WO1997026894A1

WO1997026894A1 - Intravenous magnesium gluconate for treatment of conditions caused by excessive oxidative stress due to free radical distribution

Info

Publication number: WO1997026894A1
Application number: PCT/US1997/001237
Authority: WO
Inventors: William B. Weglicki
Original assignee: Weglicki William B
Priority date: 1996-01-25
Filing date: 1997-01-24
Publication date: 1997-07-31
Also published as: EP0923379A4; AU2246397A; US5843996A; CA2244117A1; US6100297A; JP2002515863A; EP0923379A1

Abstract

The intravenous use of magnesium gluconate to substantially block free radical surge in the treatment of ischemia/reperfusion (I/R) injury due to oxidative stress.

Description

INTRAVENOUS MAGNESIUM GLUCONATE FOR TREATMENT OF

CONDITIONS CAUSED BY EXCESSIVE OXIDATIVE STRESS DUE TO

FREE RADICAL DISTRIBUTION

BACKGROUND OF THE INVENTION

It is well established that excessive oxidative stress due to free radicals may

injure biological tissues. The natural defenses of cells and tissues revolve around

antioxidant mechanisms that have evolved to protect the cells and tissues against high

levels of oxidative stress. In our oxygen rich atmosphere the presence of oxygen at

certain times of stress may be injurious; this has been termed the oxygen paradox and

relates to the role of oxygen in generating and participating in free radical processes.

In certain disease states associated with periods of restricted blood flow to tissues,

such as heart attack, stroke and restricted flow to the extremities, mtermittent episodes

of no flow followed by re-flow of blood constitute ischemia/reperfusion (I/R)

oxidative stress.

In my laboratory I have utilized cardiac cells such as endothelial cells and

cardiomyocytes and their respective membranes as in vitro models to test the

susceptibility to free radical stress. I have utilized one of the peptides (glutathione)

that is rich in sulfhydryl moieties as an indicator of oxidative stress particularly in the

cultured endothelial cell model. In me presence of high levels of superoxide and other radicals glutathione is consumed as a defense against injury to vital membranes. Glutathione is in the cytosol and to some extent in the membrane. I have used

Vitamin E or α -tocopherol and other antioxidant agents to provide "first line defense"

against excessive radical injury. I have shown that in the presence of these exogenous

antioxidant agents the endogenous protective mechanism of glutathione preservation

is maintained against levels of oxidative stress that would exhaust die glutathione

levels if higher "pharmacological levels" of antioxidants were not present. Thus, diis

in vitro assay system has enabled us to determine whether an agent has potent antioxidant properties.

Excessive Free Radical Producήon in Mg-deficiencv:

In my studies of dietary deficiency of magnesium in animals we have

established that excessive production of free radicals occurs. In these animals one of

the first indicators of depletion of normal endogenous levels of antioxidants is seen

using the red cell glutathione assay. After just a few weeks on a diet deficient in

magnesium we have found significant decreases in red cell glutathione levels. When d ese animals are supplemented with antioxidants (such as α -tocopherol and

antioxidant drugs such as probucol), I have been able to observe protection of a red

cell glutathione levels. These studies showed the absence of magnesium resulted in

such high levels of free radical production. What seems to be the causal mechanism is that neurological peptides are triggered to be released by low circulating blood

levels of magnesium and these in turn trigger production of free radicals by white

blood cells, endothelial cells, macrophages and other cells that are capable of

responding to neuropeptide stimulation by producing radicals. Further, I have discovered that nitric oxide is produced in excess in these Mg deficient animals; this

is anodier form of a free radical. Clinical Trials with Magnesium Therapy and Relevant Animal Studies:

In a large investigation, the Second Leicester Intravenous Magnesium

Intervention Trial, Woods and colleagues found that giving 2 to 3 grams of

magnesium sulfate over first five minutes of presentation and then another 5 grams

over the next 24 hours reduced mortality by myocardial infarction at 28 days by 24%

and lowered the incidence of left ventricular failure by 25% as reported in Lancet vol.

343, page 1553, 1992. The Fourth International Study of Infarct Survival (ISIS-4) clinical trials also

raised controversy in cardiology with regard to the efficacy of intravenous magnesium

administration in patients with acute myocardial infarction.

With the emerging data from the Limit 2 clinical trial and animal studies

showing protection of ischemic myocardium, the role of pharmacological levels of

magnesium (both clinically as well as in the animal laboratory) came under active

investigation. Some of the data showed that mortality was improved in patients who received intravenous magnesium for chest pain (indicating heart attack) in the

emergency room. The conclusion of Limit 2 trial was that magnesium given early to

patients with infarction was beneficial. The clinical trial by Schecter, et al. Amer.

