COMPOUNDS THAT ENHANCE THE ACTION OF METFORMIN

Abstract

The invention relates in general to a medicament comprising Metformin or a salt thereof; and a compound having the general formula (I) or a salt thereof. The invention further relates to a pharmaceutical composition comprising the medicament for use in the prevention or treatment of a metabolic disease or a cardiovascular disease.

Claims

1. A medicament comprising (i) Metformin; and (ii) a compound having the general formula (I) ##STR00003## wherein, in compound (ii), R1, R2, R3, R4, R5, R6, R7, and R8 are each independently H; OH; OMe; O-glycoside; a halogen; an aldehyde; a carboxylic acid; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; and a sulfate.

2. The medicament according to claim 1, wherein R1, R2, R3, R4, R7, and R8 are each independently H; OH; OMe; O-glycoside; a halogen; an aldehyde; a carboxylic acid; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; R5 is H; OMe; O-glycoside; a halogen; an aldehyde; a carboxylic acid; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; O-glycoside; a halogen; an aldehyde; a carboxylic acid; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; and a sulfate.

3. The medicament according to claim 1, wherein R1, R2, R3, R4, R7, and R8 are each independently H; OH; OMe; O-glycoside; a halogen; an aldehyde; a carboxylic acid; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; R5 is H; OMe; O-glycoside; a halogen; an aldehyde; a carboxylic acid; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; a sulfate; R6 is H; OH; O-glycoside; a halogen; an aldehyde; a carboxylic acid; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; and a sulfate in compound (ii).

4. The medicament according to claim 1, wherein R1, R3, and R8 are each independently H; OH; OMe; O-glycoside; a halogen; an aldehyde; a carboxylic acid; R2 is OH; OMe; O-glycoside; a halogen; an aldehyde; a carboxylic acid; R4 is H; OH; O-glycoside; a halogen; an aldehyde; a carboxylic acid; R5 is H; OMe; O-glycoside; a halogen; an aldehyde; a carboxylic acid; R6 is H; OH; O-glycoside; a halogen; an aldehyde; a carboxylic acid; and R7 is H; OH; O-glycoside; a halogen; an aldehyde; and a carboxylic acid in compound (ii).

5. The medicament according to claim 1, wherein R1, R3, and R8 are each independently H; OH; OMe; R2 is OH; OMe; R4 is H; OH; R5 is H; OMe; R6 is H; OH; and R7 is H; and OH in compound (ii).

6. The medicament according to claim 1, wherein R1, R3, R6 and R8 are each independently H; R2 is OH; OMe; R4 is OH; R5 is H; OMe; and R7 is H; and OH in compound (ii).

7. The medicament according to claim 1, wherein compound (ii) is Lusianthridin (7-Methoxy-9,10-dihydrophenanthrene-2,5-diol, CAS number 87530-30-1) and has the formula ##STR00004##

8. The medicament according to claim 1, wherein Metformin or a salt thereof, and a compound of general formula (I), or a salt thereof are combined in a single preparation.

9. The medicament according to claim 1, wherein Metformin or a salt thereof, and a compound of general formula (I), or a salt thereof are separately formulated into compositions and used in combination.

10. A pharmaceutical composition comprising a medicament comprising (i) Metformin; and (ii) a compound having the general formula (I) ##STR00005## wherein, in compound (ii), R1, R2, R3, R4, R5, R6, R7, and R8 are each independently H; OH; OMe; O-glycoside; a halogen; an aldehyde; a carboxylic acid; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; and a sulfate, in a pharmaceutically acceptable form.

11. The pharmaceutical composition of claim 10, for use in the prevention or treatment of a metabolic disease or a cardiovascular disease.

12. The pharmaceutical composition according to claim 11, wherein the metabolic disease is diabetes.

13. The pharmaceutical composition according to claim 12, wherein the prevention or treatment of diabetes is by increasing the inhibition of lipogenesis.

14. The pharmaceutical composition according to claim 13, wherein the prevention or treatment of diabetes is by increasing the inhibition of lipogenesis in liver cells and improving insulin sensitivity by activation of AMP-activated protein kinase (AMPK).

15. The pharmaceutical composition according to claim 9, wherein metformin is used at a sub-pharmalogical dose for the subject.

16. The pharmaceutical composition according to claim 15, wherein the sub-pharmacological dose is not more than about 50% of the standard pharmacological dose for the subject.

17. The pharmaceutical composition according to claim 14, wherein the subpharmacological dose of metformin reduces the incidence of side effects associated with metformin use, wherein the side effects are lactic acidosis, and/or gastro-intestinal side effects, particularly diarrhea, nausea, and vomiting.

