device, cause the computing device to perform a method comprising:

[0270] (a) Optionally receive data confirming the transport of biofluid samples from the subject;

[0271] (b) Receive data on the levels of urolithin or urolithin conjugates in biological fluids;

[0272] (c) Optionally receive subject data, such as time and date, age, sex, weight, race, sample type, etc.;

[0273] (d) Compare the level of urolithin or urolithin conjugate with data that correlates the level with a therapeutic dose of urolithin (e.g., urolithin A) to calculate the therapeutic dose;

[0274] (e) Optionally, the results may be communicated to the subjects.

[0275] The levels of urolithin or urolithin conjugates are compared to determine whether the subject will benefit from urolithin supplementation as defined above for the method of the present invention.

[0276] The levels of urolithin or urolithin conjugates are compared with data relating the levels of urolithin or urolithin conjugates to a therapeutic dose of urolithin supplements as described above with respect to the method of the present invention.

[0277] A computer device including a processor, memory, and a display, said computer device being configured to implement a computer-implemented method of the present invention or computer-executed instructions of the present invention. Such computer devices include mobile phones, tablet computers, laptop computers, etc.

[0278] Results include information extrapolated from the levels of urolithin or urolithin conjugates in the biofluid, such as whether the subject would benefit from the therapeutic dose of urolithin supplements and / or urolithin supplements.

[0279] Drug treatment

[0280] The present invention further provides urolithin compositions for use in pharmaceutical treatments. Therefore, according to another embodiment of the invention, a urolithin supplement is provided for use in a method of treating a human subject, wherein it has been determined by the method of the present invention that the subject will benefit from taking the urolithin supplement.

[0281] According to another embodiment of the invention, a treatment method is provided, which includes determining, according to the method of the invention, whether a subject will benefit from taking a urolithin supplement, and then administering the urolithin supplement to the subject.

[0282] According to another embodiment of the invention, the use of urolithin supplements in the production of medicaments for treating diseases and conditions in human subjects is provided, wherein it has been determined by the method of the invention that the subjects will benefit from taking the urolithin supplements.

[0283] According to another embodiment of the present invention, a method for treating a human subject with urolithin is provided, comprising determining a therapeutic dose of a urolithin supplement according to the method of the present invention, and then administering the urolithin supplement to the subject.

[0284] According to another embodiment of the invention, a treatment method is provided, which includes determining a therapeutic dose of a urolithin supplement according to the method of the invention, and then administering the urolithin supplement to a subject.

[0285] According to another embodiment of the invention, the use of urolithiasis supplements in the production of medicaments for treating diseases and conditions in human subjects is provided, wherein the therapeutic dose has been determined by the method of the invention.

[0286] In another embodiment, the use of a calibrated dose of urolithin for treating diseases or conditions in human subjects is provided.

[0287] In another embodiment, the use of calibrated doses of urolithin in the production of a medicament for treating diseases or conditions in human subjects is provided.

[0288] In another embodiment, a method is provided for treating a disease or condition in a human subject using a calibrated dose of urolithin.

[0289] The calibration dose is the dose determined using the method of the present invention.

[0290] In another embodiment of the invention, a urolithin supplement is provided in a method for treating human subjects, the method comprising determining a therapeutic dose of the urolithin supplement according to the method of the invention.

[0291] In another embodiment of the invention, urolithin is provided in a method for preventing or treating aging symptoms in a human subject, wherein the method includes administering a urolithin supplement to the subject, and wherein it has been determined by the method of the invention that the subject will benefit from taking the urolithin supplement, or a therapeutic dose for the subject has been calculated using the method of the invention.

[0292] This invention provides a method for treating muscle-related pathological conditions in subjects according to the method of the invention. Muscle-related pathological conditions include conditions affecting generally healthy individuals as well as pathological conditions. Such muscle conditions found in healthy individuals or diseased populations include musculoskeletal diseases or conditions; cachexia; muscle atrophy; myopathy; age-related decline in muscle function; pre-fainting; weakness; neuromuscular diseases such as Duchenne muscular dystrophy and other malnutritions; sarcopenia, such as acute sarcopenia; muscle atrophy and / or cachexia, such as muscle atrophy and / or cachexia associated with burns, bed rest, limb immobilization, or major chest, abdominal, and / or orthopedic surgery; multiple sclerosis, such as relapsing-remitting multiple sclerosis; and muscle degenerative diseases.

[0293] Examples of age-related diseases that can be treated with the compositions of the present invention include sarcopenia, pre-fainting, weakness, dysphagia or difficulty swallowing, and muscle atrophy. Generally, these treatments improve mitochondrial function associated with age-related decline in muscle function and / or activity.

[0294] In another embodiment, the present invention provides a method for treating diseases or conditions selected from the group consisting of: metabolic syndrome, decreased metabolic rate, metabolic stress, cardiovascular disease, sarcopenia, muscle degeneration, inclusion body myositis (e.g., sporadic inclusion body myositis), Duchenne muscular dystrophy, alcoholic liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), drug-induced liver injury, drug-induced cravings, anemia, alpha-1 antitrypsin deficiency, ischemia / reperfusion injury, inflammation, inflammatory bowel disease, Crohn's disease, obesity, metabolic syndrome, type II diabetes, hyperlipidemia, osteoarthritis, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, Huntington's disease, anxiety disorders, ulcers, amyotrophic lateral sclerosis (ALS), mitochondrial diseases (including, for example, dysplasia, loss of muscle coordination), Muscle weakness, vision problems, hearing problems, heart disease, liver disease, kidney disease, gastrointestinal diseases, respiratory disorders, neurological problems, autonomic dysfunction (sometimes learning disabilities), and dementia caused by mitochondrial diseases. Other diseases associated with mitochondrial dysfunction include: diabetes and deafness (DAD); Leber hereditary optic neuropathy (LHON); Leigh syndrome (subacute sclerotic encephalopathy); neuropathy, ataxia, retinitis pigmentosa, and ptosis (NARP); neurogastrointestinal encephalomyopathy (MNGIE); myoclonic epilepsy with broken red fibers (MERRF); mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS); and mtDNA loss; as well as cancer, cognitive impairment, stress, and mood disorders; for improving cognitive function; for weight management; or for increasing muscle function or mental performance. The compositions of the present invention are particularly suitable for improving muscle function, muscle strength endurance, and muscle recovery.

[0295] Specifically, this invention provides treatments for diseases or conditions selected from the group consisting of: metabolic syndrome, decreased metabolic rate, metabolic stress, cardiovascular disease, sarcopenia, pre-fainting, weakness, muscle degeneration, inclusion body myositis (e.g., sporadic inclusion body myositis), Duchenne muscular dystrophy, alcoholic liver disease, non-alcoholic fatty liver disease, drug-induced liver injury, drug-induced cravings, anemia, alpha-1 antitrypsin deficiency, ischemia / reperfusion injury, inflammation, inflammatory bowel disease, Crohn's disease, obesity, metabolic syndrome, type II diabetes, hyperlipidemia, osteoarthritis, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, anxiety disorders, ulcers, amyotrophic lateral sclerosis (ALS), and cancer, cognitive impairment, stress, and mood disorders; for improving cognitive function; for weight management; or for increasing muscle function or mental performance.

[0296] The present invention also provides treatments for diseases or conditions selected from the group consisting of: metabolic stress, sarcopenia, muscle degenerative diseases, inclusion body myositis (e.g., sporadic inclusion body myositis), Duchenne muscular dystrophy, alcoholic liver disease, non-alcoholic fatty liver disease, drug-induced liver injury, α1-antitrypsin deficiency, ischemia / reperfusion injury, inflammatory bowel disease, Crohn's disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and cancer.

[0297] The present invention further provides a method for increasing autophagy or mitophagy in cells, which includes the therapeutic methods of the present invention. For example, autophagy or mitophagy can occur in the following cells: embryonic stem cells, induced pluripotent stem cells, adult stem cells, differentiated cells, blood cells, hematopoietic cells, epithelial cells, exocrine cells, endocrine cells, connective tissue cells, adipocytes, osteocytes, smooth muscle cells, striated muscle cells, nerve cells, sensory cells, heart cells, hepatocytes, gastric cells, intestinal cells, lung cells, epidermal (i.e., skin) cells (including keratinocytes and fibroblasts), kidney cells, and germ cells. Therefore, it can, for example, treat or prevent diseases or conditions selected from the following groups: metabolic syndrome, decreased metabolic rate, metabolic stress, cardiovascular disease, sarcopenia, muscle degeneration, inclusion body myositis (e.g., sporadic inclusion body myositis), Duchenne muscular dystrophy, alcoholic liver disease, non-alcoholic fatty liver disease, drug-induced liver injury, drug-induced cravings, anemia, alpha-1 antitrypsin deficiency, ischemia / reperfusion injury, inflammation, inflammatory bowel disease, Crohn's disease, obesity, metabolic syndrome, type II diabetes, hyperlipidemia, osteoarthritis, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, anxiety disorders, ulcers, amyotrophic lateral sclerosis (ALS), and cancer, cognitive impairment, stress, and mood disorders; or it can help with weight management, or increase muscle function or mental performance.

[0298] Among neurodegenerative diseases, those that may be specifically mentioned include AIDS-related dementia syndrome, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), adrenoleukodystrophy, Alexander disease, Alper's disease, ataxia-telangiectasia, Barten's disease, bovine spongiform encephalopathy (BSE), Canavan's disease, corticobasal degeneration, Kreutzfeldt-Jacob's disease, Lewy body dementia, lethal familial insomnia, frontotemporal lobe degeneration, Huntington's disease, and Ken's disease. The list includes: Neidit disease, Crabbe disease, Lyme disease, Machado-Joseph disease, multiple sclerosis, multiple system atrophy, neuroacanthosis, Niemann-Pick disease, Parkinson's disease, Pick's disease, primary lateral sclerosis, progressive supranuclear palsy, Rifsum disease, Sandhoff's disease, diffuse sclerosis, spinocerebellar ataxia, subacute combined degeneration of the spinal cord, dorsal horn disease, Tay-Sachs disease, toxic encephalopathy, infectious spongiform encephalopathy, and hedgehog wobbling disease. In one embodiment, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, and Parkinson's disease. In another embodiment, the neurodegenerative disease is Alzheimer's disease.

