New use of carbamate ß phenylethanolamine analogues for enhancing intracellular clearance of LDL cholesterol and for combining therapy with statins to enhance the efficacy and reduce adverse effects

Abstract

The invention related to the use of carbamate-β-phenylethanolamine analogues for up-regulating of LDL receptors, facilitating uptake of extracellular LDL cholesterol and reducing intercellular total cholesterol. It also related to use of carbamate-β-phenylethanolamine analogues and statins or other lipid lowering agents as combined therapies for synergic effects in lowering LDL cholesterol as well as for reducing adverse effects.

Claims

1. Methods of use compounds of (±)-carbamate-β-phenylethanolamine analogues described in formula I and their active enantiomers as well as their pharmaceutically suitable salts in a pharmaceutical composition in the manufacture of a medicament for up-regulating the activities of LDL receptors and for facilitating the up taking, internalization and clearance of intercellular LDL cholesterol in cells and for combination therapeutics with statins or other lipid lowering agents to achieve synergistic efficacy or reduce toxicity or adverse effects to a patient in need. ##STR00005## Wherein A is selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl; B is selected from hydrogen or —CO—N(W)(X); W and X are independently selected from hydrogen, substituted or unsubstituted alkyl; C is selected from hydrogen or —CO—N(Y)(Z); Y and Z are independently selected from hydrogen, substituted or unsubstituted alkyl.

2. A method of claim 1, wherein said compounds is R-bambuterol presented in the structure formula II: ##STR00006## Wherein A is n-butyl, B is —CO—N(W)(X), W and X are methyl, C is —CO—N(Y)(Z), Y and Z are methyl according to Formula I.

3. A method of claim 1, wherein said compounds is R-mono-bambuterol presented in formula III: ##STR00007## 3-(2-(tert-butylamino)-1-hydroxyethyl)-5-hydroxyphenyl dimethylcarbamate bambuterol mono-carbamate (MONO) Wherein A is n-butyl, B is —CO—N(W)(X), W and X are methyl, C is hydrogen according to Formula I.

4. A method of claim 1, wherein the said compounds is Ethylating bambuterol Presented in the structure formula IV: ##STR00008## 5-(1-hydroxy-2-(tert-pentylamino)ethyl)-1,3-phenylene bis(ethyl(methyl) carbamate) Wherein A is tert-pentyl, B is —CO—N(W)(X), W is methyl, X is ethyl, C is —CO—N(Y)(Z), Y is methyl, Z is ethyl according to Formula I.

5. A method of claim 1, wherein the said up-regulating activities comprise of increased expression of the receptors, increased binding of LDLC-LDL receptors and increased internalization of LDLC-LDL receptor complexes from cellular spaces into cells.

6. A method of claim 1, wherein the said clearance is reduced synthesis of LDL cholesterol.

7. A method of claim 1, wherein the said clearance is increased metabolize of LDL cholesterol.

8. A method of claim 1, wherein the said clearance is secretion of cholesterol in the form of bile acids.

9. A method of claim 1, wherein, the said cells is hepatic cells, muscle cells, smooth muscle cells, cardiac cells, endothelia cells, kidney cells, retina cells, neuronal cells, glia cells, macrophages and other cells which are capable of uptake or synthesis LDL cholesterol.

10. A method of claim 1, wherein, the said up-regulating activities of LDL receptors or clearance of LDL cholesterol involve an inhibition of butyric cholinesterase.

11. A method of claim 1, wherein, the said reduced toxicity involves an inhibition of butyric cholinesterase.

12. A method of claim 1, wherein, the said lipid lowering agents are statins.

13. A method of claim 12, wherein wherein the statin is selected from the group consisting of: atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin.

14. A method of claim 1, wherein, the lipid lowering agents are cholesterol absorption or transport inhibitors selected from the group consisting of Ezetimibe, Gemfibrozil, Fenofibrate acid (fibrates), Nicotinic acid, Cholestyramine or colestipol (resins), Cholesterol ester transfer protein (CETP) inhibitors.

15. A method of claim 1, wherein the said lipid lowering agents are PCSK 9 inhibitors selected from the group consisting of evolocumab, alirocumab, bococizumab and inclisiran.

16. A method of claim 1, wherein the combination therapy comprises of at least on of compounds of formula I and at least one lipid lowering agents according claim 1 for simultaneous, sequential or separate administration to a patient in need.

17. A method of claim 1, wherein the combination therapeutics comprise of at least one of compounds of formula I in the amount of 1-99% and at least one lipid lowering agents according claim I in the amount of 1-99%.

18. The method of claim 1, wherein the said pharmaceutical compositions are selected from the group consisting of tablets, capsules, granules, suppository, ointment, time-released dosage forms, skin patch, aqueous solutions and inhaled aerosol and for oral, topical, rector, vagina, parenteral injection, lung inhalation, nasal spray, or implant use.

