Method for Inhibiting Cellular Na+-K+ ATPase Activity

Abstract

The present invention discloses an inhibitive effect of Na+-K+-ATPase caused by a compound selected from the group consisting of magnesium lithospermate B (MLB), isomer, prodrug, derivative, pharmaceutically acceptable salt, and a composition thereof. In this invention, the variations of Na+-K+-ATPase activity were monitored with increasing MLB concentrations, and the result shows the Na+-K+ ATPase activity is repressed by MLB. An outcome of the inhibitory effect, the function of cellular sodium/potassium lo exchanger is reduced and cellular calcium ion concentration is increased. That is, the MLB is useful for inhibiting the function of cellular Na+-K+ pump, and further brings the utility for cardiac stimulation, diuretic enhancement, heart failure curing, and so on.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, appended claims, and accompanying drawings where:

FIG. 1 is a diagram of measured Na⁺-K⁺ ATPase activity of a rat brain cortex treated with various MLB concentrations.

FIG. 2 is another diagram of measured Na⁺-K⁺ ATPase activity of a rat brain cortex treated with various MLB or ouabain concentrations.

FIG. 3 shows two molecular structures of MLB and cardiac glycoside.

FIG. 4 is a diagram of measured Na⁺-K⁺ ATPase activity of a rat brain cortex treated for various MLB concentrations.

FIG. 5 is another diagram of measured Na⁺-K⁺ ATPase activity of a rat myocardium cell membrane treated for various MLB concentrations.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to the present preferred embodiments of the invention, examples of which are illustrated in the lo accompanying drawings. Wherever possible, the same reference numbers are used in the drawings and the description to refer to the same or like parts.

In the present invention, the action of MLB as an innovative cellular Na⁺-K⁺ ATPase inhibitor is disclosed. FIG. 1 is a diagram of measured Na⁺-K⁺ ATPase activity of rat brain cortex treated for various MLB concentrations, which shows the inhibitory effect of increasingly MLB concentration. Results are determined using the following steps:

First, Male Sprague-Dawley (NarII: SD) rats (3-month-old) were purchased from National Laboratory Animal Center (Nankang, Taipei) and raised under specific pathogen-free conditions. Animals were provided with rat chow (Rodent Laboratory Chow 5001, Purina, Mo.) and tap water throughout the studies. The rats received humane care in accordance with the guidelines of a guidebook for the care and use of laboratory animals. The animals were sacrificed by decapitation, and the brain and heart organs of the rats were removed immediately after complete exsanguination.

Next, the plasma membrane was isolated from the rat brain and heart at 4° C. The brain and heart homogenate were prepared respectively with homogenized plasma membrane in 10-20 volumes of 0.32 mM sucrose solution containing 5.0 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 1.0 mM EDTA, pH 7.5. Then, the brain or heart homogenate was centrifuged at 1000×g for 10 min, and kept the resultant supernatant for further centrifuge at 17000×g for 30 min to obtain a crude plasma membrane fraction.

Then, the fraction was washed and suspended twice in 0.32 M sucrose HEPES-buffer, which was subjected to a discontinuous sucrose density gradient consisting of successive layers of 0.3, 0.8 and 1.0 mM, and centrifuged at 63000×g for 1 hour. The plasma membrane was collected at the interface between 0.8 and 1.0 mM sucrose to be further suspended in 0.32 M sucrose solution for enzyme assays within 2 hours.

Na⁺-K⁺ ATPase activity was determined by measuring the amount of inorganic phosphate (Pi) liberated from ATP hydrolysis.

First, commercial Na⁺-K⁺ ATPase from porcine cerebral cortex (Sigma, 0.3 units/mg) or a purified plasma membrane fraction incorporated into a reaction mixture of 1 ml containing 3 mM ATP, 5 mM MgCl₂, 80 mM NaCl, 20 mM KCl, and 40 mM Tris-HCl, pH 7.4, was prepared. The enzymatic reaction was terminated 15 min after Na⁺-K⁺ ATPase incorporation by adding 200 μl of 30% (W/V) trichloroacetic acid.

After centrifugation at 6000 rpm for 15 min, supernatant of 500 μl measured the inorganic phosphate using spectrophotometric methods. Enzyme activity was expressed as μmol Pi liberated from ATP by 1 mg of Na⁺-K⁺ ATPase during 1 hour. Protein content was quantified using a Bradford protein assay kit (Sigma). For the observation of inhibitory effects on Na⁺-K⁺ ATPase activity, ouabain or MLB of various concentrations was incubated with commercial Na⁺-K⁺ ATPase or the purified plasma membrane fraction at 37° C. for 10 min prior to incorporation into the reaction mixture.