[0299] One aspect of the present invention is to improve cognitive function. In one embodiment, cognitive function is selected from the group consisting of free perception, memory, attention, speech comprehension, speech generation, reading comprehension, image creation, learning, and reasoning. In another embodiment, cognitive function is selected from the group consisting of free perception, memory, attention, and reasoning. In yet another embodiment, cognitive function is memory.

[0300] One aspect of the present invention is the treatment of stress-induced or stress-related cognitive deficits. Another aspect of the present invention is the treatment of mood disorders. In one embodiment, the mood disorder is selected from the group consisting of depression, postpartum depression, dysphoric mood, and bipolar disorder. In one embodiment, the mood disorder is depressive disorder. In one embodiment, the mood disorder is dysphoric mood.

[0301] One aspect of the present invention is the treatment of stress-induced or stress-related mood disorders, such as dysphoric mood. Another aspect of the present invention is the treatment of anxiety disorders. In one embodiment, the anxiety disorder is selected from the group consisting of generalized anxiety disorder, panic disorder, agoraphobic panic disorder, agoraphobia, social anxiety disorder, obsessive-compulsive disorder, and post-traumatic stress disorder. In one embodiment, the anxiety disorder is generalized anxiety disorder. In one embodiment, the anxiety disorder is post-traumatic stress disorder.

[0302] One aspect of the present invention is the treatment of stress-induced or stress-related anxiety.

[0303] One aspect of the present invention is the treatment of muscle or neuromuscular diseases. In one embodiment, the muscle or neuromuscular disease is myopathy. In one embodiment, the muscle or neuromuscular disease is sarcopenia. In one embodiment, the muscle or neuromuscular disease is muscular dystrophy. In one embodiment, the muscle or neuromuscular disease is Duchenne muscular dystrophy. In one embodiment, the muscle or neuromuscular disease is inclusion body myositis, such as sporadic inclusion body myositis.

[0304] One aspect of this invention is the treatment of mitochondrial diseases. For example, subjects may need treatment for loss of muscle coordination, muscle weakness, vision problems, hearing problems, heart disease, liver disease, kidney disease, gastrointestinal disorders, respiratory disorders, neurological problems, autonomic dysfunction (sometimes learning disabilities), and dementia caused by mitochondrial diseases.

[0305] One aspect of this invention is enhancing muscle function. In one embodiment, muscle function is selected from the group consisting of strength, speed, endurance, and recovery. In humans, muscle function typically declines with age, beginning around age thirty; generally, the rate of decline accelerates after age 65. Therefore, one aspect of this invention is maintaining muscle function during the aging process. Enhancing muscle function can be part of the application of the said compounds in sports nutrition, aiding healthy aging (e.g., from age 45 to 65), and slowing the rate of muscle decline in individuals over 65 (pre-frailty stage).

[0306] Non-pharmacological treatment:

[0307] The compositions of this invention can be used to improve muscle function, prevent muscle decline, increase muscle mass, and / or reduce muscle atrophy. Improving muscle function, increasing muscle mass, and / or reducing muscle atrophy can be part of a pharmaceutical treatment, or for personal preference (“lifestyle”) or cosmetic reasons, or as part of personal over-the-counter management of nutritional or physiological health. The compositions of this invention can be used as pharmaceuticals. The compositions can be used as dietary supplements, functional foods, functional beverages, special nutritional products, or medical foods.

[0308] This invention provides therapies for diseases and disease states. The compositions can be used to manage the normal physiological functions of healthy individuals suffering from diseases characterized by poor physical function, impaired endurance, and impaired muscle function. The treatment methods of this invention can improve physical function in individuals suffering from diseases, including young and old. The treatment methods of this invention can improve physical function, such as short-term or long-term function in healthy individuals, including active individuals, non-active individuals, sedentary individuals, and older adults. Such improvements in function can be measured by the time taken to walk or run a certain distance (e.g., improved performance in the 6-minute walk test (MWT), improved time to run a certain distance, improved IPAQ scores on the International Physical Activity Questionnaire, an increase in the number of chairs used within a certain time period, or other tests designed to measure physical function.

[0309] The treatment methods of the present invention can also be used to manage and maintain normal physiological functions (e.g., physical function, endurance, and muscle function) in healthy individuals.

[0310] The treatment method of the present invention can also be used to manage nutritional status, thereby improving mitochondrial function. This is important, for example, for patients or hospitalized patients, where urolithiasis supplements are administered not as a treatment for disease, but as a nutritional supplement.

[0311] The treatment method of the present invention further provides for the improvement of endurance. Endurance refers to the time to fatigue during exercise under a constant workload (typically at intensity <80% VO2max). The treatment method of the present invention can improve the endurance of individuals with disease, including young and old. The treatment method of the present invention can improve the endurance of healthy individuals, including athletic individuals, non-athletic individuals, sedentary individuals, and older adults. The present invention provides a method for increasing the time to fatigue during specific activities, such as fitness training, walking, running, swimming, or cycling. This improvement in endurance can be assessed by objective measurements (e.g., speed, oxygen consumption, or heart rate) or by self-reported measurements (e.g., using validated questionnaires).

[0312] This invention further provides treatment methods for improving, maintaining, or reducing muscle function loss. The compositions of this invention can improve, maintain, or reduce muscle function loss in affected individuals (including young and old). The compositions of this invention can improve, maintain, or reduce muscle function loss in healthy individuals, including active individuals, non-active individuals, sedentary individuals, and the elderly. For example, the compositions of this invention can improve, maintain, or reduce muscle function loss in frail or pre-frail individuals. For example, the compositions of this invention can increase muscle strength, as evidenced by improvements in physical activity (such as exercise) (e.g., increased weightlifting ability or increased grip strength). Furthermore, the compositions of this invention can improve muscle structure, for example by increasing or maintaining muscle mass in cases of normal muscle function, decreased muscle function, or impaired muscle function.

[0313] The present invention further provides therapeutic methods to improve an individual's perceived physical function or endurance. For example, such as a reduction in perceived exertion or effort during exercise or activity, as determined by using a self-report questionnaire.

[0314] In another embodiment of the invention, a method for maintaining adequate nutritional levels in human subjects is provided, wherein the method includes administering a urolithin supplement to the subject, and wherein the subject has been determined, as described herein, to be able to benefit from taking a urolithin supplement or the dosage of the urolithin supplement for the subject has been determined.

[0315] Muscle function:

[0316] The treatment methods of the present invention can be used to enhance muscle and / or physical function. Therefore, the present invention provides a composition for enhancing muscle and / or physical function. The present invention also provides a method for enhancing muscle and / or physical function by administering an effective amount of the composition of the present invention to a subject. Administerment may be self-administered.

[0317] Muscle function enhancement can be one or more improvements in muscle function, reduction of muscle function decline, maintenance of muscle function, improvement of muscle strength, improvement or maintenance of muscle endurance, and improvement of muscle recovery.

[0318] Therefore, the treatment methods of the present invention can be used in methods for improving physical endurance (e.g., the ability to perform physical activities such as exercise, manual labor, sports activities), inhibiting or delaying physical fatigue, enhancing work capacity and endurance, and reducing muscle fatigue.

[0319] Improving muscle function is particularly beneficial for older subjects whose muscle function declines due to age-related conditions such as sarcopenia and muscle atrophy. The treatment methods of this invention can be used to enhance muscle function by administering the compositions of this invention to subjects who are sedentary, frail, or in the pre-frailty stage.

[0320] Muscle function can be categorized as motor function, which refers to the performance of muscles in athletic individuals during physical activity. Improvements in motor function, strength, speed, and endurance are measured by increases in muscle contraction intensity, amplitude of muscle contraction, or shortening of the muscle response time between stimulus and contraction. An athletic individual is defined as someone who participates in any level of physical activity and seeks to improve their strength, speed, or endurance, such as fitness enthusiasts, cyclists, long-distance runners, and sprinters. Enhanced motor function manifests as the ability to overcome muscle fatigue, maintain activity levels for longer periods, and achieve more effective training.

[0321] Urolithin

[0322] Urolithin is a metabolite of ellagitannins and ellagic acid produced by the gut microbiota of mammals (including humans). Ellagannins and ellagic acid are compounds commonly found in foods such as pomegranates, nuts, and berries. Ellagannins are poorly absorbed in the gut itself. Urolithin is a class of compounds with the representative structure (I) described above. Referring to structure (I), Table 1 below describes the structures of some particularly common urolithins.

[0323]

[0324]

[0325]

[0326] In fact, for commercial-scale products, the synthesis of urolithiasis is quite convenient. Synthetic routes are described, for example, in WO 2014 / 004902 and WO 2015 / 100213.

[0327] Urolithin of any structure conforming to structure (I) can be used in the methods of this disclosure.

[0328] In one aspect of the uses and methods of this disclosure, a suitable compound is a compound of formula (I), wherein A, C, D and Z are individually selected from H and OH, and B, W, X and Y are all H, preferably at least one of A, C, D and Z is OH.

[0329] A particularly suitable compound is naturally occurring urolithin. Therefore, Z is preferably OH, and W, X, and Y are preferably all H. When W, X, and Y are all H, and A and B are all H, and C, D, and Z are all OH, the compound is urolithin C. When W, X, and Y are all H, and A, B, and C are all H, and D and Z are all OH, the compound is urolithin A. Preferably, the urolithin used in the method of this disclosure is urolithin A, urolithin B, urolithin C, or urolithin D. Most preferably, the urolithin used is urolithin A.