19. The method of claim, wherein the pharmaceutical acceptable salts are selected from the groups of inorganic or organic acids consisting of hydrochloride, hydrobromide, sulphate, hydrogen sulphate, dihydrogen phosphate, methanesulphonate, bromide, methyl sulphate, acetate, oxalate, maleate, fumarate, succinate, 2-naphthalene-sulphonate, glyconate, gluconate, citrate, tartaric, lactic, pyruvic isethionate, benzenesulphonate or para-toluenesulphonate.

Description

EXAMPLES

Example 1

[0052] R-bambuterol increases expression of LDL receptors and facilitates up-taking of LDL-Cholesterol

[0053] Test Method:

[0054] Mouse liver AML12 cells were seeded in 24 well plates in DMEM/F12 and supplemented with 10% heat-inactivated fetal bovine serum. Cells were incubated with florescence labeled LDL to study the up-taking and metabolite of LDL cholesterol by the cells. LDL specific antibody were used to study LDL receptors expressed by the cells. Cells were divided into different groups and loaded with R-bambuterol (R-BM, Atorvastatin (Statin) or R-BM+Statin separately including: control, LDL, LDL+statin10 μM, LDL+R-BM 10 μM or 20 μM, LDL+R-BM10 μM+Statin10 μM LDL+R-BM20 μM+Statin20 μM groups. After 24 hours of treatments, the cells were washed and fixed with paraformaldehyde. Cells were then incubated with anti-LDLR primary antibody with 1:200 dilution at room temperature for 4 hours. After washed, the second antibody Alexa Fluor 488 with 1:200 dilutions was added and incubated at room temperature for 2 hours. The cells were washed again before examined under fluorescent microscope. The fluorescence anti-body bound LDL receptors in green and either bound or internalized fluoresces labeled LDL in red were examined separately or after merging. Fluorescent microscope equipped with a digital camera (Axio Observer 7). Computer software was used to quantify the intensity of fluorescence, which is correlated with the amount of LDL and LDL receptors bounded to antibodies.

Test Results:

[0055] 1) there are significantly more bound and intracellular fluorescence labeled LDL in groups treated with both 10 μM and 20 μM R-bambuterol in comparison of either control and LDL, LDL+Statin10 μM and LDL+statin20 μM. There is more bound and intracellular labeled LDL in R-bambuterol 20 μM than R-bambuterol 10 μM. This results indicate that R-bambuterol enhanced the transporting of LDL cholesterol into hepatic cells in a dose depend fashion. Hepatic cells is the major metabolic pathway for LDL cholesterol. Therefore, R-bambuterol facilitates the clearance of LDL from extracellular spaces or blood.

[0056] 2), The bound and intracellular fluorescence labeled LDL in Atorvastatin treated group is more than LDL alone group but less than R-bambuterol treated groups. This indicates that Atorvastatin can also facilitate the LDL transport into cell.

[0057] 3), The bound and intracellular fluorescence labeled LDL in group treated with combination of Statin and R-bambuterol are significantly more than either R-bambuterol or statin along treated groups. This results indicate a synergistic effects of R-bambuterol and Statin in LDL transporting into cells.

[0058] 4), By quantification of the fluorescence intensity in randomly selected areas in the fields under microscope, the amount of LDL receptors in each group can be quantified as list in table below. LDL receptors were significantly unregulated with LDL loaded media but there is further increases of LDL receptors in R-bambuterol treated groups. The increases in LDL receptors expression in R-bambuterol treated cells was twice as much as seen in statin treated groups. These results indicate that R-bambuterol can up-regulate the expression of LDL receptors and it is more potent than statins in this regard.

[0059] However, the effects of statin were greatly enhanced when combined with R-bambuterol in comparison of statin alone.

TABLE-US-00001 TABLE 1 Up-regulating of LDL receptors by R-BM and Statin in Hepatic cells (Intensity of fluoresces) Treatment (μM) MEAN % SD control 100.0 2.8 R-BM 10 202.9 3.7 R-BM 20 337.2 4.9 Statin 10 159.1 5.4 Statin 20 140.2 7.1 R-BM20 + Statin20 226.5 7.3

Example 2

[0060] R-bambuterol enhances the clearance or turnover LDL cholesterol and has synergistic effects with statin.

[0061] Human liver HepG2 cells were seeded in six well plates in DMEM/F12 supplemented with 10% heat-inactivated fetal bovine serum. Cells were then treated with different doses of R-bambuterol (R-BM) in the present of LDL (40 μg/ml). After 24 hours of incubation, total intracellular and extracellular cholesterol content were determined using Total Cholesterol Assay Kit E105 (Applygen Technologies Inc., Beijing, China) according to the manufacturer's protocol.

