CHALCONES AND DERIVATIVES FOR USE IN MEDICAMENTS AND NUTRACEUTICALS

Abstract

Provided herein are means and methods for inhibiting processes and/or facilitating processes in cells by means of a compound of structural formula I

##STR00001##

wherein R1, R2, R3, R4, R5 and R6 are each independently H, OH, CH3, OCH3, a monosaccharide, an oligosaccharide or Cl; with the proviso that at least two of R1-R6 are H; and at least one other of R1-R6 is OH. The compound can be used among others in the prophylactic or curative treatment of an elevated blood interleukin-1β level and/or the treatment of low grade inflammation in an animal subject in need thereof. Also provided is a method for increasing longevity or increasing the health span in a non-diseased animal subject, the method including administering to the animal subject an effective amount of a hydroxychalcone of formula I as indicated herein. Also provided are food and food supplements including a compound of formula I as indicated herein.

Claims

1. A method for the prophylactic or curative treatment of an elevated blood interleukin-1β level, an elevated level of IL-18 and/or the treatment of low grade inflammation in an animal subject comprising administering a hydroxychalcone of formula I ##STR00013## wherein R1=OH, R2=H, R3=OH, R4=H, R5=OH and R6=H; to animal subject in need thereof.

2. The method of claim 1, wherein the elevated blood interleukin-1β level, an elevated level of IL-18 and/or the treatment of low grade inflammation is caused by a disease selected from the group consisting of multiple sclerosis, amyotrophic lateral sclerosis, gout, COPD, macular degeneration, hypertension, osteoporosis, osteoarthritis, rheumatoid arthritis, Duchenne muscular dystrophy, chronic fatigue syndrome (also referred to as myalgic encephalomyelitis), depression, non-fatty acid liver disease (NAFLD) and/or fibrosis.

3-4. (canceled)

5. A method of inhibiting caspase-1, interleukin-1β and/or IL-18 production by a cell, the method comprising contacting the cell with a hydroxychalcone of formula I ##STR00014## wherein R1=OH, R2=H, R3=OH, R4=H, R5=OH and R6=H.

6.-8. (canceled)

9. A method for increasing longevity and/or increasing health span in a non-diseased animal subject, the method comprising administering to the animal subject an effective amount of a hydroxychalcone of formula I ##STR00015## wherein R1, R2, R3, R4, R5 and R6 are each independently H, OH, CH3, OCH3, a monosaccharide, an oligosaccharide or Cl; with the proviso that at least two of R1-R6 are H; and at least one other of R1-R6 is OH.

10. The method of claim 9, wherein R1 is CH.sub.3; R2 is H; R3 is OH; R4 is H; R5 is CH.sub.3; R6 is H; or R1 is OH; R2 is H; R3 is CH.sub.3; R4 is H; R5 is CH.sub.3; R6 is H; or R1 is H; R2 is CH.sub.3; R3 is H; R4 is OH; R5 is CH.sub.3; R6 is H; or R1 is CH.sub.3; R2 is H; R3 is OH; R4 is H; R5 is OH; R6 is H; or R1 is OCH3; R2 is H; R3 is OH; R4 is H; R5 is OH; R6 is H; or R1 is OH; R2 is H; R3 is OH; R4 is H; R5 is CH.sub.3; R6 is H; or R1 is OH; R2 is H; R3 is OH; R4 is H; R5 is OH; R6 is OH; or R1 is OH; R2 is H; R3 is OH; R4 is H; R5 is OH; R6 is H.

11. The method of claim 10, wherein R1=OH, R2=H, R3=OH, R4=H, R5=OH and R6=H.

12. The method of claim 9, wherein the animal subject is a human.

13-15. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0082] FIG. 1: IL-1β levels in LPS/ATP stimulated, and PMA differentiated THP-1 cells.

[0083] FIG. 2: IL-18 levels in LPS/ATP stimulated, and PMA differentiated THP-1 cells

[0084] FIG. 3: mTOR and autophagy activity in LPS/ATP stimulated and unstimulated PMA differentiated THP-1 cells.

[0085] FIG. 4: Caspase-1 level in LPS/ATP stimulated PMA differentiated THP-1 cells. The active form of caspase-1 can be measured in the supernatant of cells.

[0086] FIG. 5: The effect of 2,2′,4′-trihydroxychalcone treatment on the survival of nematodes.

[0087] FIG. 6: Table of suitable chalcones. The chalcones are numbered in order estimated activity. The chalcone with the highest expected activity received number 1. The one with the next highest expected activity number 2 etc.

[0088] FIG. 7: Estimated affinity, ligand efficiency (LE), Lipophilic ligand efficiency (LLE) and torsion angle (TOR) of several hydroxychalcone, compared to Glyburide, a known NLRP3 inhibitor. The drawing on the right shows the position of the 2,2′-dimethyl-4″-hydroxychalcone within the NLRP3 PYD domain.