[0330] According to one embodiment, a method of the present invention is provided, wherein the compound of formula (I) is urolithin A.

[0331] According to one embodiment, a method of the present invention is provided, wherein the compound of formula (I) is urolithin B.

[0332] According to one embodiment, a method of the present invention is provided, wherein the compound of formula (I) is urolithin C.

[0333] According to one embodiment, a method of the present invention is provided, wherein the compound of formula (I) is urolithin D.

[0334] Composition form:

[0335] The compositions used in the treatment methods of the present invention can take any suitable physical form. They can be in the form of solids (e.g., tablets or strips), semi-solids (e.g., soft capsules, capsules (e.g., hard capsules), or sugar-coated pills), powders, or liquids (including emulsions). The compositions of the present invention can be nutritional compositions. The compositions of the present invention can be pharmaceutical compositions. The compositions can be in the form of dietary supplements, functional foods, functional beverages, or medical foods or medical nutritional products.

[0336] The tablet form composition can be of any suitable type and may contain excipients commonly used in the art. Excipients may, for example, provide desired hardness, shelf life, and flavor, resulting in a composition with an acceptable taste, an appealing appearance, and good storage stability. The strips can be of any suitable type and may contain ingredients commonly used in the production of snack bars.

[0337] The semi-solid form may also contain excipients commonly used in the art. Excipients can, for example, provide the desired hardness, shelf life, and flavor, resulting in a composition with an acceptable taste, an appealing appearance, and good storage stability. Semi-solid forms for oral or topical application are available.

[0338] Powders are commonly used in nutritional supplements and also in pharmaceutical compositions. The advantage of powders is that multiple doses can be provided in a single container, and different sized doses can be dispensed from the same supply container. Powders generally have good storage properties. Powder compositions may also contain excipients commonly used in the art. Excipients can, for example, provide shelf life, flavor, and moisture resistance, resulting in a composition with an acceptable taste, an appealing appearance, and good storage stability. The present invention can be in the form of a kit comprising a nicotinamide ribose composition and a separate composition containing urolithin, such as a nicotinamide ribose powder composition and a separate solid or liquid composition containing urolithin. The solid or liquid composition containing urolithin (e.g., tablets or beverages, or other forms described herein) may be provided with instructions for use along with the nicotinamide ribose powder. For example, both nicotinamide ribose and urolithin can be in powder form.

[0339] Liquid compositions can be in pharmaceutical or beverage form. Liquid formulations can be solutions, emulsions, slurries, or other semi-liquids. Excipients in liquid compositions can, for example, provide shelf life, visual appearance, flavor, and mouthfeel, giving the composition an acceptable taste, an appealing appearance, and good storage stability. Liquid compositions for oral administration are available. Liquid compositions for topical application are available, for example, in the form of creams, ointments, or emulsions.

[0340] For certain applications, the compositions of the present invention may also be in solution form suitable for injection or intravenous administration.

[0341] Additional components in the composition of this invention:

[0342] The composition used in the treatment methods of the present invention may contain ingredients other than urolithin and nicotinamide ribose. Additional ingredients may be compounds that provide health benefits, such as those selected from vitamins, minerals, polyunsaturated fatty acids, functional amino acids, and other compounds.

[0343] Among vitamins, vitamins A, C, D, E, B12, and K2 may be specifically mentioned. As used herein, “vitamin D” refers to any known form of vitamin D, specifically including vitamin D2 (ergocalciferol), vitamin D3 (cholecalciferol), vitamin D precursors, metabolites, and other analogs and combinations thereof, as well as various active and inactive forms of vitamin D. For example, vitamin D3 can be provided in its unhydroxylated inactive form (such as cholecalciferol) or its hydroxylated active form (such as calcitriol).

[0344] Creatine is considered to have beneficial effects in treating muscle diseases. It can be included in the compositions of the present invention. β-hydroxy-β-methylbutyrate (HMB) is considered to have beneficial effects in treating muscle diseases. It can be included in the compositions of the present invention.

[0345] Among the minerals, those that may be specifically mentioned are calcium salts (such as calcium phosphate), selenium, zinc salts, magnesium salts, and iron salts.

[0346] For many muscle growth and / or muscle enhancement treatments, providing certain specific amino acids is beneficial. For example, L-arginine, L-glutamine, lysine, and branched-chain amino acids are considered important. These amino acids are sometimes referred to as "functional amino acids." The compositions of the present invention may contain one or more branched-chain amino acids (leucine, isoleucine, and valine). The compositions of the present invention may contain one or both of L-arginine and L-glutamine. The compositions of the present invention may contain lysine.

[0347] The pharmaceutical compositions of the present invention may contain other pharmaceutically active compounds. For example, they may contain statins. The present invention may be provided in the form of a kit comprising a composition of urolithin and nicotinamide sugar; and a pharmaceutically active compound, such as a statin.

[0348] The compositions of the present invention may contain one or more other agents for mitochondrial biogenesis or for the treatment of mitochondrial diseases. Such compounds include, but are not limited to, resveratrol, pyrrolidone, ubiquinone, sulforaphane, coenzyme Q10, genistein, hydroxytyrosol, quercetin, L-carnitine, alpha-lipoic acid, and folinic acid (e.g., as hydroxylophylic acid).

[0349] The compositions of the present invention may further (or optionally) contain other compounds, including, for example, tomatine, ursolic acid, curcumin, capsaicin, menthol, triethanolamine salicylate, and methyl salicylate.

[0350] In some exemplary embodiments, in addition to nicotinamide ribose and urolithin, the compositions disclosed herein may also include one or more additional macronutrients, typically proteins, fats or carbohydrates, or two or more of proteins, fats and carbohydrates.

[0351] In the compositions of the present invention, any suitable source of fat or oil commonly used in the manufacture of food and pharmaceuticals may be used. Non-limiting examples of suitable sources of fat for use in the compositions described herein also include polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), arachidonic acid (ARA), eicosapentaenoic acid (EPA), and combinations thereof.

[0352] Non-limiting examples of suitable carbohydrates or sources thereof for use in the compositions described herein may include maltodextrin, hydrolyzed or modified starch or corn starch, glucose polymers, corn syrup, corn syrup solids, rice-derived carbohydrates, glucose, fructose, lactose, trehalose, high-fructose corn syrup, tapioca dextrin, isomaltulose, sucromalt, maltitol powder, glycerol, fructooligosaccharides, soybean fiber, corn fiber, guar gum, konjac flour, polydextrose, honey, sugar alcohols (e.g., maltitol, erythritol, sorbitol), and combinations thereof. Maltodextrin, sucrose, and fructose are particularly preferred.

[0353] Non-limiting examples of suitable proteins or their sources for use in the compositions described herein may include hydrolyzed, partially hydrolyzed, or non-hydrolyzed proteins or protein sources. They may be derived from any known or other suitable source, such as milk (e.g., casein, whey), animal (e.g., meat, fish), cereal (e.g., rice, corn), or vegetable (e.g., soybean, pea) sources. Combinations of different sources or types of proteins may be used. Non-limiting examples of proteins or their sources include whole pea protein, whole pea protein isolate, whole pea protein concentrate, milk protein isolate, milk protein concentrate, casein isolate, casein concentrate, whey protein concentrate, whey protein isolate, sodium caseinate or calcium caseinate, whole milk, partially or completely skimmed milk, yogurt, soy protein isolate and soy protein concentrate, and combinations thereof. Combinations of different sources or types of proteins may be used. For example, Greek and Icelandic yogurts are known to be particularly high in protein, making them particularly suitable for use in the formulations of this invention. For example, yogurt used in the compositions of this invention may contain 2g to 15g of protein per 100g. Yogurt with a particularly high protein level is preferred, for example, 6g to 15g per 100g, 7g to 15g per 100g, or 8g to 15g per 100g. Optionally, protein supplements may be added to the yogurt formula to increase the protein content in the formula. The yogurt of the present invention may contain active cultures, such as Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus, or Lactobacillus acidophilus.

[0354] The total concentration or amount of protein, fat, carbohydrates and other components varies depending on the intended user's nutritional needs.

[0355] Other components in the compositions of the present invention may be compounds that improve the composition in other ways without providing health benefits to the subjects, such as, as described above, taste, texture, or shelf life. Therefore, the compositions of the present invention may further comprise one or more compounds selected from emulsifiers, colorants, preservatives, gums, curing agents, thickeners, sweeteners, and flavoring agents.

[0356] Suitable emulsifiers, colorants, preservatives, gums, curing agents, and thickeners are well known in the field of emulsion and other semi-liquid manufacturing. For example, preservatives such as benzoic acid, sorbic acid, phosphoric acid, lactic acid, acetic acid, hydrochloric acid, and their soluble salts can be used.

[0357] Sweeteners may be particularly beneficial in the compositions of this invention. Highly effective non-nutritive carbohydrate sweeteners, such as those selected from aspartame, sucrose, acesulfame potassium, saccharin, cyclamate, stevia, sweet protein, and mixtures thereof, can be used. Aspartame is particularly suitable.

[0358] Flavoring agents may be particularly beneficial in the compositions of this invention. In liquid or semi-liquid compositions, fruit flavoring can be provided by adding fruit sauces or purees. Typical flavoring agents include strawberry, raspberry, blueberry, apricot, pomegranate, peach, pineapple, lemon, orange, and apple. Generally, fruit flavoring agents comprise fruit extracts, fruit preserves, or fruit purees, and any combination thereof with sweeteners, starches, stabilizers, natural and / or artificial flavorings, colorings, preservatives, water, and citric acid or other suitable acids to control the pH.