[0062] 1) Enhancement of Clearance of Intracellular LDL Cholesterol

[0063] The intracellular cholesterol is similar in control and LDL groups. Neither statin of 10 μM nor R-bambuterol of 10 μM had any effects on the levels of intracellular cholesterol. However, in another experiment, mouse liver AML12 cells were treated with R-BM 10 μM and Statin 10 μM were used separately or in combination in the present of LDL (40 μg/ml), similar cell culture media and measurement methods of cholesterol were used.

Test Results

[0064] There was a significant reduction of intracellular cholesterol when the same dose of R-bambuterol and Atorvastatin were added in combining. These indicate synergistic effects on the clearance or turnover of intracellular LDL cholesterol by the combining treatment of R-bambuterol and Atorvastatin.

TABLE-US-00002 TABLE 2 Reduction of intercellular LDL cholesterol by R-bambuterol Treatment (μM) MEAN % SD LDL Control 100 LDL + RBM 5 94.07 18.3 LDL + RBM 10 69.26 13.6 LDL + RBM 20 28.54 3.5 LDL + RBM 40 36.04 12.2 LDL + RBM 80 21.02 10.7

2) Synergistic Effects of R-Bambuterol and Statin in Reduction of Cholesterol

[0065] Unlike in HepG2 cells, R-BM 10 μM induce less reduction of intercellular cholesterol in Mouse liver AML12 cells. Similarly, Atorvastatin 10 μM also showed little effects on intercellular cholesterol. However, there was a significant more reduction in total cholesterol when cells were treated with a combination of both R-BM and Atorvastatin (Statin) at the same doses above. The reduction effects is more than an addition of both, therefore, there is a synergy for R-BM and Statin in the clearance of intracellular LDL cholesterol.

TABLE-US-00003 TABLE 3 Synergistic Effects of Combining R-bambuterol and Statin Treatment (μM) MEAN % SD LDL Ctrl 100.0 1.1 LDL + Statin 10 95.3 7.8 LDL + RBM 10 94.5 10.6 LDL + Statin10 + RBM10 70.8 8.3

Examples 3

[0066] Protective effects of R-bambuterol against the toxicities induced by statin.

Test Method

[0067] Either HepG2 cell or Pulmonary artery smooth muscle cell (PASMC) were seeded evenly in 96-well plates. when cells grow about 50%, culture medium was replaced with FBS-free medium and Atorvastatin (Statin) or/and R-bambuterol (R-BM). Cells were cultivated for 24 hours at room temperature. The cells then replaced again with FBS-free medium containing 10% CCK-8 reagent and cultivated for 3 hours in the dark.

[0068] A Multi-function micro plate reader (TriStar2S LB942) was used to measure the cell viability.

[0069] Cells added no drugs (0 μM) were used as control. The viabilities from R-BM or statin or both were normalized as percent of control. For the combination of statin and R-BM treatment, R-BM was 20 μM for all groups except in control (OpM) in which no R-BM was added.

Test Results

1) Hepatic Cells

[0070] R-bambuterol showed no toxicity to the viability of hepatic cells (HepG2) at all doses. However, Statin has significant toxic effects at initial dose of 5 μM, the viability of cells was inhibited by Statin in a dose-dependent fashion. The toxicity of statins was significantly reduced by combing with R-bambuterol, and completely abolished at lower dose of statin groups.

TABLE-US-00004 TABLE 4 Effects of R-bambuterol and Atorvastatin on the Viabilities of Hepatic cells (Percent of control %) Treatment 0 μM 5 μM 10 μM 20 μM 40 μM 80 μM Statin 100 ± 9 81 ± 9 77 ± 8 76 ± 6 67 ± 1 51 ± 5 R-BMB 100 ± 7 103 ± 13  98 ± 12 105 ± 12 103 ± 7  101 ± 7  Statin + R-BMB(20 μM) 100 ± 5 98 ± 8 102 ± 4  93 ± 3 85 ± 5 63 ± 4

2) Pulmonary Artery Smooth Muscle Cell

[0071] R-bambuterol showed no toxicity to the viability of pulmonary artery smooth muscle cell (PASMC), PASMC seems more tolerable to Atorvastantin than HepG2 cells. However, there were also a dose-dependent inhibition of cell viability of PASMC when exposed to statin. Similarly, these toxicity of statins were significantly reduced by combining with R-bambuterol as in HepG2 cells.

TABLE-US-00005 TABLE 5 Effects of R-bambuterol and Atorvastatin on the Viabilities of PASMC cells (Percent of control %) Treatment 0 μM 5 μM 10 μM 20 μM 40 μM 80 μM Statin 100 ± 6  92 ± 8  93 ± 2 88 ± 3 79 ± 2 68 ± 2 Statin + 100 ± 4 108 ± 2 100 ± 4 99 ± 6 84 ± 2 72 ± 3 R-BM(20 μM)