Diagnostic Method

Abstract

The current application relates to personalised nutrition methods, particularly to methods of determining whether a human subject would benefit from taking a urolithin supplement and methods for determining the treatment dose of a urolithin supplement for a human subject. Particularly methods comprising determining the level of urolithin or a urolithin conjugate in a biological fluid, such as a dried whole blood spot sample, a dried plasma spot, a m spot sample or a urine sample The current application also relates to systems for presenting whether a human subject would benefit from taking a urolithin supplement and for presenting the treatment dose of a urolithin supplement for a human subject. The current application also relates to computer implementation of methods of the invention.

Claims

1. A method of determining whether a human subject will benefit from taking a urolithin supplement, comprising the steps of analysing a sample of a biological fluid collected from the subject, ascertaining whether the biological fluid contains a urolithin or a urolithin conjugate, then determining whether the subject is one who will benefit from ingesting a urolithin supplement from the level of the urolithin or urolithin conjugate in the biological fluid; wherein the sample of biological fluid is selected from urine or blood and in the case of blood is analysed in the form of a dried blood spot.

2. The method of claim 1, wherein the urolithin conjugate is urolithin A glucuronide and the biological fluid is blood and wherein if the level of urolithin A glucuronide is under 100 ng/mL, then the subject is one who will benefit from a dose of a urolithin supplement.

3. The method of claim 1, the urolithin conjugate is urolithin A glucuronide and the biological fluid is urine and wherein if the level of urolithin A glucuronide is under 50,000 ng/mL, then the subject is one who will benefit from a dose of a urolithin supplement.

4. A method for determining a treatment dose of a urolithin supplement for a human subject, comprising a) Measuring the level of a urolithin or a urolithin conjugate in a biological fluid; and b) Determining the treatment dose of the urolithin supplement from the level of urolithin or urolithin conjugate in the biological fluid sample wherein the sample of biological fluid is selected from urine or a dried blood spot.

5. The method of claim 4, wherein the urolithin conjugate is urolithin A glucuronide and the biological fluid is a dried blood spot and: a) if the level of urolithin A glucuronide is less than 5 ng/mL then the treatment dose of urolithin supplement is 1500 mg or 1000 mg per day, for example, 1000 mg per day; b) if the level of urolithin A glucuronide is between 5 ng/mL and 50 ng/mL then the treatment dose of urolithin supplement is 500 mg per day; and c) if the level of urolithin A glucuronide is between 50 ng/mL and 100 ng/mL the treatment dose of urolithin supplement is 250 mg/day.

6. The method of claim 4, wherein the urolithin conjugate is urolithin A glucuronide and the biological fluid is urine and: a) if the level of urolithin A glucuronide is less than 10,000 ng/mL then the treatment dose of urolithin supplement is 1500 mg or 1000 mg per day, for example, 1000 mg per day; b) if the level of urolithin A glucuronide is between 10,000 ng/mL and 25,000 ng/mL then the treatment dose of urolithin supplement is 500 mg per day; and c) if the level of urolithin A glucuronide is between 25,000 ng/mL and 50,000 ng/mL the treatment dose of urolithin supplement is 250 mg/day.

7. A method for determining whether a human subject will benefit from taking a urolithin supplement, comprising a) Measuring the level of a urolithin or a urolithin conjugate in a first biological fluid sample, collected prior to administration of a test dose of a urolithin or a urolithin precursor; b) Administering a test dose of a urolithin or a urolithin precursor, for example pomegranate juice; c) Measuring the level of a urolithin or a urolithin conjugate in a second biological fluid sample; and d) Determining whether the subject is one who will benefit from ingesting a urolithin supplement from the increase in level of urolithin or urolithin conjugate in the biological fluid sample; wherein the sample of biological fluid is selected from urine or a dried blood spot.

8. The method of claim 7, wherein a urolithin is administered, the urolithin conjugate is urolithin A glucuronide and the biological fluid is a dried drug spot, and wherein if the level of urolithin glucuronide is above 5 ng/mL and under 100 ng/mL, then the subject is one who will benefit from a dose of a urolithin supplement.

9. The method of claim 7, wherein a urolithin precursor is administered, the urolithin conjugate is urolithin A glucuronide and the biological fluid is a dried drug spot, further comprising calculating the dose of the urolithin supplement.

10. The method of claim 7, wherein a urolithin is administered, the urolithin conjugate is urolithin A glucuronide and the biological fluid is urine, and wherein if the level of urolithin glucuronide is above 10,000 ng/mL and under 50,000 ng/mL, then the subject is one who will benefit from a dose of a urolithin supplement.

11. The method of claim 7, wherein a urolithin precursor is administered, the urolithin conjugate is urolithin A glucuronide and the biological fluid is urine, further comprising calculating the dose of the urolithin supplement.

12. The method of claim 7, wherein the biological fluid sample is collected either; (a) between about 6 to about 8 hours after administration of the urolithin or urolithin precursor; (b) between about 18 to about 30 hours after the administration of the urolithin or urolithin precursor; or (c) at about 24 hours after the administration of the urolithin or the urolithin precursor.

13. The method of claim 7, wherein the test dose of urolithin is selected from 250 mg, 500 mg or 1000 mg.

14. The method of claim 1, wherein the conjugate is a urolithin glucuronide or a urolithin sulphate.

15. The method of claim 1, wherein the dose of urolithin supplement is selected from 250 mg per day, 500 mg per day or 1000 mg per day or 1500 mg per day.