Reference is made to FIG. 1, which shows the inhibitory effect of Na⁺-K⁺ ATPase activity with increasing MLB concentration. The X-axis represents the variations of MLB concentration incorporated with the reaction mixture, and the Y-axis represents the variations amount of inorganic phosphate (Pi) liberated from ATP hydrolysis. As shown as FIG. 1, the measured free inorganic phosphate (Pi) decreases with increased MLB concentration showing the ATPase inhibition effect is dependent on raised MLB dosage. According to the proportion phenomenon the MLB is a Na⁺-K⁺ ATPase inhibitor.

The results of FIG. 1 disclose MLB provides a mechanism similar to the cardiac glycoside. FIG. 1 shows MLB represses the Na⁺-K⁺ ATPase activity. Accordingly, the Na⁺-K⁺ ATPase activity is depressed when treated with different MLB concentration. MLB activity Na⁺-K⁺ ATPase activity depression is compared with a cardiac glycoside, ouabain. As shown in FIG. 2, the inhibitory behavior of MLB is consistent with the ouabain, and the amount of free inorganic phosphate (Pi) is dependent on the raised inhibitor (MLB or ouabain) dosage.

Reference is made to FIG. 3, which shows the molecular structures of the MLB and a cardiac glycoside. The molecular structure of cardiac glycoside 310 consists of a steroid core and at least one glycoside group. The molecular structure of MLB 320 is a compound with a metal ion located in the center of the MLB structure. In consideration, according to the results shown in FIG. 2, the inhibitory behavior of MLB is consistent with ouabain, and the enzyme activation is a “key and lock” model. MLB conformation plays a similar role as cardiac glycoside in inhibiting Na⁺-K⁺ ATPase activity. That is, MLB conformation is similar to cardiac glycoside and inhibits the Na⁺-K⁺ ATPase activity by way of inter-molecule affinity (secondary bond), such as electrostatic bond, hydrogen bond, hydrophobic bond or van der Waals bond.

According to the preferred embodiment of the present invention, the central MLB metal ion is a two-valence metal cation, such as magnesium, iron, manganese, calcium, zinc, copper or cobalt. Furthermore, the functional group (side chain) “R” of the MLB 320 comprises hydrogen, hydroxyl group, alkane, alkene, alkyne, aromatic group, glycosyl group or combined thereof.

References are made to FIG. 4 and FIG. 5, which show the inhibitory effect of the Na⁺-K⁺ ATPase activity of MLB in cardiac and cortex cell membrane, respectively. FIG. 4 is the result of Na⁺-K⁺ ATPase activity of brain cortex cells treated with various MLB concentrations. The X-axis represents the variations MLB concentration incorporated with the reaction mixture, and the Y-axis represents the variations amount of inorganic phosphate (Pi) liberated from ATP hydrolysis. The result of FIG. 4 shows the measured free inorganic phosphate (Pi) decreasing with increased MLB concentration showing Na⁺-K⁺-ATPase activity is repressed by MLB, and the ATPase inhibition effect is dependent on raised MLB dosage. Hence, the MLB is able to inhibit the Na⁺-K⁺ ATPase activity of the cortex cell membrane.

FIG. 5 is another result of Na⁺-K⁺ ATPase activity of cardiac cells membrane treated with various MLB concentrations that shows a consistent result with FIG. 4. The X-axis represents the variations in MLB concentration incorporated with the reaction mixture, and the Y-axis represents variations in the amount of inorganic phosphate (Pi) liberated from ATP hydrolysis. The result of FIG. 5 shows the measured free inorganic phosphate (Pi) decreases with increased MLB concentration indicating Na⁺-K⁺-ATPase activity is repressed by MLB, and the ATPase inhibition effect is dependent on raised MLB dosage. Hence, MLB is able to inhibit the Na⁺-K⁺ ATPase activity of cardiac cell membranes.

According to the results of FIG. 1-5, the MLB is able to inhibit the Na⁺-K⁺ ATPase activity of cardiac cell membrane, and therefore bring the physical effects to reduce the function of the cellular sodium/potassium exchanger and increases cellular calcium ion concentration. For this reason, the MLB provides an identical mechanism as the cardiac glycoside, that is MLB stimulates the systole for the purpose of cardiac stimulation and another purpose of diuretic enhancement.

In accordance with the preferred embodiment of the present invention, the MLB is applied to treat disease selected from a group consisting of congestive heart failure (CHF), arrhythmia (such as atrial fibrillation, atrial flutter, and paroxysmal tachycardia), hypertension, edema, coronary heart disease (such as angina pectoris and myocardial infarction) and diseases related to the foregoing disease.

