Patent ID: 12220424

EXAMPLES

The following Examples illustrate the invention.

Example 1: Compounds

Urolithin A was prepared as follows:

Urolithin A (4) was prepared in two steps starting from bromide 1 and resorcinol 2. The pure compound was obtained as a pale yellow powder.

Step 1:

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 an additional 30 minutes. The mixture was allowed to cool to room temperature and the solid was filtered on a Büchner 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:

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 and13C NMR were in accordance with the structure of 4.

Example 2: Powder Formula Composition Targeting Healthy Aging and Age-Related Muscle Loss Containing High Protein, Nicotinamide Riboside and Urolithin A

TABLE 8CompositionPer 100 g (single serving)Protein51.5 gWhey(45 g)Leucine(3.5 g)Isoleucine(1.5 g)L-Arginine(1.5 g)Carbohydrates28 gFat11.5 gPolyunsaturated Fatty Acids2.5 gFiber2.0 gVitamin A50 μgVitamin D350 μgVitamin E20 mgVitamin C100 mgVitamin B62 mgVitamin B1210 μgFolic Acid500 μgNiacin50 mgZinc5 mgCalcium100 mgSelenium40 μgIron20 mgMagnesium100 mgCreatine1.5 gUrolithin A500 mgNicotinamide Riboside500 mg

The compositon with the nutrient profile shown in Table 8 is given to a subject to counteract age related muscle loss.

Example 3: An Enteral Nutrition Liquid Composition Targeting Immobilized Subject in Intensive Care or Hospital Settings Containing Nicotinamide Riboside and Urolithin A

TABLE 9CompositionPer 100 mLProtein10.5 g100% Hydrolyzed WheyCarbohydrates15.4 gFat9.9 gPolyunsaturated Fatty Acids3.8 gFiber0 gVitamin A170 μgVitamin D32 μgVitamin E3 mgVitamin C20 mgVitamin B60.3 mgVitamin B121 μgFolic Acid50 μgNiacin5 mgZinc1.5 mgCalcium100 mgSelenium10 μgIron2 mgMagnesium40 mgUrolithin A500 mgNicotinamide Riboside500 mg

The drink compositon with the nutrient profile shown in Table 9 is given to an immobilised subject in intensive care or a hospital setting.

Example 4: A Cereal Bar Composition Targeting an Active Athlete for Optimal Muscle Function During Endurance Training Containing Nicotinamide Riboside and Urolithin A

TABLE 10CompositionPer bar of 35 gEnergy600 kcalProtein6.7 gCarbohydrates17.2 gFat7.6 gPolyunsaturated Fatty Acids3.8 gFiber1.6 gNiacin25 mgZinc2.5 mgCalcium180 mgSodium25 mgPotassium60 mgMagnesium80 mgL-Carnitine200 mgUrolithin A500 mgNicotinamide Riboside500 mg

The bar compositon with the nutrient profile shown in Table 10 is given to an active athlete for optimal muscle function during endurance.

Example 5: A Yogurt Composition

TABLE 11Nutrition value:per 100 gFat0.2 gCarbohydrates3.7 gProtein9.8 gVitamin B20.18 mg13% of RDACalcium95 mg12% of RDAPhosphorus170 mg24% of RDALive Active CulturesUrolithin A100 mg, 250 mg, 500 mg,750 mg or 1000 mgNicotinamide250 mg, 500 mg , 1000 mg,Riboside2000 mg, 3000 mg

Example 6: In Vitro Testing of Urolithin A and Nicotinamide Riboside on Mitochondrial Respiratory Subunits Proteins in Muscle Cells

C2C12 myoblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) including 4.5 g/L glucose, 10% fetal calf serum, and 50 μg/mL gentamicin. Urolithin A (UA) was dissolved in DMSO in a stock solution of 50 mM. Nicotinamide riboside (NR) in the form of the triflate salt was dissolved in double-distilled water in a stock solution of 1 M. One hundred thousand cells were plated at time 0 hours in 6-wells plates. A total of 3 wells was used per condition (n=3 per group). Cells were treated in a volume of 2 ml for a period of 48 hours at final concentrations of (a) DMSO 0.1%, (b) 25 μM UA, (c) 0.1 mM NR, (d) 1 mM NR, (e) 25 μM UA+0.1 mM NR and (f) 25 μM UA+1 mM NR.