[0359] For oral formulations, the composition may be used alone or in combination with appropriate additives to form tablets, powders, granules, or capsules, for example, in combination with conventional additives such as lactose, mannitol, corn starch, or potato starch; in combination with binders such as crystalline cellulose, cellulose derivatives, gum arabic, corn starch, or gelatin; in combination with disintegrants such as corn starch, potato starch, or sodium carboxymethyl cellulose; in combination with lubricants such as talc or magnesium stearate; and, if necessary, in combination with diluents, buffers, wetting agents, preservatives, and flavoring agents.

[0360] The compositions can be formulated into liquid formulations by dissolving, suspending, or emulsifying them in aqueous or non-aqueous solvents such as vegetable oils or other similar oils, synthetic fatty acid glycerides, higher carbon fatty acid esters, or propylene glycol; and, if desired, conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives can be added. The compositions can be used in aerosol formulations for inhalation. They can be mixed with various matrices such as emulsifying matrices or water-soluble matrices to form suppositories.

[0361] Unit dosage forms for oral administration, such as syrups, elixirs, and suspensions, are available, wherein each dose unit, such as a teaspoon, tablespoon, tablet, or capsule, contains a predetermined amount of the composition of the present invention. Similarly, unit dosage forms for injection or intravenous administration may include the compound of the present invention in the composition as a solution in sterile water, physiological saline, or other pharmaceutically acceptable carriers, wherein each dose unit, such as mL or L, contains a predetermined amount of the composition of the present invention.

[0362] The composition used in the treatment methods of the present invention may be used as a single treatment, or more commonly, as a series of treatments. In one example, the subject takes one dose of the composition before or after exercise. For subjects who cannot exercise, for example, one dose of the composition may be taken once, twice, or three times daily, or once, twice, three times, four times, five times, or six times weekly. It should also be understood that the effective dose of the compound may be increased or decreased during a particular treatment.

[0363] Dosage preparation

[0364] The appropriate dosage of urolithin supplements for treating human subjects will depend on their degree of urolithin formation as shown by the diagnostic analysis results of this invention and the optimal dosage for therapeutic effect on the patient. That is, the most suitable dosage is the optimal dosage minus the amount of urolithin produced by the subject. For example, a dosage of 50 mg to 2000 mg daily. For example, a dosage of 50 mg to 1500 mg daily. For example, a dosage of 50 mg to 1400 mg daily, for example, to 1300 mg daily, to 1200 mg daily, to 1100 mg daily, for example, 50 mg to 1000 mg daily. In another embodiment, a suitable dosage form may be 100 mg to 1000 mg daily, for example, 100 mg to 750 mg daily, for example, 100 mg to 500 mg daily, for example, 250 mg to 500 mg daily. For example, the dosage of urolithiasis supplements is selected from 100 mg, 250 mg, 500 mg, or 1000 mg or 1500 mg / day, such as 250 mg, 500 mg, or 1000 mg or 1500 mg / day.

[0365] In another embodiment, a suitable dosage of the urolithiasis supplement may be a unit dose comprising urolithiasis or multiple unit dosage forms comprising tablets. For example, unit doses may include 100 mg, 250 mg, 500 mg, 1000 mg, or 1500 mg. For the avoidance of doubt, the dosage may be multiple of the above unit doses or combinations thereof.

[0366] The appropriate dosage of urolithiasis supplements can vary depending on the method of administration, the subject's age, weight, and overall health condition. Factors such as the subject's medical condition, age, and weight may be important, and the dosage can be adjusted to achieve the best response.

[0367] In this specification, the urolithin concentration for dried blood spots is expressed in ng / ml, which is related to the concentration in the original blood sample in ng / ml before drying on a support such as filter paper.

[0368] The term “about” refers to a tolerance of ±20% of the relevant value, for example, ±15% of the relevant value, or ±10% or ±5% of the relevant value.

[0369] The phrase "biofluid sample" refers to any sample from a subject, such as a human or animal, in which urolithin or urolithin conjugates can be determined. Examples include urine or blood. In the case of blood, analysis can be performed in the form of dried blood spots.

[0370] "Dried blood spot" refers to a whole blood, plasma, or serum sample that has been dried on a material, wherein the dried sample can be used to determine the level of urolithin or urolithin conjugates in the sample. For the avoidance of doubt, "dried drug spot" refers to any dried whole blood, dried plasma, or dried serum sample adsorbed on or dried into a layer on said material, or dried whole blood, plasma, or serum scraped from said material.

[0371] The term "human subject" refers to a male, female, or child. Male or female subjects can be young adults, older adults, or middle-aged adults. The term "child" refers to anyone between approximately 1 year and approximately 18 years of age. The term "young adults" refers to anyone between approximately 18 and 40 years of age. The term "middle-aged adults" refers to anyone between approximately 40 and 65 years of age. The term "older adults" refers to anyone 65 years of age or older.

[0372] The term "urolithin conjugate" refers to any urolithin derivative formed by the metabolism of urolithin in a human subject. Examples of urolithin conjugates include urolithin glucoside and sulfated urolithin, particularly urolithin glucoside. In one embodiment, the method of the present invention includes determining the level of urolithin conjugates, particularly urolithin glucoside.

[0373] The term "urolithin precursor" refers to plant-derived materials or substances that can be metabolized in the human gut to form urolithin. Examples of plant-derived materials include: tea; fruits such as pomegranate and guava; berries such as raspberry, blackberry, cloudberry, boysonberry, strawberry, and cranberry; nuts such as pecan, walnut, and almond; and seeds such as cloudberry seeds and veggie fungi. Examples of substances include ellagitannins and ellagic acid, such as punicin. Attached Figure Description

[0374] Figure 1 The study showed the levels of urolithin glucoside in dried blood spots in subjects before treatment and after administration of pomegranate juice and 500 mg of urolithin.

[0375] Figure 2 The flowchart of the study described in Example 5 is shown.

[0376] Figure 3The table shows the levels of UA-glucuronide in plasma samples collected at 0 hours (baseline), 6 hours, and 24 hours after administration of pomegranate juice or urolithin A in all n = 100 healthy participants in Example 5. The data shown are the average of 100 samples.

[0377] Figure 4 The levels of UA-glucuronic acid in DBS (dried blood spot) samples collected at 0 hours (baseline), 6 hours, and 24 hours after taking pomegranate juice or urolithin A are shown in Example 5 for all n=100 healthy participants.

[0378] Figure 5 The correlation between UA glucuronide DBS (whole blood spot sample) measurements and plasma levels was shown for n=100 subjects (A) at t=6 hours and (B) at t=24 hours (Spearman correlation coefficient r=0.97).

[0379] Figure 6 shows...

[0380] A) Box plots showing the differences in metagenomic species (MGS) richness (left) and Shannon diversity (right) among non-UA producers, low-UA producers, and high-UA producers. Paired comparisons were performed for all groups using the Man Whitney U test (N=99). *P≤0.05; **P≤0.01; ***P≤0.001; *****P≤0.00001.

[0381] B) Principal Coordinate Analysis (PCoA), based on Bray-Curtis dissimilarity between samples calculated from MGS abundance. Samples are color-coded according to UA producer status. Each sample is connected to its group center of gravity by a thin line. The ellipse encompasses two standard errors of the mean of the group center of gravity, indicating the determinacy of the group center of gravity location. The x-axis and y-axis labels represent the microbial differences explained by the first two principal coordinates.

[0382] C) Relative abundance (percentage) of Firmicutes and Bacteroidetes, which differed significantly between UA producers and non-producers. Boxes represent interquartile ranges (IQRs), and inner horizontal lines represent the median. Box bars represent values ​​within the 1.5x IQR range for the first and third quartiles.

[0383] D) Firmicutes / Bacteroidetes ratio (F / B ratio) for each group, shown as the median (IQR). **P ≤ 0.01 (Man Whitney U test).

[0384] Figure 7Box plots are shown, illustrating differences in microbiome gene richness (left) and Shannon diversity (right) among non-UA producers, low-UA producers, and high-UA producers. Paired comparisons (N=99) were performed using the Man Whitney U test. *P≤0.05; **P≤0.01; ***P≤0.001; *****P≤0.00001.

[0385] Figure 8 The relative abundance (percentage) of the four MGS and two phyla differs significantly between low-producing and non-producing UA species. Relative abundance is shown as the area of ​​each square, scaled up to the maximum value for each species.

[0386] Figure 9 shows a comparison of physiological parameters and dietary habits among the non-UA producer, low UA producer, and high UA producer groups.

[0387] A) The BMI of non-UA producers (p=0.02) and low-UA producers (p=0.01) was significantly higher than that of high-UA producers.

[0388] B) The resting diastolic blood pressure of the non-producer group (p = 0.017) was significantly higher than that of the high-producer group.

[0389] C) Heart rate of non-producer subjects compared to high-producer subjects (p = 0.07).

[0390] D) According to the dietary questionnaire assessment, the high UA producer group had higher fruit and berry intake than the non-producer group (p = 0.07) and the low producer group (p = 0.01). N = 100. Data are presented as mean + / - SEM and analyzed using repeated measures ANOVA. *P ≤ 0.05; **P ≤ 0.01.

[0391] Example

[0392] The invention will now be described with reference to the following non-limiting examples.

[0393] Example 1: Preparation of urolithin A

[0394] Urolithin A (4) was prepared in two steps using 2-bromo-5-methoxybenzoic acid 1 and resorcinol 2 as starting materials. The pure compound was obtained as a light yellow powder.