16. A computer-implemented method comprising the steps of: (a) Optionally receiving data confirming transit of sample of a biological fluid from a subject; (b) Receiving data of the level of a urolithin or urolithin conjugate in the biological fluid; (c) Optionally receiving subject data, for example, time and date, age, sex, weight, ethnicity, sample type etc. (d) Comparing the level of urolithin or a urolithin conjugate with data correlating the level with a treatment dose of a urolithin, for example, urolithin A, to calculate the treatment; (e) Optionally communicating results to the subject.

17. A non-transitory computer-readable medium comprising computer-executable instructions that, when executed by a processor of a computing device, cause the computing device to perform a method, the method comprising: (f) Optionally receiving data confirming transit of sample of a biological fluid from a subject; (g) Receiving data of the level of a urolithin or urolithin conjugate in the biological fluid; (h) Optionally receiving subject data, for example, time and date, age, sex, weight, ethnicity, sample type etc. (i) Comparing the level of urolithin or a urolithin conjugate with data correlating the level with a treatment dose of a urolithin, for example, urolithin A, to calculate the treatment dose; (j) Optionally communicating results to the subject.

18. A non-transitory computer-readable medium comprising computer-executable instructions of claim 17, wherein the data correlating the level with the treatment dose is: the urolithin conjugate is urolithin A glucuronide and the biological fluid is a dried blood spot and: a) if the level of urolithin A glucuronide is less than 5 ng/mL then the treatment dose of urolithin supplement is 1500 mg or 1000 mg per day, for example, 1000 mg per day; b) if the level of urolithin A glucuronide is between 5 ng/mL and 50 ng/mL then the treatment dose of urolithin supplement is 500 mg per day; and c) if the level of urolithin A glucuronide is between 50 ng/mL and 100 ng/mL the treatment dose of urolithin supplement is 250 mg/day; or the urolithin conjugate is urolithin A glucuronide and the biological fluid is urine and: a) if the level of urolithin A glucuronide is less than 10,000 ng/mL then the treatment dose of urolithin supplement is 1500 mg or 1000 mg per day, for example, 1000 mg per day; b) if the level of urolithin A glucuronide is between 10,000 ng/mL and 25,000 ng/mL then the treatment dose of urolithin supplement is 500 mg per day; and c) if the level of urolithin A glucuronide is between 25,000 ng/mL and 50,000 ng/mL the treatment dose of urolithin supplement is 250 mg/day.

19. A computer device comprising a processor, memory and display, the computing device being arranged to implement a method of claim 16.

20. The method of claim 1, further comprising administering a urolithin supplement to the subject.

21. The method of claim 4, then further comprising administering a urolithin supplement to the subject.

22. A method of preventing or treating symptoms of aging in a human subject wherein the method comprises administering a urolithin supplement to the subject and wherein the subject is one who has been determined as benefiting from taking a urolithin supplement by a method of claim 1.

23. A method of maintaining adequate nutrient levels in a human subject wherein the method comprises administering a urolithin supplement to the subject and wherein the subject is one who has been determined as benefiting from taking a urolithin supplement by a method of claim 1.

24. A method of determining the health of an individual's gut microbiome comprising the steps of analysing a sample of biological fluid collected from the subject, ascertaining whether the biological fluid, contains a urolithin or a urolithin conjugate, as an indicator of the health of the individual's gut microbiome; wherein the sample of biological fluid is selected from urine or blood and in the case of blood is analysed in the form of a dried blood spot.

25. The method of determining the health of an individual's gut microbiome of claim 24, further comprising the collecting a stool sample for microbiome analysis and analysing the stool sample.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0355] FIG. 1: shows the levels of urolithin glucuronide in dried blood spots in subjects pre-treatments and after administration of pomegranate juice and 500 mg urolithin.

[0356] FIG. 2: shows a flow chart of the study described in Example 5.

[0357] FIG. 3: shows UA-glucuronide levels in plasma samples collected at the timepoints of 0 (baseline), 6 and 24 hrs. post intake after either Pomegranate juice or Urolithin A in all n=100 healthy participants in Example 5. Data shown are Mean of 100 samples.

[0358] FIG. 4 shows UA-glucuronide levels in DBS (dried blood spot) samples at the timepoints of 0 (baseline), 6 and 24 hrs. post intake after either Pomegranate juice or Urolithin A in all n=100 healthy participants in Example 5.

[0359] FIG. 5A: shows the correlation of DBS (whole blood spot samples) measurements with plasma level measurements for UA glucuronide at t=6 hrs for the n=100 subjects (Spearman correlation coefficient r=0.97).

[0360] FIG. 5B: shows the correlation of DBS (whole blood spot samples) measurements with plasma level measurements for UA glucuronide at t=24 hrs for the n=100 subjects (Spearman correlation coefficient r=0.97).

[0361] FIG. 6A shows boxplots showing differences in metagenomics species (MGS) for richness (left panel) and Shannon diversity (right panel) between groups with no-, low-, and high-UA producer status. All groups were compared pairwise by Mann-Whitney U test (N=99). *P0.05; **P0.01; ***P0.001; *****P0.00001.

[0362] FIG. 6B shows principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarities among samples, calculated based on the MGS abundances. Samples are color coded by the UA producer status. Each sample is connected to its group centroid by a thin segment line. The ellipses cover two standard errors of the mean of the group centroids, i.e. they illustrate the certainty of the group centroid positions. The x- and y-axis labels indicate the microbial variance explained by the first two principal coordinates.

[0363] FIG. 6C shows relative abundance (in per cent) of phyla Firmicutes and Bacteroidetes which significantly differed in abundance in producer of UA compared with no-producer. Boxes represent interquartile range (IQR), with the inside horizontal line representing the median. Whiskers represent values within 1.5IQR of the first and third quartiles.