At the end of the treatment, cells were lysed with cell lysis buffer (#9803, Cell signalling) containing protease (cOmplete™, Roche) and phosphatase inhibitor (PhosSTOP™, Roche) and applied to Invitrogen NuPage® Novex® Gel System (Bis-Tris Protein Gels—4-12%, Thermo fisher Scientific). Protein levels were examined for mitochondrial respiratory subunits, including mitochondrially encoded cytochrome c oxidase I (MTCO1), succinate dehydrogenase complex flavoprotein subunit A (SDHA) and B (SDHB), ubiquinol-cytochrome c reductase core protein II (UQCRC2) and ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1 (ATP5A). The housekeeping protein tubulin-α was measured as a loading control. Detection of the proteins was performed using an Azure c300 (Azure biosystem) (FIG.1B). Western Blot images were representative of the three biological replicates. Experiments were repeated at least 2 times. Bands were quantified using ImageJ software. The heatmap of the band intensities was drawn using GEN-E (Broad Institute) conditional formatting. Control DMSO values were set to one (FIG.1A).

As shown inFIG.1, UA at 25 μM alone induces an increase in SDHA (+54%), SDHB (+10%), UQCRC2 (+26%) and MT-CO1 (+14%) proteins levels, while no change is observed in ATP5A protein levels. NR has a different effect, which is not dose-dependent at the tested concentrations. Both NR 0.1 and 1 mM lead to a +≈100% increase in SDHA, no change in SDHB, +≈10% in UQCRC2 and −≈10% in MT-CO1. NR at 0.1 mM has no effect on ATP5A while NR at 1 mM decreases it by 20%. In contrast, the combination of UA and NR increases all protein levels, and to higher extent than with the single compounds. UA 25 μM+NR 0.1 mM increase SDHA by 140%, SDHB by +18%, UQCRC2 by +30%, MT-CO01 by 37% and ATP5A by 18% while UA 25 μM+NR 1 mM increase SDHA by 127%, SDHB by +34%, UQCRC2 by +63%, MT-CO1 by 66% and ATP5A by 33%.

These results show that the combination of UA and NR has a synergistic effect on mitochondrial biogenesis, and this effect is conserved across several concentrations of NR. Interestingly, there is a dose-dependent effect with NR when combined with UA, while it is not the case with the compound alone.

Example 7: In Vitro Testing of Urolithin A and Nicotinamide Riboside on Mitochondrial Biogenesis and Autophagy Markers Expression in Muscle Cells

C2C12 myoblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) including 4.5 g/L glucose, 10% fetal calf serum, and 50 μg/mL gentamicin. Urolithin was dissolved in DMSO in a stock solution of 50 mM. Nicotinamide riboside in the form of the triflate salt was dissolved in double-distilled water in a stock solution of 1 M. One hundred thousand cells were plated at time 0 hours in 6-wells plates. A total of 6 wells was used per condition (n=6 per group).

In a first batch of experiments, cells were treated in a volume of 2 ml with (a) DMSO 0.1% for 24 hours, (b) 0.1 μM UA for 24 hours, (c) 1 μM UA for 24 hours, (d) 1 mM NR for 6 hours, (e) 0.1 μM UA for 24 hours and 1 mM NR for 6 hours, (f) 1 μM for 24 hours and 1 mM NR for 6 hours (FIGS.2and3)

In a second batch of experiments, cells were treated in a volume of 2 ml with (a) DMSO 0.1% for 24 hours, (b) 25 μM UA for 24 hours, (d) 1 mM NR for 24 hours, and (e) 25 μM UA and 1 mM NR for 24 hours (FIG.4).