[0089] Ligand Efficiency LE. The ligand efficiency is a measure for the activity normalized by the number of non-H atoms. More precisely, it is the relative free binding energy in kcal/mol per non-H atom, calculated from an IC50 value. Especially in early project stages prioritizing compounds based on their ligand efficiency values is a much more favorable approach compared to judging from plain activities alone: “For the purposes of HTS follow-up, we recommend considering optimizing the hits or leads with the highest ligand efficiencies rather than the most potent.” (Ref.: A. L. Hopkins et al., Drug Disc. Today, 9 (2004), pp. 430-431).

To give an example: A compound with 30 atoms (400 MW) that binds with a Kd=10 nM has a ligand efficiency value of 0.36 kcal/mol per non-H atom. Another compound with 38 non-H atoms (500 MW) and the same ligand efficiency would have a 100 fold higher activity with Kd=0.106 nM. Let us assume an HTS screening revealed two hit compounds A and B with equal activities of IC50=10 nm, but different molecular weights of 400 and 500, respectively. Based on activities both compounds look equally attractive. Considering, however, that a synthetic introduction of a new group with 8 non-H atoms into compound A would match compound B in terms of weight, but would increase the activity by a factor of 100, if its ligand efficiency value can be maintained, it becomes clear that compound A is the by far more attractive alternative.

Lipophilic Ligand Efficiency LLE

[0090] The LLE value is a builds on the fact that the typical compounds of drug discovery projects huddle at the lipophilic side of the acceptable lipophilicity range. A gain in lipophilicity therefore is associated with a loss in bioavailability and should be compensated by higher activity on the target. To express this relationship the LLE is calculated as LLE=−log IC50−c Log P. A rough rule of thumb may be the suggestion of Jonathan S. Mason from Lundbeck Research to aim for LLE values above 3 for lead compounds and above 5 for clinical candidates.

Torsion Angle (TOR)

[0091] Every molecule has a preferred (lowest energy) shape. The color coding indicates whether the shape of the molecule in the structure deviates from this lowest energy shape. Green indicates a good fit of the molecule when in it's preferred shape. Orange and red indicates an increasing deviation from the preferred fit. All indicated chalcones have a green symbol at TOR. For glyburide the symbol is red.

EXAMPLES

Example 1

Determination of IL-1β, IL-18, Phospho P70 S6K and Caspase-1 Levels in THP-1 Cells.

[0092] Evaluation of the effect of test compounds on inflammasome induced IL-1β or IL-18 generation and mTOR or caspase-1 activity was performed using PMA-differentiated THP-1 cells stimulated with LPS (2 ng/ml, 4h (LPS (sigma, cat #L3012)) plus ATP (5 mM, 5 min. (Sigma, cat #A1852)). PMA-differentiated THP-1 cells untreated with LPS/ATP were used as control. PMA (phorbol 12-myristate 13-acetate) stimulation was done by adding 150 nM PMA to the cell culture for 24 hours. followed by 24 hours of incubation in culture medium and removal of non-adherent cells.

The supernatant was collected immediately after experimental procedures for measurement of IL-1β ELISA (R&D Systems, cat #DY201) or IL-18 ELISA (R&D Systems, cat #7620) levels.

[0093] The cell lysates were collected accordingly the manufacturer's instructions for analysis of phospho p70 S6K (Thr389) (eBioscience, cat #85-86052) levels by ELISA, to determine the mTOR activity or to determine caspase-1 (R&D Systems cat #AF6215) levels by Western blot. As secondary antibody for the Western blot a HRP-conjugated anti-IG antibody (R&D Systems cat #HAF017) was used and immunoreactive bands were visualized by the Enhanced Chemiluminescence method (Thermo Scientific, Rockford, Ill.). All assays were performed accordingly to the respective manufacturers instructions.

Test Compounds

[0094] 2,2′,4′-trihydroxychalcone (abcr cat #AB151762), isoliquiritigenin (TCI Europe cat #I0822) and resveratrol (TCI Europe cat #R0071). As control Rapamycin (APExBIO, cat #A8167) was used.

Conditions Evaluated (LPS/ATP Unstimulated and LPS/ATP Stimulated Cells):

[0095] a) Dose response curves for test compounds: 5 points of 3-fold dilutions beginning from 300 μM
b) Control conditions (all contain comparable amount of DMSO, e.g. <1%)

1. Untreated

[0096] 2. LPS+ATP only,

3. Rapamycin (25, 50 and 100 nM)

Cellular Model:

[0097] PMA-differentiated THP-1 cells, 1.5×10{circumflex over ( )}5 cells/well, 96-well format

Procedures: (in Order of Execution):

[0098] 1. Pre-treatment for 1 hour with experimental compounds.
2. LPS stimulation (2 ng/mL) for 4h of all conditions except unstimulated (LPS/ATP unstimulated) cells
3. ATP (5 mM) stimulation for 5 minutes of all conditions except unstimulated (LPS/ATP unstimulated) cells
4. Material collection: Supernatant and cell lysates (snap frozen, stored at −80° C.)