[0395]

[0396] Step 1:

[0397] A mixture of 2-bromo-5-methoxybenzoic acid 1 (27.6 g; 119 mmol; 1.0 equivalent), resorcinol 2 (26.3 g; 239 mmol; 2.0 equivalent), and sodium hydroxide (10.5 g; 263 mmol; 2.2 equivalent) was heated under reflux in water (120 mL) for 1 hour. Then, a 5% aqueous solution of copper sulfate (3.88 g CuSO4 5H2O dissolved in 50 mL of water; 15.5 mmol; 0.1 equivalent) was added, and the mixture was refluxed again for 30 minutes. The mixture was cooled to room temperature, and the solid was filtered through a Buchner filter. The residue was washed with cold water to give a light red solid, which was then ground in hot MeOH. The suspension was incubated overnight at 4 °C. The resulting precipitate was filtered and washed with cold MeOH to give the title compound 3 as a light brown solid.

[0398] Step 2:

[0399] Dry dichloromethane containing 1 M boron tribromide solution (11.93 mL pure BBr3 dissolved in 110 mL anhydrous dichloromethane; 124 mmol; 3.0 equivalent) was added dropwise to 100 mL of dry dichloromethane containing suspension 3 (10.0 g; 41 mmol; 1.0 equivalent) at 0 °C. The mixture was allowed to stand at 0 °C for 1 hour, and then heated to room temperature. The solution was stirred at the stated temperature for 17 hours. Sufficient ice was then added to the mixture. The yellow precipitate was filtered and washed with cold water to give a yellow solid, which was heated under reflux in acetic acid for 3 hours. The hot solution was rapidly filtered, the precipitate was washed with acetic acid, and then with diethyl ether to give the title compound 4 as a yellow solid. The 1H and 13C NMR spectra were consistent with the structure of 4.

[0400] Example 2: Urolithiasis A formulation

[0401] Urolithin A is formulated into soft capsules containing the following ingredients:

[0402]

[0403]

[0404] Example 3 - Study on the effects of pomegranate juice and oral administration of urolithin on urolithin levels in blood collected from dried blood spots.

[0405] A study was conducted on seven participants. At the start of the study, capillary blood samples were collected from each participant by finger pricking, and 15-20 μL of whole blood was dried on a dried blood sample (DBS) filter paper card (Whatman, Little Charfonton, Buckinghamshire, UK). At least two spots were collected for each sampling at each time point. At T0, the baseline sample, participants ingested 80 mL of pomegranate juice (freshly prepared). Twenty-four hours after ingestion, dried blood samples were collected again, and 15-20 μL of whole blood was dried on a DBS filter paper card. Subsequently, participants took a soft capsule containing 500 mg of urolithiasis A, and then, 48 hours later (or 24 hours after UA ingestion), dried blood samples were collected again, and 15-20 μL of whole blood was dried on a DBS filter paper card.

[0406] Example 3a: Determination of urolithin A glucuronide in dried blood spot samples

[0407] This analytical method was used to analyze whole blood spot samples collected on a dried blood spot sample card (DBS filter card) in Example 3.

[0408] Each dried blood spot was punched onto a card using a 6 mm diameter punch and transferred to a test tube. Then, 150 μL of methanol containing the internal standard was added. The sample was then sonicated for 5 minutes, followed by a vortex mixing step for 20 minutes. The sample was then centrifuged at 50,000 g for 2 minutes at 8 °C. Aliquots of 100 μL of the supernatant were transferred to an automated sampling bottle for analysis, and diluted with 100 μL of water before analysis.

[0409] The quantification of UA-glucuronic acid was performed by reversed-phase column chromatography, followed by detection using a three-section quadrupole MS / MS in the selected reaction monitoring mode.

[0410] The results are shown in Table 1.

[0411] Table 1

[0412]

[0413]

[0414] Example 4: Bioavailability study in healthy adults

[0415] We conducted a single-center, two-phase, crossover, randomized, open-label study to assess urolithiasis A production status in a healthy adult population in the United States and to determine the advantage of dietary supplement UA supplementation in producing higher circulating levels of urolithiasis A than dietary exposure.

[0416] Study population:Healthy men and women aged 18 to 80 years. At least 20% of participants were between 20 and 40 years old, 41 and 60 years old, and 61 and 80 years old. The remaining 40% of participants were distributed across all age groups.

[0417] Sample size: N=100; all participants completed the study.

[0418] Research products

[0419] I. Apply 237 mL of pomegranate juice (PJ) daily (POM Wonderful, Los Angeles, California, 90064).

[0420] II. Apply one 500mg berry-flavored packet daily. 1 Urolithin A (UA)

[0421] 1* The ingredients include blueberry, raspberry powder, and natural flavorings from rice husk and pomegranate.

[0422] Before administering the powder to the participants, mix the urolithiasis A powder into a vanilla-flavored commercially available yogurt (4 fl oz).

[0423] therapeutic parameters

[0424] Primary endpoint:

[0425] • Absolute change in plasma UA-glucuronide levels from T0 to T+24 hours in the urolithiasis A (UA) group compared to the pomegranate juice (PJ) group.

[0426] Secondary endpoint:

[0427] • The absolute change in UA-glucuronide blood spot levels from T0 to T+24 hours in the UA group compared to the PJ group.

[0428] • The absolute change in plasma UA-glucuronide levels from T0 to T+6 hours in the UA group compared to the PJ group.

[0429] • The absolute change in UA-glucuronide blood spot levels from T0 to T+6 hours in the UA group compared to the PJ group.

[0430] • Compared with the PJ group, the change in AUC of UA glucuronide levels from T0 to T+6 hours and / or T+24 hours in the UA group

[0431] • Compared with the PJ group, the absolute changes and AUC changes in the following variables from T0 to T6 hours and / or T24 hours in the UA group:

[0432] -UA sulfate plasma levels

[0433] -UA aglycone (maternal) plasma levels

[0434] • Assess the prevalence of UA producers in healthy U.S. populations after they consume a fixed amount of pomegranate juice.

[0435] Note: "UA Producer" status is defined as a person whose UA (and its conjugates) levels are detectable in plasma / dried blood spots after ingestion of pomegranate juice. "UA Producer" status is determined after review of results on T+24.

[0436] • Differences between UA producers and non-producers in the following baseline variables (if any):

[0437] -Age and BMI

[0438] - Grip strength

[0439] Walking speed

[0440] -Based on the following criteria for physical activity and energy levels:

[0441] -International Physical Activity Questionnaire (IPAQ)

[0442] - Fatigue VAS score

[0443] - Intake of foods rich in ellagitannins and punicin (i.e., nuts and berries)

[0444] • Differences between UA producers and non-producers in the following aspects (if any):

[0445] - Plasma and / or DBS metabolomics analysis

[0446] - Any blood biochemical parameters measured at the first visit: HbA1c, creatinine, AST, ALT, total bilirubin, total cholesterol, HDL, LDL, and triglycerides.

[0447] - Stool analysis via microbiome sequencing, including:

[0448] ο Sample-specific relative abundance and taxonomic identifiers of all OTUs (operational taxonomic units)

[0449] ο Sample-specific relative abundance from phylum to genus level

[0450] α and β diversity measures (e.g., Shannon index and UniFrac distance)

[0451] ο Identify phylogenetic groups where significant changes in UA abundance occur between producers and non-producers.

[0452] Statistical analysis of differences in microbial community composition and diversity measures between producers and non-producers of OTA.

[0453] Selection criteria:

[0454] Participants must meet all of the following inclusion criteria:

[0455] 1. Men and women aged 18 to 80;

[0456] 2. As determined by the clinical research team, the overall health condition is good;

[0457] 3. Willing to consume the research product, complete the questionnaire, and complete all outpatient visits;

[0458] 4. Voluntary written informed consent forms have been provided to research participants;

[0459] Exclusion criteria:

[0460] Subjects will be excluded from the study if they have any of the following conditions:

[0461] 1. Women who become pregnant, are breastfeeding, or are planning to become pregnant during the trial;

[0462] 2. Has a history of alcohol abuse or drug misuse within the past 6 months;

[0463] 3. Volunteers who plan to donate blood during the study period or within 30 days after the study ends;

[0464] 4. It is known that the subject is allergic to the active or inactive components of the test material;

[0465] 5. The subject's condition was unstable;

[0466] 6. Abnormal laboratory results that are clinically significant during screening;

[0467] 7. Participate in a clinical research trial within 30 days prior to randomization;

[0468] 8. Allergy or sensitivity to the research ingredient; lactose intolerance.

[0469] 9. Individuals with cognitive impairment and / or unable to provide informed consent;

[0470] 10. Any other situation that the researcher believes may adversely affect the subject's ability to complete the study or its measurement, or may pose a significant risk to the subject;

[0471] 11. You have taken antibiotics in the past 30 days.

[0472] The research flowchart can be found here. Figure 2 .

[0473] plasma collection

[0474] At each time point shown in the study evaluation form, aspirate 12 mL of blood into a K2-EDTA-coated tube. Gently invert the blood several times to ensure complete mixing with the anticoagulant. Record the exact time of sample collection on the tube and in the electronic case report form (eCRF). Within 30 minutes of collection, centrifuge each blood sample at 1500 g for 10 minutes at 4°C. Within 30 minutes of centrifugation, transfer the top layer of human plasma to two pre-labeled polypropylene tubes, each containing approximately 1500 μL of plasma (two aliquots at each time point). Immediately cap the tubes at each time point and freeze the plasma upright at approximately -80°C.

[0475] Plasma samples were collected to assess UA-glucuronide levels in all n = 100 healthy participants at 0 hours (baseline), 6 hours, and 24 hours after administration of pomegranate juice or urolithin A (Mitopure). Data are as follows: Figure 3 As shown, the average value is represented by 100 samples.