At the end of treatment, total RNA was prepared using TRIzol (Invitrogen). cDNA was prepared using the QuantiTect Reverse Transcription Kit (Qiagen) following the manufacturer's instructions. The RT-qPCR reactions were performed using the Light-Cycler system (Roche Applied Science) and a qPCR Supermix (Qiagen) with the indicated primers (Table 12). Genes that belong to mitochondrial biogenesis pathway (succinate dehydrogenase complex flavoprotein subunit A, SDHA; mitochondrially encoded cytochrome c oxidase II, MT-CO2; ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1, ATP5A and NADH:ubiquinone oxidoreductase subunit B2, NDUFB2) and autophagy pathway (Autophagy related 5, ATG5; GABA type A receptor associated protein like 1, GABARAPL1 and sequestosome 1, p62) were analysed and normalized over the housekeeping genes actin beta (ACTB) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).FIG.2shows the combination of UA at 0.1 μM for 24 hours with NR at 1 mM for 6 hours, whileFIG.3shows the combination of UA at 1 μM for 24 hours with NR at 1 mM for 6 hours.FIG.4shows the combination of UA at 25 μM with NR at 1 mM for 24 hours. Bargraphs represent mean±SEM. *P<0.05; **P<0.01; ***P<0.001 is for the statistical difference between the combination of UA and NR and other treatments after one-way ANOVA followed by Bartlett's test and Dunnett's multiple comparisons test. Experiments were repeated at least 2 times.

As shown inFIGS.2and3, the combination of UA 0.1 or 1 μM 24 hours and NR 1 mM 6 hours leads to a significantly higher increase of mitochondrial respiratory subunits (FIG.2A-CandFIG.3A-C) and autophagy genes (FIGS.2D-Eand3D-E) than with UA alone or NR alone. Likewise, the combination of UA 25 μM and NR 1 mM for 24 hours induces significantly more mitochondrial biogenesis (FIG.4A-C) and autophagy genes (FIG.4D-E) than with UA alone or NR alone. These results show that the combination of UA and NR has a synergistic effect on mitochondrial biogenesis and autophagy, and this effect is conserved across several concentrations of UA and several treatment timings of NR. These results are surprising as both mitochondrial biogenesis and autophagy genes are upregulated upon the combination of urolithin A and nicotinamide riboside.

The primers used for the RT-qPCR analyses were the literature primers known from the references indicated in the Table 12 below.

TABLE 12GeneGene IDReferenceActb11461Ryu et al., Nature Medicine 2016,22 (8):879-88 (Pubmed ID 27400265)Hprt115452Hruz et al., BMC Genomics. 2011; 12:156.(Pubmed ID 21418615)SDHA66945Al-Sawaf et al., Sci Rep. 2014; 4:3625.(Pubmed ID 24406502)MT-CO217709Gaignard et al., Endocrinology, 2015, 156(8), pp. 2893-2904, also; Ryu et alATP5A11946Mohamed et al., J Transl Med. 2016; 14: 149.(Pubmed ID 27234427)NDUFB268198Hwang et al, Biochemistry, 2015, 54(24), pp 3739-3748 (Pubmed ID 26030260)GABARAPL157436Lee and Goldberg, J Biol Chem.Dec. 18, 2015; 290(51): 30269-30279(PMCID 4683253), also; Ryu et alp6218412Ryu et al., Nature Medicine 2016, 22(8):879-88 (Pubmed ID 27400265)Atg511793Ryu et al., Nature Medicine 2016, 22(8):879-88 (Pubmed ID 27400265)

Example 8a: In Vitro Testing of Urolithin a and Nicotinamide Riboside on Autophagy and Mitochondrial Biogenesis in Muscle Cells

C2C12 myoblast are cultured in Dulbecco's modified Eagle's medium (DMEM) including 4.5 g/L glucose, 20% fetal calf serum, and 50m/mL gentamicin. Urolithin is dissolved in DMSO in a stock solution of 50 mM. Nicotinamide riboside is dissolved in DMSO in a stock solution of 50 mM. Cells are treated at final concentrations of 50 μM urolithin, 1 mM nicotinamide riboside, or 50 μM urolithin and 1 mM nicotinamide riboside for a period 24 hours. Control cells are treated with DMSO at an equivalent final concentration for the same period and serves as the untreated control.

Human primary skeletal myocytes are cultured in vitro and exposed to concentrations of 50 μM urolithin, 1 mM nicotinamide riboside, or 50 μM urolithin and 1 mM nicotinamide riboside for 24 hours. Human skeletal myoblasts are grown in DMEM plus 2% horse serum. Control cells are treated with DMSO at an equivalent final concentration for the same period and served as the untreated control.