[0364] FIG. 6D shows firmicutes/Bacteroidetes ratio (F/B ratio) for each group shown as median (IQR). **P0.01 (Mann-Whitney U test).

[0365] FIG. 7: shows Boxplots showing differences in microbiome genes for richness (left panel) and Shannon diversity (right panel) between groups with no-, low-, and high-UA producer status. All groups were compared pairwise by Mann-Whitney U test (N=99). *P0.05; **P0.01; ***P0.001, *****P0.00001.

[0366] FIG. 8: Relative abundance (in per cent) of four MGS and two phyla which significantly differed in abundance or prevalence in UA low-producer compared with no-producer. The relative abundance is shown as the area of each square, scaled to the maximum within each species.

[0367] FIG. 9A shows comparison of physiological parameters and food habits between groups with no-, low-, and high-UA producer status. BMI was significantly higher in no- (p=0.02) and low- (p=0.01) UA producers compared with high producers.

[0368] FIG. 9B shows comparison of physiological parameters and food habits between groups with no-, low-, and high-UA producer status. Resting diastolic blood pressure was significantly higher in the no-producer group (p=0.017) than the high-producer group.

[0369] FIG. 9C shows comparison of physiological parameters and food habits between groups with no-, low-, and high-UA producer status. Heart rate in subjects who were non-producers compared to the high producers (p=0.07).

[0370] FIG. 9D shows comparison of physiological parameters and food habits between groups with no-, low-, and high-UA producer status. Fruits and berry intake were higher in the high UA producer group than both the no-producer group (p=0.07) and the low-producer group (p=0.01) as assessed via a dietary questionnaire. N=100. Data are expressed as mean+/SEM and analyzed using repeated measure ANOVA. *P0.05; **P0.01.

EXAMPLES

[0371] The invention will now be illustrated with respect to the following non-limiting Examples

Example 1: Preparation of Urolithin A

[0372] Urolithin A (4) was prepared in two steps starting from 2-bromo-5-methoxybenzoic acid 1 and resorcinol 2. The pure compound was obtained as a pale yellow powder.

##STR00002##

Step 1:

[0373] A mixture of 2-bromo-5-methoxybenzoic acid 1 (27.6 g; 119 mmol; 1.0 eq.), resorcinol 2 (26.3 g; 239 mmol; 2.0 eq.) and sodium hydroxide (10.5 g; 263 mmol; 2.2 eq.) in water (120 mL) was heated under reflux for 1 hour. A 5% aqueous solution of copper sulphate (3.88 g of CuSO4.5H2O in 50 mL water; 15.5 mmol; 0.1 eq.) was then added and the mixture was refluxed for additional 30 minutes. The mixture was allowed to cool to room temperature and the solid was filtered on a Bchner filter. The residue was washed with cold water to give a pale red solid which was triturated in hot MeOH. The suspension was left overnight at 4 C. The resultant precipitate was filtered and washed with cold MeOH to yield the title compound 3 as a pale brown solid.

Step 2:

[0374] To a suspension of 3 (10.0 g; 41 mmol; 1.0 eq.) in dry dichloromethane (100 mL) was added dropwise at 0 C. a 1 M solution of boron tribromide in dry dichloromethane (11.93 mL of pure BBr3 in 110 mL of anhydrous dichloromethane; 124 mmol; 3.0 eq.). The mixture was left at 0 C. for 1 hour and was then allowed to warm up to room temperature. The solution was stirred at that temperature for 17 hours. Then ice was added thoroughly to the mixture. The yellow precipitate was filtered and washed with cold water to give a yellow solid which was heated to reflux in acetic acid for 3 hours. The hot solution was filtered quickly and the precipitate was washed with acetic acid, then with diethyl ether to yield the title compound 4 as a yellow solid. 1H and 13C NMR were in accordance with the structure of 4.

Example 2: Urolithin a Dosage Form

[0375] Urolithin A was formulated into a soft gel capsule containing the following components:

TABLE-US-00002 Fill Ingredients Amount (mg)/Cap % Total Urolithin A 250 22.73% Lecithin NF (35% Total PC) 284.25 25.84% (Epikuron 135 F IP) - E322 Medium Chain Triglycerides 284.25 25.84% (MCT) Glycerol Monostearate (40-55) 11.5 1.06% EP, Mono- and Diglycerides NF Fill Weight 830 mg 75.47%

TABLE-US-00003 Shell Ingredients Amount (mg)/Cap % Total Gelatin EP, NF 165.97 15.09% Glycerol - E422 80.01 7.27% Water 21.62 1.96% Titanium Dioxide EP - E171 1.96 0.18% DualDustmaster FD&C Blue 0.234 0.021% #1 (Brilliant Blue FCF - E133) Sodium Copper Chlorophyllin 0.196 0.018% Powder (min 95%) - E141 Shell Weight 270 mg 24.539% Total Capsule Weight 1100 mg .sup.100%

Example 3Study of the Effect of Pomegranate Juice and Oral Urolithin on Levels of Blood, Collected in Dried Blood Spots

[0376] A study was conducted in seven subjects. At the beginning of the study a sample of capillary blood via a finger prick was taken from each subject and 15-20 uL of whole blood was dried onto a dried blood sample (DBS) filter paper sample card (Whatman, Little Chalfont, Buckinghamshire, UK). A minimum of 2 spots per sampling were collected for each time-point. At T0 i.e. baseline sample, subjects ingested 80 ml pomegranate juice (freshly prepared/). At 24 hours following pomegranate juice intake, a further dried blood spot sample was taken and 15-20 uL of whole blood was dried onto a DBS filter paper card. Following this, subjects ingested 500 mg of Urolithin A in a softgel capsule and then at 48 hours (or 24 hours following UA intake), a further dried blood spot sample was taken and 15-20 uL of whole blood was dried onto DBS filter paper sample card.