[0476] Dry blood spot (DBS) collection:

[0477] The following items were used:

[0478] • Blood collection card (filter paper - Whatman Filter Paper 903 5-position paper card);

[0479] • Sealable foil bags for biohazardous materials

[0480] Desiccant pack

[0481] Samples are collected by pricking the finger with a lancet. Each blood collection card is used for only one subject. Apply blood to the card, creating at least 3 to 4 blood spots on the DBS card. Ensure the entire circle is evenly saturated and that each card is clearly and appropriately labeled. After pricking the finger with the lancet, gently press the finger to collect a large drop of free-flowing blood from the puncture site. Wipe away the first drop of blood. Quickly pinch the edge of the filter paper to allow it to lightly touch the large drop of blood, allowing enough blood to saturate and completely fill or saturate a circle in one step. A complete saturated spot contains 20 to 40 μl of blood. Repeat the sampling until enough blood is collected to fill at least 3 circles on the blood collection card. Complete filling of the blood spot circles is crucial because the laboratory uses a hole punch to perforate a portion of the blood circles for testing. DBS samples are stored at room temperature in clearly labeled biohazard foil bags.

[0482] DBS (dried blood spot) samples were collected to assess UA-glucuronide levels at 0 hours (baseline), 6 hours, and 24 hours after administration of pomegranate juice or urolithin A (Mitopure) in all n = 100 healthy participants. Data are as follows: Figure 4As shown.

[0483] UA bioavailability measurement

[0484] As previously described, measurements were performed in accordance with FDA industry guidance and EMA bioanalytical method validation guidelines (Toney et al. (2019) Obesity (Silver Spring) 27(4):612-20). Quantification of UA and its metabolites (i.e., UA-glucuronide and UA-sulfate in plasma) was performed by reversed-phase column separation followed by detection using a three-segment quadrupole MS / MS in a selected reaction monitoring mode. Plasma levels were assessed at T0, T6, and T24 hours after oral administration of Mitopure or PJ to measure bioavailability. The plasma limits of quantification for maternal UA were 5.00 pg / ml, and for UA-glucuronide and UA-sulfate and their metabolites, the plasma limits of quantification were 5.00 ng / ml. An analytical method for determining UA-glucuronide in human whole blood collected from dried blood spots (DBS) using liquid chromatography-tandem mass spectrometry (LC MS / MS) was also developed and validated. Based on the analysis of human dried blood spot samples (6 mm spots), the limit of quantification (LOQ) for UA-glucuronic acid in human dried blood spot samples (6 mm spots) ranged from 5.00 ng / mL to 5000 ng / mL. Quantification of UA-glucuronic acid was performed by reversed-phase liquid chromatography column separation, followed by detection using three-section quadrupole MS / MS in the selected reaction monitoring mode.

[0485] result

[0486] The results showed that, in both plasma and DBS methods, very few subjects (12%) had circulating UA-glucuronide at baseline derived from natural exposure to dietary precursors of urolithin A.

[0487] Following a dietary challenge with 100% pomegranate juice (rich in dietary ellagitannins), these levels showed no significant change 6 hours after ingestion. However, after 24 hours, approximately 40% of the subjects had metabolized the precursor, and fairly high levels of UA-glucuronide were detected in a few drops of dried whole blood. These subjects were considered “high producers.” The remaining 60% of the subjects either could not convert the precursor (33%) or had poor conversion ability (27%). Subjects were classified as low producers (plasma circulating urolithin A glucuronide levels <100 ng / ml) or high producers (plasma circulating urolithin A glucuronide levels ≥100 ng / ml).

[0488] Compared to the PJ group, similar baseline levels were observed in the urolithin A (Mitopure product) group at baseline (after a 10- to 12-day washout period). Following oral administration of urolithin A (Mitopure), UA-glucuronide concentrations significantly increased (maximum concentration - Cmax) in all 100 participants within 6 hours. These levels stabilized after 24 hours, and UA-glucuronide levels were detectable in all subjects (when comparing population mean, levels in the urolithin A (Mitopure) group were more than 2 times higher than in the pomegranate juice group, p < 0.0001 at the 6- and 24-hour time points). UA exposure (area under the curve - AUC) after urolithin A (Mitopure) ingestion was more than 6 times higher than in the 100% pomegranate juice group.

[0489] Table 1: PK parameters (mean plasma concentration) of plasma UA-glucuronide levels at different time points (0, 6 and 24 hours) for urolithiasis A (Mitopure) and 100% pomegranate juice.

[0490]

[0491] Table 2: Changes in plasma UA-glucuronide concentration over time (PK increment, AUC, area under the curve)

[0492] 100% Pomegranate Juice 68728.35 9188.13 UA Mitopure supplement 471857.30 20840.53

[0493] Table 3: DBS UA-glucuronide levels (Mitopure A) vs. 100% pomegranate juice at different time points (0, 6, and 24 hours) using pharmacokinetic parameters (mean DBS concentration).

[0494]

[0495] Comparison of urolithin A glucuronide levels measured in plasma and dried blood spots

[0496] The DBS level of UA-glucuronide was compared with that of plasma to observe whether the DBS method (spotting 20 μL of dried capillary whole blood onto a DBS card) had similar accuracy, sensitivity and specificity to plasma collection (12 mL of venous blood). Figure 5 The results showed a very high correlation between the two methods at the collection time points of 6 hours (A) and 24 hours (B) after urolithiasis A (Mitopure) ingestion (Spilman correlation coefficient r = 0.975). Therefore, the DBS method can be readily used as an alternative to more invasive methods of plasma venous collection.

[0497] Fecal sample collection

[0498] Each participant also received a stool sample collection kit (OMNIGene OMR-200, DNAGenotek, Kanata, ON, K2V 1C2, Canada) at the screening visit to collect samples at home prior to study visit V2. Upon return to the research center, the stool samples were stored at -80°C.

[0499] Example 5A - Microbiome Shotgun Metagenomic Sequencing and Analysis

[0500] use The 96Soil kit (Macherey Nagel) extracted DNA from approximately 0.1 g aliquots of fecal samples collected in Example 5. Throughout the experiment (including sequencing), each batch of DNA extraction samples included at least one negative control. ZymoBIOMICS TMMicrobial community standard samples (Zymo Research) were also included in the analysis as positive (dummy) controls. Before sequencing, DNA sample quality was assessed using agarose gel electrophoresis, and DNA quantity was assessed using Qubit 2.0 quantitative PCR. The prepared DNA library was assessed using Qubit 2.0 quantitative PCR, and fragment size distribution was assessed using an Agilent 2100 bioanalyzer. The concentration of the final DNA library was determined using real-time quantitative PCR (qPCR) before sequencing. The DNA library was sequenced on the Illumina platform using 2×150bp paired-end sequencing. A total of 99 fecal samples were sequenced, with an average depth of 19.9M read pairs per sample (Illumina 2×150PE). 96.5% of the high-quality microbiome reads were mapped to a reference human gut gene catalog, with an average of 200 metagenomic species (MGS) detected per sample. To perform MGS abundance analysis, we detected 1273 metagenomic species (MGS) with highly consistent abundance and base composition in 1776 independent reference human gut samples. The analysis was based on the metagenomic species concept (17). For taxonomic annotation of MGS, all catalog genes were transferred to the NCBI RefSeq Genome Database (October 1, 2018). Different similarity levels were required for annotation at different taxonomic levels (95%, 95%, 85%, 75%, 65%, 55%, 50%, and 45% similarity for subspecies, species, genus, family, order, class, phylum, and superkingdom, respectively) and a minimum sequence coverage of 80%. The percentage of genes mapped to each MGS for each species was calculated, and a species-level classification was defined as MGS if >75% of the genes could be annotated to a single species. Consistency levels of 60%, 50%, 40%, 30%, and 25% were used for genus, family, order, class, and phylum, respectively. In addition, for species and genera, if more than 10% of the genes in another group belong to a substitute species / genus, then MGS is not assigned.

[0501] result

[0502] The diversity and richness of the gut microbiome play a crucial role in defining UA producers and non-producers.

[0503] Following pomegranate juice ingestion, shotgun sequencing was used to investigate the composition of the microbiome in fecal samples from individuals producing different levels of UA. Metagenomic analysis measured the abundance of metagenomic species (MGS) and genes and compared them among subjects in non-producer, low-producer, and high-producer groups. Metagenomic analysis was first used to determine microbiome α-diversity, an indicator of microbial variation in a single sample. α-diversity was assessed by microbiome richness (the number of species or genes observed in a sample) and microbiome variability and quantified using the Shannon index (18). The Shannon index reflects the number of species or genes in a community and their relative abundance. For MGS ( Figure 6A ) or based on genes ( Figure 7 When measured, the richness of the Shannon index of the microbiome was significantly higher in both the low-producer and high-producer groups compared to the non-producer group. Secondly, the Bray-Curtis dissimilarity method was used to assess changes in β-diversity, reflecting differences in the relative abundance of MGS among samples. The Bray-Curtis dissimilarity ranges from 0 to 1, where 0 indicates that two samples have the same composition (they share all species at the same relative abundance), and 1 indicates that two samples are completely different (they do not share any species). Principal coordinate analysis (PCoA) of Bray-Curtis dissimilarity was performed. Figure 6B The results showed that the overall microbiome composition changed significantly when comparing non-producers with low-producers (p = 0.048) and high-producers (p = 0.001), as indicated by the clear segregation between the groups. No clear segregation was observed between the low-producer and high-producer groups. In summary, the α and β diversity results suggest that the ability to convert UA from its precursors is significantly associated with higher microbiome richness and overall composition.

[0504] UA producers and non-producers exhibit different abundances of gut microbiota taxa.