Results

[0099] IL-1β secretion in LPS/ATP stimulated, PMA differentiated THP-1 cells.
FIG. 1 shows the results of an assay with PMA differentiated THP-1 cells which were LPS/ATP stimulated. It can be seen that the increasing concentrations of the test compounds: 2,2′,4′-trihydroxychalcone; Isoliquiritigenin; Resveratrol inhibited the production of IL-1β in these cells. Rapamycin had no effect.
The test compounds inhibit the secretion of IL-1β by PMA differentiated THP-1 cells, that are stimulated with LPS/ATP, in a dose dependent fashion. The IC50 of the tested compounds is: [0100] 6.1 μM for 2, 2′, 4′-trihydroxychalcone [0101] 11.9 μM for Isoliquiritigenin [0102] 87.3 μM for Resveratrol
Since IL-1β secretion, under these conditions, is the result of activation of inflammasomes it can be concluded that the test compounds inhibit inflammasome induced IL-1β production. 2,2′,4′-trihydroxychalcone was the most effective inhibitor. Furthermore, since inhibition of mTOR by rapamycin did not inhibit inflammasome induced IL-1β production it can be concluded that these two processes can operate through independent mechanisms.
Unstimulated PMA differentiated THP-1 cells did not produce IL-1β above the detection limit of the procedure (4 pg/ml).
IL-18 secretion in LPS/ATP stimulated, PMA differentiated THP-1 cells
FIG. 2 shows the results of an assay with PMA differentiated THP-1 cells which were LPS/ATP stimulated.
2,2′,4′-trihydroxychalcone inhibits the secretion of IL-18 by PMA differentiated THP-1 cells, that are stimulated with LPS/ATP, in a dose dependent fashion. Since, under these conditions, IL-18 secretion is the result of activated inflammasomes, it can be concluded that 2,2′,4′-trihydroxychalcone inhibits inflammasome induced IL-18 production

[0103] Measurement of mTOR and autophagy activity by determination of the phosphorylation status of p70-56K in LPS/ATP stimulated and unstimulated PMA differentiated THP-1 cells.

[0104] The compounds 2,2′,4′-trihydroxychalcone; Isoliquiritigenin and rapamycin were added in various concentrations to LPS/ATP stimulated and unstimulated PMA-differentiated THP-1 cells.

[0105] The results show that the test compounds lead to dephosphorization the P70 S6K protein in a dose dependent fashion, both in PMA differentiated THP-1 cells, that are stimulated with LPS/ATP or in unstimulated PMA differentiated THP-1 cells. Rapamycin 25 nM was used as positive control. Inhibition in both unstimulated and stimulated cells is indicative for an increase in autophagy. Since inhibition was seen in both unstimulated and stimulated cells it is clear that mTOR regulated autophagy acts independently from the processes involved in inflammasome formation.

[0106] Since inhibition of mTOR leads to dephosphorization of the P70 S6K protein and subsequently to an upregulation of autophagy activity it can be concluded that the test compounds inhibit mTOR activity and upregulate autophagy. The IC50 of the test compounds in LPS/ATP stimulated and PMA differentiated THP-1 cells is found to be 98.8 μM for 2,2′,4′-trihydroxychalcone and 174.2 μM for Isoliquiritigenin The compound of formula I is thus a more active inhibitor of mTOR than Isoliquiritigenin.

Determination of Caspase-1 Levels in LPS/ATP Stimulated, PMA Differentiated THP-1 Cells

[0107] LPS/ATP stimulated, PMA differentiated THP-1 cells were incubated with a compound of formula I and caspase-I formation was analyzed and compared to a control without the compound. The result of a western blot for caspase-I is indicated in FIG. 4.

[0108] The results show that 2,2′,4′-trihydroxychalcone inhibits caspase-1 production in LPS/ATP stimulated, PMA differentiated THP-1 cells. Since, under these conditions, caspase-1 production is the result of activated inflammasomes, it can be concluded that 2,2′,4′-trihydroxychalcone inhibits inflammasome induced caspase-1 production.