Example 3a: Measurement of Urolithin A Glucuronide in Dried Blood Spot Samples

[0377] This analytical method was developed to analyse whole blood spot samples collected on dry blood spot sample cards (DBS Filter card) in Example 3.

[0378] Each dried blood spot was punched from cards using a 6 mm diameter puncher and transferred into a tube. Afterwards 150 L of methanol containing the internal standard was added. Subsequently the samples were undergoing sonication for 5 minutes followed by a vortex mixing step for 20 minutes. Then samples were centrifuged for 2 minutes at 50000 g and 8 C. An aliquot of 100 L of the supernatant was transferred in an autosampler vial for analysis and diluted with 100 L of water prior to analysis.

[0379] The quantification of UA-glucuronide was performed by column separation with reversed-phase chromatography followed by detection with triple-stage quadrupole MS/MS in the selected reaction monitoring mode.

[0380] Results are shown in Table 1

TABLE-US-00004 TABLE 1 Baseline-Free Pomegranate 500 mg single UA Living Juice dosing Pre UA levels 24 h UA levels 24 h UA levels Subject (ng/mL) (ng/mL) (ng/mL) 1 0 125 2 37.9 38.8 134 3 0 22.4 173 4 0 45.2 153 5 0 0 6 0.0 0.0 93.0 7 0.0 0.0 98.0

Example 4: Bioavailability Study in Healthy Adults

[0381] A single-centre, two-period, crossover, randomized, open labelled study was performed to evaluate the Urolithin A producer status in a healthy US based adult population and to establish the superiority of dietary supplementation UA supplementation in establishing higher Urolithin A circulating levels over dietary exposure.

STUDY POPULATION: Healthy males and females aged between 18-80 years old. At least 20% participants will be between 20-40 years; 41-60 years; and 61-80 years. The remaining 40% distributed among any age group.
SAMPLE SIZE: N=100; all participants completed the study

Investigational Product

[0382] I. 237 mL of Pomegranate Juice (PJ)/day of administration (POM Wonderful, Los Angeles, Calif., USA, 90064). [0383] II. 500 mg Urolithin A (UA) in berry flavoured.sup.1 food sachet/day of administration 1* ingredients that include blueberry, raspberry fruit powder along with rice hull and pomegranate natural flavouring [0384] The urolithin A powder was admixed into a vanilla flavoured commercial yogurt (4 fl. oz) prior to administration to the participants

Efficacy Parameters

Primary Endpoint:

[0385] Absolute change from T.sub.0 to T+24 hours in the Urolithin A (UA) group as compared to pomegranate juice (PJ) group in UA-glucuronide plasma levels.

Secondary Endpoints:

[0386] Absolute change from T.sub.0 to T+24 hours in the UA group as compared to PJ group in UA-glucuronide blood spot levels. [0387] Absolute change from T.sub.0 to T+6 hours in the UA group as compared to PJ group in UA-glucuronide plasma levels. [0388] Absolute change from T.sub.0 to T+6 hours in the UA group as compared to PJ group in UA-glucuronide blood spot levels. [0389] Change in AUC from T.sub.0 to T+6 and/or T+24 in the UA group as compared to the PJ group in UA glucuronide levels [0390] Absolute change from T.sub.0 to T6 hours and/or to T24 hours and change in AUC in the UA group as compared to PJ group in the following variables: [0391] UA sulfate plasma levels [0392] UA aglycone (parent) plasma levels [0393] To assess prevalence of UA producers following consumption of fixed volume of Pomegranate juice in a healthy US population

[0394] Note: UA producer status is defined as someone who has detectable UA (and its conjugates) levels in plasma/dried blood spots following pomegranate juice intake. UA producer status was decided following review of results at T+24. [0395] Differences, if any, between UA producers and non-producers for the following variables at baseline: [0396] Age and BMI [0397] Hand grip strength [0398] Gait speed [0399] Physical activity and energy levels via: [0400] International Physical Activity Questionnaire (IPAQ) [0401] Fatigue VAS Score [0402] Dietary intake of foods high in ellagitannins and punicalagins i.e. Nuts and berries [0403] Differences, if any, between UA producers and non-producers in: [0404] Metabolomic analysis of plasma and/or DBS [0405] Any of the blood biochemistry parameters measured at visit 1: HbA1c, Creatinine, AST, ALT, Total bilirubin, total cholesterol, HDL, LDL and triglycerides) [0406] Faecal Analysis via microbiome sequencing to include: [0407] Sample-specific relative abundance and taxonomic identity of all OTUs (operational taxonomical units) [0408] Sample-specific relative abundance of bacteria from phylum to genus level [0409] Alpha and beta diversity measures (e.g. Shannon index and UniFrac distances) [0410] Identification of phylogenetic groups that change significantly in abundance between UA producer's and non-producers [0411] Statistical analysis of differences in microbiome community composition and diversity measures between UA producer's and non-producers

Inclusion Criteria:

[0412] To be eligible for inclusion, the subject must have fulfilled all of the following criteria:

1. Males and females 18 to 80 years of age;
2. Is in general good health, as determined by the clinical research team;
3. Willingness to consume investigation product, complete questionnaires and to complete all clinic visits;
4. Have given voluntary, written, informed consent to participant in the study;

Exclusion Criteria:

[0413] The presence of any of the following criteria excluded the subject from participating in the study: [0414] 1. Women who were pregnant, breastfeeding, or planning to become pregnant during the course of the trial; [0415] 2. Alcohol or drug abuse within the last 6 months; [0416] 3. Volunteers who planned to donate blood during the study or within 30 days of completing the study; [0417] 4. Subject having a known allergy to the test material's active or inactive ingredients; [0418] 5. Subjects with unstable medical conditions; [0419] 6. Clinically significant abnormal laboratory results at screening; [0420] 7. Participation in a clinical research trial within 30 days prior to randomization; [0421] 8. Allergy or sensitivity to study ingredients; lactose intolerance [0422] 9. Individuals who were cognitively impaired and/or who were unable to give informed consent; [0423] 10. Any other condition which in the Investigator's opinion may adversely affect the subject's ability to complete the study or its measures or which may pose significant risk to the subject; [0424] 11. Had taken antibiotics within the previous 30 days.

[0425] The study flowchart can be seen in FIG. 2.

Plasma Collection

[0426] At each time point indicated in the study assessments table, a 12 mL blood sample was drawn into K2-EDTA coated tube. The blood samples were gently inverted a few times for complete mixing with the anticoagulant. The exact time of sample collection was be recorded on the tube and in the electronic case report form (eCRF). Within 30 minutes following blood collection, each blood sample was centrifuged at 1500 g for 10 minutes at 4 C. Within 30 minutes after the centrifugation, the top layer of human plasma was transferred into two prelabelled polypropylene tubes, containing approximately 1500 L of plasma each (2 aliquots per time-point). Tubes were capped immediately from each time point and the plasma was frozen in an upright position at approximately 80 C. for storage.

[0427] Plasma samples were collected to assess UA-glucuronide levels at the time points of 0 (baseline), 6 and 24 hrs. post intake after either Pomegranate juice or Urolithin A (Mitopure) in all n=100 healthy participants. Data is shown in FIG. 3 as is expressed as the mean of 100 samples.

Dried Blood Spot (DBS) Collection:

[0428] The following items were used: [0429] Blood collection card (filter paperWhatman Filter Paper 903 5-spots card); [0430] Sealable biohazard foil bags [0431] Desiccant packs

[0432] Samples were collected by a finger-prick with a lancet. Each collection card was used for only one subjects. Blood was applied to the card with at least 3-4 blood spots on the DBS card. Care was taken to ensure the entire circle was uniformly saturated and each card. clearly labelled with appropriate identification. Follow the finger-prick with the lancet, gentle pressure was applied to the finger to allow a large drop of free-flowing blood to collect at the puncture site. The first drop was wiped away. Working quickly, the filter paper was held by the edges and the filter paper touched gently against the large drop of blood and in one step a sufficient quantity of blood was allowed to soak through and completely fill or saturate a circle. A completed saturated spot contained 20-40 l of blood. Spotting was repeated, until enough blood was collected to fill at least 3 circles on the blood collection card. Completing filling of the blood spot circle was important as the laboratory used a hole puncher to punch a section of the circle of blood for testing. DBS samples were stored at room temperature in biohazard foil bags with clear labels

[0433] DBS (dried blood spot) samples were collected to assess UA-glucuronide levels at the timepoints of 0 (baseline), 6 and 24 hrs. post intake after either Pomegranate juice or Urolithin A (Mitopure) in all n=100 healthy participants. Data is shown in FIG. 4.

UA Bioavailability Measurements

[0434] Measurements were performed according to the FDA Guidance for Industry and EMA guidelines on bioanalytical method validation as previously described (Toney et al (2019) Obesity (Silver Spring) 27(4):612-20). The quantification of UA and its metabolitesi.e. UA-glucuronide and UA-sulfate in plasmawas performed by column separation with reverse phase chromatography followed by detection with triple stage quadrupole MS/MS in the selected reaction monitoring mode. Plasma levels were assessed following oral intervention with either Mitopure or PJ at the T0, T6 and T24 hrs. time points for bioavailability assessments. The limit of quantification in plasma was 5.00 pg/mL for parent-UA and 5.00 ng/mL for both the UA-glucuronide and UA-sulfate its metabolites. An analytical method to determine UA-glucuronide in human whole blood collected in dried blood spots (DBS) using liquid chromatography coupled to mass spectrometry (LC MS/MS) was also developed and validated. The limit of quantification for UA-glucuronide in human dried blood spots samples (6-mm spots) range was 5.00 to 5000 ng/mL for the analysis of human dried blood spots samples (6-mm spots). The quantification of UA-glucuronide was performed by column separation with reversed phase liquid chromatography followed by detection with triple stage quadrupole MS/MS in the selected reaction monitoring mode.

Results

[0435] The results show that in both plasma and DBS method that very few subjects (12%) with circulating UA-glucuronide at baseline from natural exposure to dietary precursors of Urolithin A.

[0436] Following dietary challenge with 100% pomegranate juice (rich in dietary ellagitannins), these levels did not change drastically at 6 hours post intake, but by 24 hours approx. 40% of subjects had metabolized the precursors and considerable levels of UA-glucuronide that were detected in a few drops of dried whole blood. These subjects were considered as High producers. The remaining 60% were either unable to convert (33%) or were poor convertors (27%). Subjects were categorized as low-producers with <100 ng/mL circulating urolithin A glucuronide levels in plasma or high-producers with 100 ng/mL circulating urolithin A glucuronide levels in plasma.