18. A method of preventing or treating a metabolic disease or a cardiovascular disease comprising the administration of a medicament comprising (i) Metformin; and (ii) a compound having the general formula (I) ##STR00006## wherein, in compound (ii), R1, R2, R3, R4, R5, R6, R7, and R8 are each independently H; OH; OMe; O-glycoside; a halogen; an aldehyde; a carboxylic acid; a primary, secondary, or tertiary amine; a primary or secondary amide; a cyano; a nitro; a sulfonate; and a sulfate to a subject in need thereof.

19. The method according to claim 18, wherein the disease is diabetes.

Description

BRIEF DESCRIPTION OF FIGURES

[0118] FIG. 1. AMP and Lusianthridin activation of bacterially-expressed AMPKα2β1γ1. Lusianthridin (7-Methoxy-9,10-dihydrophenanthrene-2,5-diol, CAS number 87530-30-1) in combination with AMP result in additive activation of AMPK. AMP binding through the AMPKγ subunit causes a dose-dependent increase in AMPK activity and addition of various concentrations of Lusianthridin cause an additive activation of AMPK, without affecting the EC50 of AMP activation.

[0119] FIG. 2. Low concentrations of Lusianthridin alone and Metformin alone, cause an inhibition of lipogenesis. There is a further increase in lipogenesis when these two activators are used in combination.

EXAMPLES

Example 1

[0120] Lusianthridin Causes an Additive Effect on AMP Activation of Activate Bacterially-Expressed AMPKα2β1γ1 Complex.

[0121] The AMPK heterotrimers were expressed in bacteria and purified through the His-α subunit by nickel purification, further purified through gel filtration and finally phosphorylated by incubation with CaMKKβ. This phosphorylated AMPK was incubated with varying concentrations of AMP for 30 mins using substrate and reagents from the HTRF-KinEASE Cisbio assay kit (STK S1 Kit). Phosphorylation of the substrate was measured by incubating with donor and acceptor antibodies for 2 h at room temperature as per the manufacturer's protocol (and Coulerie et al., (2016), see below) and phosphorylated peptide detected by performing HTRF. The 665 nm/620 nm ratio was determined and displayed in the graph. In the presence of fixed concentrations of Lusianthridin, there was an additive activation of AMPK by AMP.

[0122] Reference 1: Standardized LC×LC-ELSD Fractionation Procedure for the Identification of Minor Bioactives via the Enzymatic Screening of Natural Extracts. Coulerie P, Ratinaud Y, Moco S, Merminod L, Naranjo Pinta M, Boccard J, Bultot L, Deak M, Sakamoto K, Queiroz E F, Wolfender J L, Barron D. J Nat Prod. 2016 Nov. 23; 79(11):2856-2864. Epub 2016 Oct. 28.

Example 2

[0123] Lusianthridin and Metformin have an Additive Effect on Inhibition of Lipogenesis in Primary Hepatocytes.

[0124] Hepatocyte isolation: The liver was first perfused with 50 ml perfusion buffer (Krebs-Hepes buffer with 0.5 μM EDTA), followed with 50 ml collagenase A buffer (Krebs-Hepes buffer with 5 mM CaCl.sub.2) and 0.5 mg/ml collagenase). After passage through a 100 μm mesh, the cell solution was washed several times with cold media and finally the cell culture pellet was resuspended in culture medium (medium 199 (M199)+GlutaMAX, 100 U/ml penicillin G, and 100 μg/ml streptomycin, 0.1% (wt/vol) BSA, 10% FCS, 10 nM insulin, 200 nM triiodothyronine and 500 nM dexamethasone). Hepatocytes were left to attach (3-4 h) and cultured overnight in M199 supplemented with antibiotics and 100 nM dexamethasone. Cells were used for experiments the following morning.

[0125] For lipogenesis measurements in primary hepatocytes, cells were seeded at 600 K cells per well in a 6-well plate overnight. Media was replaced with fresh M199 media alone for 2 hours prior to incubation with varying concentrations of Lusianthridin or compound 1 for 1 h at 37 C, in the presence of [1-.sup.14C]-acetate. The incorporation of [.sup.14C] into fatty acids was determined in the lower organic layer after separation from the aqueous phase. The results are displayed as the disintegrations per min (DPM) per μg of protein.

[0126] Lipogenesis is controlled by the AMPK substrate ACC, and phosphorylation and inhibition of ACC by AMPK, leads to a decrease in lipogenesis. Lipogenesis was measured in primary hepatocytes by determining the incorporation of .sup.14C-labelled acetate into fatty acids.

[0127] Lipogenesis was monitored in the presence or absence of a low/non-saturating concentration of Lusianthridin or metformin for 1 h at 37 C. Furthermore, cells were incubated in the presence of both of these compounds. The results shown in FIG. 2, displayed as the % lipogenesis (compared to control untreated cells), that Lusianthridin and metformin are able to inhibit lipogenesis. When combined, Lusianthridin and metformin cause a further inhibition of lipogenesis. These data suggest that when low concentrations of AMPK activators are combined, there is a greater inhibition of lipogenesis compared to the effect of these activators alone.