Example 2

Materials and Methods

[0109] In this study the N2, bristol (wild-type) strain was used. Nematodes were maintained at 15° C. on nematode growth medium (NGM) seeded with Escherichia coli feeding strain 0P50.
For the treatment group, 2,2′,4′-trihydroxychalcone was dissolved in DMSO (final concentration 50 μM) and added to the NGM medium and the OP50 feeding suspension. For the control group, DMSO was added to the NGM medium and OP50 feeding suspension. Both the 2,2′,4′-trihydroxychalcone and the control plates contained a final DMSO concentration of 0.3% (v/v) during the whole experiment. For the aging assay, synchronous populations were obtained by allowing 5-10 hermaphrodites to lay eggs for 4 h. Lifespan scoring was initiated after hermaphrodites completed the final larval molt (day 1 of experiment). The starting number of nematodes was 100 per group. During the reproductive period, adult nematodes were transferred daily to new treatment plates to avoid overcrowding. Following post-reproduction, transfer occurred every third day, until the impact of aging disallowed handling of the nematodes. Survival was scored as the number of animals responsive to gentle touch as a fraction of the original number of animals on the plate.

[0110] The results of the experiment are presented in FIG. 5.

Example 3

[0111] Methods to formulate a chalcone as indicated herein.

1. Nano Particle Formulation

[0112] at least one emulsifier is dissolved in water. The emulsifier can for instance be a polysaccharide (gum ghatti), a lecithin, an ester of monoglyceride and a fatty acid, a mono- or diglycerides of a fatty acids. [0113] 2,2′,4′-trihydroxychalcone powder (crystals) are mixed into the solution. Subsequently an alcohol (for example glycerin or butanediol) is added to the emulsion. The size of the 2,2′,4′-trihydroxychalcone powder (crystals) in the emulsion is milled to on average <1 μm with a wet mill grinding (DYNO-MILL® KDL, Willy A Bachofen AG), high pressure dispersion or sonification. This will generate a stable nanosolution of 2,2′,4′-trihydroxychalcone, which contains 1-25% 2,2′,4′-trihydroxychalcone, 20-80% alcohol (glycerin) 0.5-15% emulsifier (gum ghatti) and 10-70% water.

2. Niosome Entrapment Formulation

[0114] Niosomes are nonionic surfactant-based vesicles with a similar structure to that of liposomes and can carry both hydrophilic and hydrophobic drugs within the same system.

Example Preparation of Spray-Dried Niosomes

[0115] A surfactant mixture (Tween 80 and Span 80, (ratio (mol/mol) 1:0.01-0.01:1) and cholesterol (ratio (mol/mol) 1:0.5-1:1) was dissolved in 80 mL of methanol/dichloromethane (ratio 4:1-2:1, v/v). The solvent was evaporated at 37° C. under vacuum by a rotary evaporator. The resulting dried film was redissolved in 60 mL of ethyl ether, and a solution containing 2,2′,4′-trihydroxychalcone in a mixture of dehydrated alcohol and phosphate-buffered solution (pH 6.5) was then added (ratio 2,2′,4′-trihydroxychalcone: carriers (w/w) 4:100-25:100) Next, 20 mL of phosphate-buffered solution was added after 10 minutes of sonication. Rotary evaporation was performed again at 60° C. until hydration was achieved and the residual ethyl ether was removed. The niosomal suspension was left to mature overnight at 30° C. in a thermostatic water bath shaker. In order to obtain a uniform particle size, the niosomal suspension was homogenized using a high pressure homogenizer

Preparation of Niosomal Powder

[0116] Powder-derived niosomes are superior to conventional niosomes in terms of convenience of storage, transport, and dosing. To prevent degradation, fusion, and leakage, the niosomal powder was prepared using spray-drying method. Mannitol was added into the niosomal suspension as a protectant to prevent drug leakage upon dehydration using each drying method. Six grams of mannitol was added into 100 mL of niosomal suspension before the drying method was performed.

Spray-Drying Method

[0117] The spray-drying process was done using a spray-dryer. The niosomal suspension was fed to the spray chamber by a pump. The aspirator setting, airflow rate, inlet temperature, and speed of the pump were kept at the scale of 100%, 357 L/hour, 130° C., and 1.5 mL/minute, respectively. Finally, the resulting powder was separated from the hot air stream with cyclone and collected in the bottom of the chamber.

Food Product

[0118] 2,2′,4′-trihydroxychalcone is added to a dairy product, for example milk, yoghurt or butter. Final concentration between 0.01 and 2.5%. [0119] 2,2′,4′-trihydroxychalcone is added to a beverage for instance tea, coffee, a herbal drink or an energy drink
The 2,2′,4′-trihydroxychalcone is preferably added as a freeze dried powder.
Hydroxychalcone (nano) particles are preferably provided with a coating that dissolves after ingestion. In other words a coating that dissolves, disintegrates and/or disrupts after the material has been swallowed. The coating is preferably a gel coating or an enteric coating.