[0437] In comparison with consumption of PJ, with Urolithin A (Mitopure product), at baseline (after the washout period of 10-12 days) similar baseline levels were observed. Following oral administration of Urolithin A (Mitopure), within 6 hours all 100 participants had significantly higher concentration of UA-glucuronide (maximal concentration-Cmax). These levels came close to steady state levels at 24 hours with all subjects showing detectable levels of UA-glucuronide (levels with Urolithin A (Mitopure) were significantly higher in comparison to pomegranate juice >2 fold when comparing mean of population, p<0.0001 at both 6 and 24-hour time-points). Exposure to UA following intake (i.e. area under the curve-AUC) was >6-fold higher with Urolithin A (Mitopure) compared to 100% pomegranate juice.

TABLE-US-00005 TABLE 1 PK Parameters (Plasma Mean Concentrations) on Plasma UA-Glucuronide levels Urollithin A (Mitopure) Vs. 100% Pomegranate juice at different time points of 0, 6 and 24 hrs. Product Time (hours) Mean Std. Error 100% Pomegranate Juice T0 5.481 2.04 T6 12.840 17.026 T24 110.473 13.049 UA Mitopure supplement T0 9.568 4.862 T6 480.750 23.66 T24 255.525 12.943

TABLE-US-00006 TABLE 2 Table PK incremental AUC (area under the curve) over time on Plasma UA-Glucuronide concentrations Product Mean Std. Error 100% Pomegranate Juice 68728.35 9188.13 UA Mitopure supplement 471857.30 20840.53

TABLE-US-00007 TABLE 3 Table PK Parameters (DBS Mean Concentrations) on DBS UA-Glucuronide levels (Urolithin A (Mitopure) Vs. 100% Pomegranate juice at different time points of 0, 6 and 24 hrs. Product Time Mean Std. Error 100% Pomegranate Juice T0 1.502 .952 T6 3.521 4.452 T24 32.790 3.365 UA Mitopure supplement T0 2.423 .948 T6 127.646 4.429 T24 66.866 3.348

Comparison of the Levels of Urolithin a Glucuronide Measured in Plasma and Dry Blood Spots

[0438] DBS levels of UA-Glucuronide were compared to plasma levels to see if the DBS method (20 ul of dried capillary whole blood spotted on a DBS card) had similar accurate sensitivity and specificity compared to the plasma collection (12 mL of venous blood). The results in FIG. 5A and FIG. 5B show a very high correlation between the two methods (Spearman correlation co-efficient r=0.975) at both the 6 hours (FIG. 5A) and 24 hours (FIG. 5B) collection timepoint following Urolithin A (Mitopure) intake. As such the DBS method can easily be utilized to replace the more invasive venous collection of plasma.

Faecal Sample Collection

[0439] Each participant was also supplied with a stool sample collection kit (OMNIGene OMR-200, DNA Genotek, Kanata, ON, K2V 1C2, Canada) at their screening visit to collect a sample at home prior to study visit V2. Upon return to the study site, the stool samples were stored in a 80 C. freezer.

Example 5AShotgun Metagenomic Sequencing and Analysis of Microbiome

[0440] DNA was extracted from 0.1 g aliquots of the faecal samples, collected in Example 5) using the NucleoSpin 96 Soil kit (Macherey Nagel). A minimum of one negative control was included per batch of samples from the DNA extraction and throughout the laboratory process (including sequencing). A ZymoBIOMICS Microbial Community Standard (Zymo Research) was also included in the analysis as a positive (mock) control. Before sequencing, the quality of the DNA samples was evaluated using agarose gel electrophoresis, and the quantity of the DNA was evaluated by Qubit 2.0 fluorometer quantitation. The prepared DNA libraries were evaluated using Qubit 2.0 fluorometer quantitation and Agilent 2100 Bioanalyzer for the fragment size distribution. Quantitative real-time PCR (qPCR) was used to determine the concentration of the final library before sequencing. The library was sequenced using 2150 bp paired-end sequencing on an Illumina platform. A total of 99 fecal samples were sequenced to an average depth of 19.9 M read pairs (IIlumina 2150 PE) per sample. 96.5% of the high-quality microbiome reads from a sample were mapped to a reference human gut gene catalog, and an average of 200 metagenomic species (MGS) were detected per sample. For MGS abundance profiling, a set of 1273 metagenomic species (MGS), which have highly coherent abundance and base composition in a set of 1776 independent reference human gut samples were detected. The analysis is based on the metagenomic species concept (17). To taxonomically annotate the MGSs, all the catalog genes were blasted to the NCBI RefSeq genome database (2018 Oct. 1). To annotate at the various taxonomic ranks, different levels of similarity were required (95, 95, 85, 75, 65, 55, 50 and 45% for subspecies, species, genus, family, order, class, phylum, super kingdom, respectively) and a minimum of 80% sequence coverage. The percentage of genes of each MGS that mapped to each species were calculated, and species level taxonomy were assigned to an MGS if >75% of its genes could be annotated to a single species. For genus, family, order, class and phylum, 60, 50, 40, 30 and 25% consistency levels were used, respectively. Furthermore, at species and at genus level, the MGS was not assigned if another set of more than 10% of the genes belonged to a single alternative species/genus.