[0505] Consistent with the α and β diversity results, we also found taxa with varying abundances when comparing UA producers (low and high production states) with non-producers. We first analyzed the abundance of Firmicutes (F) and Bacteroidetes (B) at the phylum level, as an increased F / B ratio is associated with several markers of gut and organismal health (Mariat et al. (2009) BMC Microbiol 2009; 9:123; Wills et al. (2014) PLoS One 9(3):e 90981; Verdam et al. (2013) Silver Spring 21(12):E607-15). Firmicutes abundance was higher than Bacteroidetes in the two UA-producing groups (low and high producers), while the opposite was observed in the non-producer group. Figure 6C The F / B ratio for UA producers is significantly higher than that for non-producers. Figure 6D Next, we analyzed all the variations in the three UA producer state groups (MGS) and higher taxa. We found that the abundance or prevalence of four MGS and two phyla in the low producer group differed significantly from that in the non-producer group. Figure 8 The differences were even greater among high-producer groups, as the abundance or prevalence of 57 MGS species and 33 taxa (14 genera, 15 families, and 4 phyla) in the high-producer group differed significantly from that in the non-producer group (Supplementary Figure 6). Notably, all taxa that showed significant differences between the non-producer and low-producer groups also showed significant differences between the non-producer and high-producer groups.

[0506] Physiological differences between UA producers and non-producers

[0507] To better assess the potential impact of UA producer status on overall health, we performed a correlation analysis of participants' BMI and cardiac function (HR and DBP). A key observation was that the BMI of high-producers (27 ± 6.1 kg / m²) was significantly lower than that of low-producers (31.7 ± 8 kg / m²; p = 0.01) and non-producers (31.2 ± 8.4 kg / m²; p = 0.02) (Figure 9A). The mean resting diastolic blood pressure (78.4 ± 8 mm Hg) in the high-producer group (p = 0.017) was also significantly lower than that in the non-producer group (83.25 ± 8.66 mm Hg) (Figure 9B). The resting heart rate was also lower in high-producers compared to non-producers (p = 0.07), although this was not statistically significant (Figure 9C). There were no significant differences in hematological and blood biochemical parameters among the groups. FFQs of dietary habits across groups showed that the high-producer group consumed more fruits and berries than the low-producer and non-producer groups (Figure 9D).

[0508] Example 6: Determination of urolithin A and its metabolites in urine samples

[0509] We conducted single-dose (Part A) and multiple-dose (Part B) studies of urolithin A to evaluate its safety, tolerability, pharmacokinetics, and pharmacodynamics in healthy elderly subjects.

[0510] Research Design

[0511] Part A: This study was a double-blind, randomized, single-dose escalation trial involving 24 healthy elderly male and female volunteers. Each participant in the three cohorts was randomized to receive two follow-up doses.

[0512] Part B: This study was a double-blind, randomized, multiple-dose escalation study involving 36 healthy elderly male and female volunteers. Each participant was randomized to receive either the study product or a placebo for 28 days.

[0513] Research objective:

[0514] To determine the safety and tolerability of urolithiasis A in healthy elderly subjects after multiple 28-day administrations.

[0515] Determine the pharmacokinetic characteristics of urolithiasis A after single and multiple administrations.

[0516] The pharmacokinetic characteristics of urolithiasis A in a single 250 mg soft capsule formulation were compared with those of urolithiasis A in single, progressively higher doses of 500 mg, 1000 mg, and 2000 mg.

[0517] The pharmacokinetic characteristics of urolithiasis A in soft capsule form administered repeatedly over 28 days at a dose of 250 mg were compared with those of urolithiasis A administered repeatedly over 28 days at escalating doses of 500 mg and 1000 mg.

[0518] Research products

[0519] 1100mg soft capsules containing 250mg of urolithiasis A (as described in Example 2 above). The soft capsules are in bulk blister packaging, and the labeling complies with local regulatory norms and requirements.

[0520] Dosage per dose:

[0521] Part A: 250mg, 500mg, 1000mg or 2000mg (1 capsule, 2 capsules, 4 capsules or 8 capsules)

[0522] Part B: 250 mg, 500 mg, or 1000 mg daily (1, 2, or 4 capsules daily).

[0523] placebo

[0524] Soft capsules containing lecithin, triglycerides, and diglycerides

[0525] Dosage time:

[0526] Part A: Based on randomization, a single oral administration was given on day 1 of each period. At approximately 8:00 AM, on an empty stomach, the medication was taken in a seated position with about 200 mL of tap water.

[0527] Part B: Repeated oral administration from day 1 to day 28, according to randomization. At approximately 8:00 AM, on a fasting basis, take approximately 200 mL of tap water while seated.

[0528] Subjects

[0529] Part A:

[0530] The study included 24 healthy older men and women aged 61 to 85.

[0531] Cohort 1 (8 subjects): 250 mg urolithiasis A (6 subjects) or placebo (2 subjects) soft capsule formulation, followed by 2000 mg urolithiasis A or placebo soft capsule formulation.

[0532] Cohort 2 (8 participants): 500 mg of urolithiasis A (6 participants) or placebo (2 participants) in soft capsule formulation.

[0533] Cohort 3 (8 participants): 1000 mg of urolithiasis A (6 participants) or placebo (2 participants) in soft capsule formulation.

[0534] Part B:

[0535] The study included 36 healthy older men and women aged 61 to 85.

[0536] Cohort 1 (12 participants): 250 mg of urolithiasis A (9 participants) or placebo (3 participants) in soft capsule formulation for 28 days.

[0537] Cohort 2 (12 participants): 500 mg urolithiasis A (9 participants) or placebo (3 participants) soft capsule formulation for 28 days.

[0538] Cohort 3 (12 participants): 1000 mg of urolithiasis A (9 participants) or placebo (3 participants) in soft capsule formulation for 28 days.

[0539] Study duration

[0540] Part A:

[0541] Screening should be performed within 21 days prior to the first application.

[0542] Each period of hospitalization lasts 48 hours (from the night of D-1 to the night of D2).

[0543] Outpatient visits were conducted on days 4 and 5 of each period.

[0544] Wash-off: Each application should be spaced at least 21 days apart.

[0545] Research visit completed: P2D5.

[0546] Telephone follow-up was conducted at P2D7(±2).

[0547] Expected follow-up duration: approximately 8 weeks for each participating subject.

[0548] Part B:

[0549] Screening should be performed within 21 days prior to the first application.

[0550] Outpatient visits were conducted on day 1 (V1), day 7 (V2), and day 14 (V3).

[0551] The hospital stay lasted from day 27 (V4) (around 4 PM) to day 29 (V6) (around 10 AM).

[0552] Outpatient visits were conducted on the 31st and 32nd days of each period.

[0553] A telephone follow-up was conducted on day 35 (+ / - 2 days).

[0554] Expected follow-up duration: approximately 8 weeks for each participating subject.

[0555] At the final visit, subjects underwent the same full clinical-biological examination as at the start of the study. All adverse events (AEs) were recorded, and if any AEs were occurring, further follow-up was scheduled. Follow-up continued until the event was resolved, the condition was unlikely to change, or the subject was lost to follow-up.

[0556] Random grouping

[0557] The sponsor representative provided a list of randomization groups. The product was allocated to subjects in group A at P1D1 and to subjects in group B at D-1(V1).

[0558] Setting up blind spots

[0559] To avoid bias, the following measures were taken:

[0560] - Double-blind studies; and

[0561] - Soft capsules containing the active product and placebo are indistinguishable in appearance.

[0562] The analysis center, researchers, team, and participants were all blinded. For each participant, a sponsor representative provided a coded list containing the product identifier (urgent letter), which was kept in a secure location throughout the clinical study. In cases requiring drug formulation, a sealed coded list was used, which was provided to the representative's pharmacist. This sealed coded list was kept in a secure location and accessible to anyone not blinded.

[0563] Pharmacokinetic variables

[0564] urine

[0565] Dosage administered between 0 and 36 hours after drug administration, and the dose excreted within that timeframe.

[0566] Percentage: Ae 0-36小时 (Applicable only to part A)

[0567] Total dose excreted in urine % (for part A only)

[0568] Urine collection

[0569] In Part A of the study, total urine volume was collected at the following time intervals: 0-4 hours, 4-8 hours, 8-12 hours, 12-24 hours, and 24-36 hours post-administration. Urine volume was carefully measured by weight for each collection period, and the exact start and stop times of the collection interval were recorded in the eCRF. Each urine sample was homogenized, and two 5 mL aliquots were preserved at each interval and stored below -80°C. With a revised protocol extending blood collection from 36 hours to 96 hours post-administration, additional urine samples were collected at 72 and 96 hours post-administration, but these were not quantified.

[0570] Bioanalysis

[0571] The concentrations of urolithin A and its metabolites in urine were determined using a validated LC-MS / MS method. The limit of quantification (LOQ) for urolithin A in urine was 10 ng / mL, and the limits of quantification for urolithin A glucuronide and sulfated urolithin A in urine were 50 ng / mL.

[0572] Table 4 shows the levels (ng / ml) of urolithiasis A glucuronide in urine within 96 hours after administration of the 250 mg, 500 mg, 1000 mg, and 2000 mg dose groups. Data are presented as mean ± standard error.

[0573]

Claims

1. A non-diagnostic and non-treatment method for determining whether a human subject will benefit from taking urolithiasis A supplements, comprising the following steps: Analyze the biofluid samples collected from the subjects to determine whether the biofluid contains urolithin A glucuronide, and then determine whether the subjects will benefit from taking urolithin A supplements based on the level of urolithin A glucuronide in the biofluid. The biofluid sample is selected from urine or blood, and in the case of blood, it is analyzed in the form of dried blood spots; and wherein a) If the biofluid is blood, then if the level of urolithiasis A glucuronide is below 100 ng / ml, the subject will benefit from the dose of urolithiasis A supplement; or b) If the biofluid is urine, and if the level of urolithiasis A glucuronide is below 50,000 ng / ml, the subject will benefit from the dose of urolithiasis A supplement.