At the end of treatment, RNA is extracted from the cells and converted to cDNA for qPCR analysis. Genes that belong to NAD+ synthesis pathway (Nampt), mitochondrial biogenesis pathway (Pgc1α, Sirt1, Nrf1, Tfam, Mrps5), mitochondrial respiratory chain subunits (Ndufb5, Sdha, CytC, CoxIV, Atp5 g1), autophagy pathway (LC3B, Pik3c3, p62, Gabarapl1) and mitophagy pathway (Parkin, PINK1) are analysed and normalized over the housekeeping genes Actb and Hprt1. The results show the effect of urolithin A combined with nicotinamide riboside on the expression of genes belonging to NAD+ synthesis pathway, mitochondrial biogenesis and respiratory subunits, autophagy and mitophagy.

Also, at the end of the treatment, cells are lysed with RIPA buffer and applied to SDS-PAGE and protein levels are examined for autophagy-related proteins (LC3-I and LC3-II, p62, AMPKa, and p-AMPKa), mitophagy related protein (Parkin), mitochondrial respiratory subunits (MTCO1, NDUFS3, SDHA, SDHB, UQCRC2, ATP5A). The housekeeping protein for total protein load β-actin is measured as a loading control. The mitochondrial protein VDAC1 is used as a housekeeping protein for mitochondrial abundance. The results show the effect of the urolithins A combined with nicotinamide riboside on the level of autophagy, mitophagy and mitochondrial abundance in the cells in question.

Example 8b: In Vitro Testing of Urolithin a and Nicotinamide Riboside on Respiratory Capacity in Muscle Cells

C2C12 myoblast are cultured in Dulbecco's modified Eagle's medium (DMEM) including 4.5 g/L glucose, 20% fetal calf serum, and 50 μg/mL gentamicin. Urolithin is dissolved in DMSO in a stock solution of 50 mM. Nicotinamide riboside is dissolved in DMSO in a stock solution of 50 mM. Cells are treated at final concentrations of 50 μM urolithin, 1 mM nicotinamide riboside, or 50 μM urolithin and 1 mM nicotinamide riboside for a period 24 hours. Control cells are treated with DMSO at an equivalent final concentration for the same period and serves as the untreated control.

Human primary skeletal myocytes are cultured in vitro and exposed to concentrations of 50 μM urolithin, 1 mM nicotinamide riboside, or 50 μM urolithin and 1 mM nicotinamide riboside for 24 hours. Human skeletal myoblasts are grown in DMEM plus 2% horse serum. Control cells are treated with DMSO at an equivalent final concentration for the same period and served as the untreated control.

At the end of the treatment, respiratory capacity is determined by measuring basal oxygen consumption and following the additions of the uncoupler Carbonyl cyanide m-chlorophenyl hydrazine (CCCP) at a final concentration of 10 μM. The results show the effect of urolithin A combined with nicotinamide riboside on respiratory capacity in the cells in question.

Example 8c: Experimental Trial on Muscle Function

22 month old C57BL/6J old mice that would be equivalent to a 65 yr to 75 yr old elderly human are used in a model of aging. The mice are treated with (i) diet without any supplement, (ii) diet supplemented with urolithin, (iii) diet supplemented with nicotinamide riboside; or (iv) diet supplemented with urolithin and nicotinamide riboside. In addition to the supplements, the diets contain protein, fat, carbohydrate essential nutrients, vitamins and minerals.

At the end of the treatment with the diet, muscles are weighed and collected. RNA is extracted from the cells and converted to cDNA for qPCR analysis. Genes that belong to NAD+ synthesis pathway (Nampt), mitochondrial biogenesis pathway (Pgc1α, Sirt1, Nrf1, Tfam, Mrps5), mitochondrial respiratory chain subunits (Ndufb5, Sdha, CytC, CoxIV, Atp5 g1), autophagy pathway (LC3B, Pik3c3, p62, Gabarapl1) and mitophagy pathway (Parkin, PINK1) are analysed and normalized over the housekeeping genes Actb and Hprt1. The results show the effect of urolithin A combined with nicotinamide riboside on the expression of genes belonging to NAD+ synthesis pathway, mitochondrial biogenesis and respiratory subunits, autophagy and mitophagy.