Results

Gut Microbiome Diversity and Richness Play an Essential Role in Defining UA-Producers Versus Non-Producers

[0441] The microbiome composition from the faecal samples of individuals producing UA at different levels was investigated following pomegranate juice intake, using shotgun sequencing. This metagenomic analysis measured the abundance of both metagenomic species (MGS) and genes and compared them in subjects belonging to the no-, low- and high producer group. The metagenomic profiles were first used to determine the microbiome alpha diversity that indicates the variation of microbes in a single sample. Alpha diversity was assessed both as microbiome richness (number of species or genes observed in a sample) and microbiome variability, and was quantified using the Shannon index (18). The Shannon index accounts for the number of species or genes in a community, and also their relative abundance. A significantly higher richness was found in the microbiome Shannon index in both the low-producers and high-producers, when comparing each with the no-producer group, for both MGS (FIG. 6A) and gene-based (FIG. 7) measures. Secondly, changes in beta diversity was evaluated, which accounts for differences in relative abundance of MGSs among samples, using Bray-Curtis dissimilarity. Bray-Curtis dissimilarity can range between 0-1, where 0 means that the two samples have identical compositions (they share all species at the same relative abundance), and 1 means that the two samples are completely different (they do not share any species). A principal coordinate analysis (PCoA) of the Bray-Curtis dissimilarities (FIG. 6B) showed a striking shift in the overall microbiome composition when comparing non-producers with low (p=0.048) and high-producers (p=0.001), as shown by the clear segregation of the groups. No clear separation was observed between the groups of low-producer and high-producer. In summary, the alpha and beta diversity results indicate that the ability to convert UA from its precursors is significantly associated with a higher microbiome richness and overall composition.

UA-Producer and Non-Producer Exhibit Differentially Abundant Gut Microbiome Taxa

[0442] In line with the alpha and beta diversity results, we also found differentially abundant taxa when comparing UA producers (low and high production status) with non-producers. We first analyzed, at the phylum level, the abundancy of Firmicutes (F) and Bacteroidetes (B), as increased F/B ratio has been associated with several markers of gut and organismal health (Mariat et al (2009) BMC Microbiol 2009; 9:123; Wills et al (2014) PLoS One9(3):e90981; Verdam et al (2013) Obesity (Silver Spring) 21(12):E607-15). Both groups (low and high producers) capable of producing UA showed a higher abundance of Firmicutes with respect to Bacteroidetes, while the opposite was observed for the no-producer group (FIG. 6C). The F/B ratio was significantly higher in UA producers compared with non-producers (FIG. 6D). Next, we analyzed all changes at the levels of species (MGS) and of higher taxa among the three UA-producer status groups. We found 4 MGSs and 2 phyla with significantly different abundance or prevalence between the low-producer group and the no-producer group (FIG. 8). High-producers showed even larger differences, as 57 MGSs and 33 taxa (14 genera, 15 families, 4 phyla) had significantly different abundance or prevalence in the high-producer group compared with the no-producer group (Supplementary FIG. 6). Notably, all taxa that were significantly different between the no-producer and low-producer group were also significantly different between the no-producer and high-producer group.

Physiological Differences Between UA Producers and Non Producers

[0443] To better appreciate the potential impact of UA-producer status on general health, we performed a correlation analysis with participants' BMI and cardiac function (HR and DBP). One key observation was that high-producers had significantly lower BMI (27+6.1 kg/m2) compared with both low-producers (31.7+8 kg/m2; p=0.01) and non-producers (31.2+8.4 kg/m2; p=0.02) (FIG. 9A). The high-producers group also had significantly (p=0.017) lower mean resting diastolic blood pressure (78.4+8 mm Hg) compared with the non-producer's group (83.25+8.66 mm Hg) (FIG. 9B). Resting heart rate was also lower although not statistically significant in the high-producers compared with the non-producers (p=0.07) (FIG. 9C). There were no major differences in hematological and blood biochemistry parameters between the groups. The FFQ on dietary habits of the different groups revealed that the high-producer group consumed greater quantities of fruits and berries than the low and no-producer groups (FIG. 9D).

Example 6: Measurement of Urolithin A and its Metabolites in Urine Samples

[0444] A single (Part A) and multiple (Part B) dose study of urolithin A was conducted to evaluate the safety, tolerability, pharmacokinetics and pharmacodynamics profile in healthy elderly subjects.

Study Design

[0445] Part A: The study was a double-blind, randomized, single ascending doses, study in 24 healthy elderly male and female volunteers. Each subject was randomized for two subsequent doses in three cohorts.

[0446] Part B: The study was a double-blinded, randomized, multiple ascending dose study in 36 healthy elderly male and female volunteers. Each subject was randomised to receive study product or placebo for 28 days.

Study Objectives:

[0447] To determine the safety and tolerability of urolithin A in healthy elderly subjects following multiple 28 days dosing.

[0448] To determine the pharmacokinetic profiles of urolithin A following a single and a multiple dose.

[0449] To compare the pharmacokinetic profiles of urolithin A delivered as soft gel formulation in a single 250 mg dose to ascending single higher doses administration at doses: 500 mg, 1000 mg and 2000 mg.

[0450] To compare the pharmacokinetic profiles of urolithin A delivered as a softgel formulation in repeated multiple 28 days 250 mg dose to ascending repeat multiple 28 days administration at doses: 500 mg and 1000 mg.

Investigational Product:

[0451] 1100 mg soft gel capsule containing 250 mg of urolithin A (as described in Example 2 above). Soft gel capsules were blister-packed in bulk, Labelling was in accordance with local regulatory specifications and requirements.

Dose Per Intake:

[0452] Part A: 250 mg, 500 mg, 1000 mg or 2000 mg (1, 2, 4 or 8 capsules)

[0453] Part B: 250 mg per day, 500 mg per day or 1000 mg per day (1, 2, or 4 capsules per day)

Placebo:

[0454] Soft gel capsule containing lecithin, triglycerides, diglycerides

Timing For Intake:

[0455] Part A: Single oral dose administration on D1 of each period according to the randomisation. The administration took place around 8:00 am with around 200 mL tap water, in sitting position, and under fasting conditions.