Consequently, an effective dosage of MLB provides a utility for cardiac stimulation and diuretic enhancement that is equivalent to the cardiac glycoside mechanism and contributes to an alternative medicine different from cardiac glycoside to develop a pharmaceutical agent or functional food. For example, it can be used to produce an active pharmaceutical ingredient or dietary supplement

In accordance with the preferred embodiment of the present invention, the structure shown in FIG. 3, named MLB 320, represents the MLB and derivatives thereof. Wherein, the MLB and derivatives thereof comprise the isomer, prodrug, and pharmaceutical acceptable salt thereof.

In another preferred embodiment of the present invention, a composition that comprise the compound structure of MLB 320 as an active principle is used to repress the cell membrane's Na⁺-K⁺ ATPase activity. Wherein, the active principle of the composition comprises of pharmaceutically acceptable salt, solvate, solvate of the pharmaceutically acceptable salt, polymorphism, and a prodrug of the MLB 320. Furthermore, the composition further comprises a pharmaceutical/food acceptable carrier, such as pharmaceutical/food acceptable assisting agent, thinner, excipient, or combination thereof. The MLB and the original herb “Danshen” and extract thereof, can be used to produce an active pharmaceutical ingredient or dietary supplement.

Moreover, the above-mentioned has shown the inhibitory effect of MLB on Na⁺-K⁺ ATPase activity. The cardiac stimulation and diuretic enhancement function of MLB and derivates thereof are equivalent to the cardiac glycoside. The present invention discloses that MLB is an alternative medicine differing from cardiac glycoside without the danger of toxication.

Although the present invention has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible. Therefore, the spirit and scope of the appended claims should not be limited to the description of the preferred embodiments contained herein.

It will be apparent to those skilled in the art that various modifications and variations can be made to the structure of the present invention without departing from the scope or spirit of the invention. In view of the foregoing, it is intended that the present invention cover modifications and variations of this invention provided they fall within the scope of the following claims and their equivalents.

Claims

1. A method to inhibit cellular Na+-K+ ATPase activity comprising of: administering a magnesium lithospermate B (MLB) and derivatives thereof to animal cells, which represented by following formula:

2. The method of claim 1, wherein the metal cation comprises magnesium, iron, manganese, calcium, zinc, copper or cobalt.

3. The method of claim 1, wherein the functional group comprises hydrogen, hydroxyl group, alkane, alkene, alkyne, aromatic group, glycosyl group or combined thereof.

4. The method of claim 1, wherein the MLB derivatives comprise a isomer, prodrug, pharmaceutically acceptable salt, and composition thereof.

5. The method of claim 4, wherein the pharmaceutically acceptable salt comprises magnesium salt, potassium salt, ammonium salt, or calcium salt.

6. The method of claim 1, wherein the ATP hydrolysis repressed by administering MLB and derivatives thereof as an effective dosage sufficient for inhibiting the Na+-K+ ATPase activity.

7. A composition for repressing the cell membrane's Na+-K+ ATPase activity, which comprises the compound structure cited in claim 1 as an active principle.

8. The composition of claim 7, wherein the active principle comprises pharmaceutical acceptable salt, solvate, solvate of the pharmaceutical acceptable salt, polymorphism, and prodrug of said compound.

9. The composition of claim 7, wherein the composition further comprises a pharmaceutical/food acceptable carrier.

10. The method of claim 9, wherein the pharmaceutical/food acceptable carrier comprises pharmaceutical/food acceptable assisting agent, thinner, excipient, or combination thereof.

11. The composition of claim 7, wherein the composition is original herb Danshen, and extract thereof.

12. The composition of claim 7, wherein the composition is an active pharmaceutical ingredient.

13. The composition of claim 7, wherein the composition is a dietary supplement.

14. The composition of claim 7, wherein the composition is a cardiac stimulation agent.

15. The composition of claim 7, wherein the composition is a diuretic agent.

16. The composition of claim 7, wherein the composition, the original herb Danshen of the active principle is applied to treat diseases selected from a group consisting of: a) Congestive heart failure (CHF);b) Arrhythmia, which comprise atrial fibrillation, atrial flutter, and paroxysmal tachycardia;c) Hypertension;d) Edema;e) Coronary heart disease, which comprise angina pectoris, myocardial infarction and diseases related to the foregoing disease.

17. A pharmaceutical derivative with a steroid structure as a core, characterized in another compound substitute for the core, wherein the compound is represented by the following formula:

18. The method of claim 17, wherein the “M” represents a metal ion, and the “R” represents any functional group.

19. The method of claim 18, wherein the metal ion comprises two-valence metal cation.

20. The method of claim 18, wherein the functional group comprises hydrogen, hydroxyl group, alkane, alkene, alkyne, aromatic group, or combined thereof.