2. A non-diagnostic and non-treatment method for determining the dosage of urolithiasis A supplement for human subjects, comprising: a) Determining the level of urolithin A glucuronide in biological fluids; and b) Determine the dosage of the urolithiasis A supplement based on the level of urolithiasis A glucuronide in the biofluid sample. The biofluid sample is selected from urine or dried blood spots; and wherein i) If the biofluid is dried blood spot, then: a) If the level of urolithiasis A glucuronide is less than 5 ng / ml, the dose of the urolithiasis A supplement is 1500 mg or 1000 mg per day. b) If the level of urolithiasis A glucuronide is between 5 ng / ml and 50 ng / ml, the dosage of the urolithiasis A supplement is 500 mg daily; and c) If the level of urolithiasis A glucuronide is between 50 ng / ml and 100 ng / ml, then the dose of the urolithiasis A supplement is 250 mg / day; or ii) If the biological fluid is urine, then: a) If the level of urolithiasis A glucuronide is below 10,000 ng / ml, the dose of the urolithiasis A supplement is 1500 mg or 1000 mg per day. b) If the level of urolithiasis A glucuronide is between 10,000 ng / ml and 25,000 ng / ml, the dosage of the urolithiasis A supplement is 500 mg daily; and c) If the level of urolithiasis A glucuronide is between 25,000 ng / ml and 50,000 ng / ml, the dose of the urolithiasis A supplement is 250 mg / day.

3. A non-diagnostic and non-treatment method for determining whether a human subject will benefit from taking urolithiasis A supplements, comprising: a) Determine the level of urolithiasis A glucuronide in a first biofluid sample collected prior to administration of the test dose of urolithiasis A or urolithiasis A precursor; b) Determine the level of urolithin A glucuronide in a second biofluid sample obtained after administration of the test dose of urolithin A; as well as c) Determine whether the subject will benefit from taking urolithiasis A supplements based on the increase in urolithiasis A glucuronide levels in the biofluid sample; The biofluid sample is selected from urine or dried blood spots; and wherein i) If the biofluid is a dry drug spot, then if the level of urolithiasis A glucuronide is higher than 5 ng / ml and lower than 100 ng / ml, then the subject will benefit from the dose of urolithiasis A supplement; and ii) If the biofluid is urine, the subject will benefit from the dose of urolithiasis A supplement if the level of urolithiasis A glucuronide is above 10,000 ng / ml and below 50,000 ng / ml.

4. The method according to claim 3, in i) If the biofluid is a dried drug spot, then if the level of urolithiasis A glucuronide is higher than 5 ng / ml and lower than 100 ng / ml, the subject will benefit from the dose of urolithiasis A supplement; and The method further includes calculating the dosage of the urolithiasis A supplement: a) If the level of urolithiasis A glucuronide is less than 5 ng / ml, the dose of the urolithiasis A supplement is 1500 mg or 1000 mg per day. b) If the level of urolithiasis A glucuronide is between 5 ng / ml and 50 ng / ml, the dosage of the urolithiasis A supplement is 500 mg daily; and c) If the level of urolithiasis A glucuronide is between 50 ng / ml and 100 ng / ml, the dose of the urolithiasis A supplement is 250 mg / day; and ii) If the biofluid is urine, then if the level of urolithin A glucuronide is higher than 10,000 ng / ml and lower than 50,000 ng / ml, the subject will benefit from the dose of urolithin A supplement; and The method further includes calculating the dosage of the urolithiasis A supplement: a) If the level of urolithiasis A glucuronide is below 10,000 ng / ml, the dose of the urolithiasis A supplement is 1500 mg or 1000 mg per day. b) If the level of urolithiasis A glucuronide is between 10,000 ng / ml and 25,000 ng / ml, the dosage of the urolithiasis A supplement is 500 mg daily; and c) If the level of urolithiasis A glucuronide is between 25,000 ng / ml and 50,000 ng / ml, the dose of the urolithiasis A supplement is 250 mg / day.

5. The method according to claim 3, wherein the biological fluid sample is collected within the following time period; (a) Between 6 and 8 hours after administration of the urolithin A or urolithin A precursor; (b) Between 18 and 30 hours after administration of the urolithin A or its precursor; or (c) 24 hours after administration of the urolithin A or urolithin A precursor.

6. The method according to any one of claims 3-5, wherein the test dose of urolithin A is selected from 250 mg, 500 mg or 1000 mg.

7. The method according to any one of claims 3-5, wherein the dosage of the urolithiasis A supplement is selected from 250 mg per day, 500 mg per day, 1000 mg per day, or 1500 mg per day.

8. A computer-implemented non-diagnostic and non-treatment method for determining whether a human subject will benefit from taking urolithiasis A supplements, comprising the following steps: (b) Receive data on the level of urolithin A glucuronide in biological fluids; (d) Compare the level of urolithin A glucuronide with data relating the level to a dose of urolithin A to calculate the dose: The biofluid sample is selected from urine or blood, and in the case of blood, it is analyzed as a dried blood spot; and in a) If the biofluid is blood, then if the level of urolithiasis A glucuronide is below 100 ng / ml, the subject will benefit from the dose of urolithiasis A supplement; or b) If the biofluid is urine, and if the level of urolithiasis A glucuronide is below 50,000 ng / ml, the subject will benefit from the dose of urolithiasis A supplement.

9. The method according to claim 8, further comprising the following step: (a) Receiving and confirming data on the transport of biological fluid samples from subjects; and (c) Receive subject data, which is selected from time and date, age, sex, weight, ethnicity and sample type.

10. A computer-implemented non-diagnostic and non-treatment method for determining the dosage of urolithiasis A supplement for human subjects, comprising the following steps: (b) Receive data on the level of urolithin A glucuronide in biological fluids; (d) Compare the level of urolithin A glucuronide with data relating the level to a dose of urolithin A to calculate the dose: The biofluid sample is selected from urine or dried blood spots; and wherein i) If the biofluid is dried blood spot, then: a) If the level of urolithiasis A glucuronide is less than 5 ng / ml, the dose of the urolithiasis A supplement is 1500 mg or 1000 mg per day. b) If the level of urolithiasis A glucuronide is between 5 ng / ml and 50 ng / ml, the dosage of the urolithiasis A supplement is 500 mg daily; and c) If the level of urolithiasis A glucuronide is between 50 ng / ml and 100 ng / ml, then the dose of the urolithiasis A supplement is 250 mg / day; or ii) If the biological fluid is urine, then: a) If the level of urolithiasis A glucuronide is below 10,000 ng / ml, the dose of the urolithiasis A supplement is 1500 mg or 1000 mg per day. b) If the level of urolithiasis A glucuronide is between 10,000 ng / ml and 25,000 ng / ml, the dosage of the urolithiasis A supplement is 500 mg daily; and c) If the level of urolithiasis A glucuronide is between 25,000 ng / ml and 50,000 ng / ml, the dose of the urolithiasis A supplement is 250 mg / day.

11. The method of claim 10, further comprising the step of: (a) Receiving and confirming data on the transport of biological fluid samples from subjects; (c) Receiving subject data, the data being selected from time and date, age, sex, weight, ethnicity, and sample type; and (e) The results are communicated to the subject.

12. A non-transitory computer-readable medium comprising computer-executable instructions, which, when executed by a processor of a computing device, cause the computing device to perform a non-diagnostic and non-therapeutic method for determining whether a human subject will benefit from taking a urolithiasis A supplement, the method comprising: (b) Receive data on the level of urolithin A glucuronide in biological fluids; (d) Compare the level of urolithin A glucuronide with data relating the level to a dose of urolithin A to calculate the dose: The biofluid sample is selected from urine or blood, and in the case of blood, it is analyzed as a dried blood spot; and in a) If the biofluid is blood, then if the level of urolithiasis A glucuronide is below 100 ng / ml, the subject will benefit from the dose of urolithiasis A supplement; or b) If the biofluid is urine, and if the level of urolithiasis A glucuronide is below 50,000 ng / ml, the subject will benefit from the dose of urolithiasis A supplement.

13. The medium according to claim 12, the method further comprising: (a) Receiving and confirming data on the transport of biological fluid samples from subjects; and (c) Receive subject data, which is selected from time and date, age, sex, weight, ethnicity and sample type.

14. A non-transitory computer-readable medium comprising computer-executable instructions, which, when executed by a processor of a computing device, cause the computing device to perform a non-diagnostic and non-therapeutic method for determining a dosage of urolithin A supplement for a human subject, the method comprising: (b) Receive data on the level of urolithin A glucuronide in biological fluids; (d) Compare the level of urolithin A glucuronide with data relating the level to a dose of urolithin A to calculate the dose: The biofluid sample is selected from urine or dried blood spots; and wherein i) If the biofluid is dried blood spot, then: a) If the level of urolithiasis A glucuronide is less than 5 ng / ml, the dose of the urolithiasis A supplement is 1500 mg or 1000 mg per day. b) If the level of urolithiasis A glucuronide is between 5 ng / ml and 50 ng / ml, the dosage of the urolithiasis A supplement is 500 mg daily; and c) If the level of urolithiasis A glucuronide is between 50 ng / ml and 100 ng / ml, then the dose of the urolithiasis A supplement is 250 mg / day; or ii) If the biological fluid is urine, then: a) If the level of urolithiasis A glucuronide is below 10,000 ng / ml, the dose of the urolithiasis A supplement is 1500 mg or 1000 mg per day. b) If the level of urolithiasis A glucuronide is between 10,000 ng / ml and 25,000 ng / ml, the dosage of the urolithiasis A supplement is 500 mg daily; and c) If the level of urolithiasis A glucuronide is between 25,000 ng / ml and 50,000 ng / ml, the dose of the urolithiasis A supplement is 250 mg / day.

15. The medium according to claim 14, the method further comprising: (a) Receiving and confirming data on the transport of biological fluid samples from subjects; (c) Receiving subject data, the data being selected from time and date, age, sex, weight, ethnicity, and sample type; and (e) The results are communicated to the subject.

16. A computing device comprising a processor, a memory, and a display, said computing device being configured to implement the method according to any one of claims 8-11.

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