[0456] Part B: Repeated oral dose administration from day 1 to day 28 according to the randomization. The administration took place around 8:00 am with around 200 mL tap water, in a sitting position, and under fasting conditions.

Subjects:

Part A:

[0457] 24 healthy elderly male and female subjects were included in the study, within the age range 61 to 85 years.

[0458] Cohort 1 (8 subjects): 250 mg urolithin A (6 subjects) or placebo (2 subjects) capsule soft gel formulation then 2000 mg urolithin A or placebo capsule soft gel formulation. Cohort 2 (8 subjects): 500 mg urolithin A (6 subjects) or placebo (2 subjects) capsule soft gel formulation. Cohort 3 (8 subjects): 1000 mg urolithin A (6 subjects) or placebo (2 subjects) capsule soft gel formulation.

Part B:

[0459] 36 healthy elderly male and female subjects were included in the study, within the age range 61 to 85 years.

[0460] Cohort 1 (12 subjects): 250 mg urolithin A (9 subjects) or placebo (3 subjects) soft gel capsule formulation for 28 days.

[0461] Cohort 2 (12 subjects): 500 mg urolithin A (9 subjects) or placebo (3 subjects) soft gel capsule formulation for 28 days.

[0462] Cohort 3 (12 subjects): 1000 mg urolithin A (9 subjects) or placebo (3 subjects) soft gel capsule formulation for 28 days.

Study Duration

Part A:

[0463] Screening within 21 days prior to the first administration.

[0464] Hospitalization for 48h (D-1 evening to D2 evening) for each period.

[0465] Ambulatory visit at D4 and D5 for each period

[0466] Wash-out: at least 21 days between each administration

[0467] End of study visit: P2D5.

[0468] Follow up phone call at P2D7 (2).

[0469] Expected duration: approximately 8 weeks for each participating subject

Part B:

[0470] Screening within 21 days prior to the first administration

[0471] Ambulatory visits at day 1 (V1), day 7 (V2), day 14 (V3).

[0472] Hospitalisation from day 27 (V4) (around 4 pm) to day 29 (V6) (around 10 am)

[0473] Ambulatory visit at day 31 and day 32 for each period

[0474] Follow up phone call at day 35 (+/2)

[0475] Expected duration: approximately 8 weeks for each participating subject.

[0476] During the last visit, subjects underwent a complete clinical biological examination, identical to an examination at the start of the study. Any (AEs) were recorded, and if they were ongoing a further follow-up was arranged. Follow up continued until the event was resolved or the condition was unlikely to change or the subject was lost to follow-up.

Randomization

[0477] A randomisation list was provided by the sponsor's representative. The product was allocated at P1D1 for part A and on D-1 (V1) on part B.

Blinding

[0478] The following measures were taken to avoid bias: [0479] double-blind study; and [0480] soft-gel capsules containing active product and placebo were indistinguishable in appearance.

[0481] The analytical centre as well as the Investigator and the team and the subject were in blind conditions. For each subject, a coding list containing the identification of the product (emergency envelopes) was supplied by the sponsor's representative and kept in a safe place during the whole clinical study period. In the case of a pharmaceutical preparation being required, the decoding system used was a sealed coding list to be given to the representative's pharmacist. The sealed coding list was kept in a safe place and was accessible to any person authorised to unblind.

Pharmacokinetic Variables

Urine

[0482] The percentage of an administered dose excreted from time zero to 36 h after dosing: Ae.sub.0-36h (Part A only)

[0483] The % of total dose excreted in urine (Part A only)

Urine Collection

[0484] In Part A of the study, total urine was collected during the following intervals: 0-4 h, 4-8 h, 8-12 h, 12-24 h and 24-36 h post-dose. The collected urine volume of each collection period was carefully measured by weight and recorded in the eCRF, as well as the exact start and stop time of the collection interval. Each fraction of collected urine was homogenized and 2 aliquots of 5 mL per interval were kept and stored below 80 C. Following a protocol amendment to extend the blood sampling scheme from 36 to 96 h post-dose, additional spot urine samples were taken 72 and 96 h post-dose but urine was not collected quantitatively.

Bioanalysis

[0485] The concentrations of Urolithin A and its metabolites in urine was determined using validated LC-MS/MS assays. The limit of quantification (LOQ) was 10 ng/mL for Urolithin A in urine and 50 ng/mL for Urolithin A glucuronide and Urolithin A sulphate in urine.

TABLE-US-00008 TABLE 4 shows the levels of Urolithin A glucuronide (ng/mL) in urine for 250 mg, 500 mg, 1000 mg and 2000 mg up to 96 hours after administration. Data is expressed as mean standard error. Time after Urolithin A Urolithin A dose (mg) administration 250 500 1000 2000 Predose 4,114 1,261 4,114 1,261 4,114 1,261 4,114 1,261 T0-T4 h 8,536 1,394 40,167 6,802 70,492 12,094 39,500 6,033 T4 h-T8 h 31,617 7,637 86,758 12,051 76,417 10,635 65,317 10,853 T8 h-T12 h 40,453 9,275 63,800 9,043 64,967 8,075 50,450 5,793 T12 h-T24 h 62,817 8,454 81,125 10,792 107,983 17,683 114,817 12,123 T24 h-T36 h 33,962 8,708 44,242 4,631 59,575 9,560 60,533 12,216 T72 h N.D. 15,706 3,751 29,412 5,717 70,567 17,054 T96 N.D. 8,720 3,113 16,335 3,897 38,487 12,858