Heart J. vol. 132, No. 2, part 2 483-486 (1996) also confirmed the efficacy of

magnesium at pharmacological levels when given intravenously to patients having heart attacks. Using animal models Herzog, et al. Lancet vol. 343, pages 1285-1286 (1994) provided convincing evidence that magnesium, when present during

reperfusion (minute to hour) of previously ischemic myocardial tissue, was protective;

indeed, a decrease in the size of the anticipated myocardial infarction was reported in these animal studies. These essential observation of these animal studies (which were

more tightly controlled in their design than previous clinical trials such as Limit 2 and

ISIS 4) pointed to the protective effect of pharmacological levels of magnesium during

the early stage of reperfusion injury. In my previous studies with the isolated perfused

rat heart and in the in vivo pig heart, as well as in coronary bypass patients, I have

shown a burst of oxygen derived free radicals and free radical derived products in the

effluent from hearts perfused after periods of I/R. The earliest burst occurs within seconds to minutes of reperfusion and is not observed beyond 30 minutes of the

reperfusion period. The hypothesis that emerged from these observations is that

oxygen derived free radicals participate in the I/R injury and that if magnesium at

pharmacological levels is able to protect the myocardium during reperfusion it may

have an antiradical effect.

Intravenous Therapy with Magnesium:

Clinically Mg sulfate has been utilized for a number of years for patients with

toxemia of pregnancy. The intravenous use of Mg sulfate in these patients is

efficacious in lowering hypertension which is life threatening in some of these

patients. Recent data suggest that the health of the fetus also benefits from magnesium therapy in the peripartum period of time. Another clinical use of intravenous Mg

sulfate is in the coronary care unit where patients who have life threatening arrhythmias particularly Torsade de Pointe are given intravenous infusions of Mg

sulfate to block these arrhythmias; some of these patients may be deficient in

magnesium and repletion is effective in controlling the disordered heart beat. It is

curious that only intravenous Mg sulfate is utilized in this country. Other countries

have preparations of Mg chloride which can be given intravenously. Early studies by

Selye, et al. Amer. Heart J. 55: 163-173 (1958) suggested that Mg sulfate might not

be as effective as Mg chloride or other magnesium preparations with different anions.

No data exist on Mg gluconate in such clinical studies.

Potential Mechanisms for Cxtoprotection hy Mg gluconate and Proposed Clinical EjrjScflO

The present invention relates to the fact that Mg gluconate has therapeutic

efficacy greater than that of Mg sulfate in pathobiological conditions that result from

excessive free radical production in vivo. In particular, I/R injury of the myocardium

and cerebral tissues is one area of efficacy. Another area is that of cardioplegia at the

time of bypass surgery. I/R is also observed in organ preservation; the harvesting of

cardiac, renal and hepatic tissues is associated with a prolonged period of no flow or anoxia; prior to implantation in the recipient reinstitution of flow occurs with an

oxygenated solution that may result in free radical induced membrane injury. The

present invention relates to the use of intravenous Mg gluconate in the early phases

of myocardial infarction, bypass cardioplegia, stroke, organ preservation for

transplantation and other acute I/R injury conditions. Mg gluconate has greater efficacy than the use of Mg sulfate. SUMMARY OF THE INVENTION

Briefly, the present invention comprises a method of treating ischemia/reperfusion I/R injury due to oxidative stress by the administration of

intravenous magnesium gluconate to a patient in need thereof to substantially block

free radical surge in the patient.

It is an object of the present invention to provide a treatment for ischemia/reperfusion injury.

It is another object of the present invention to provide treatment for patients

suffering from myocardial infarction.

It is another object of the present invention to provide treatment for patients

suffering from stroke.

It is another object of the present invention to provide prevention of

reperfusion injury in mammalian tissue that has had its blood flow stopped and

reinstituted.

It is another object of the present invention to provide an improved

cardioplegic solution for use in bypass surgery.

It is another object of the present invention to provide a method of preserving

organs before or after harvesting for transplantation. These and other objects and advantages will be apparent from the more detailed

description which follows. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 represents the results of an assay to assess whether or not magnesium

sulfate, magnesium chloride or magnesium gluconate would affect the site-specific

iron mediated oxidation of deoxyribose.

Figure 2 represents the results of a test wherein magnesium gluconate protects

against iron mediated membrane lipid peroxidation.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, magnesium gluconate in a sterile aqueous solution is utilized for the treatment of conditions caused by excessive

oxidative stress due to free radical distribution and more specifically, magnesium

gluconate, is used in a method of treating ischemia/reperfusion injury.

Ischemia/reperfusion injury is defined as the loss of tissue function or viability due to the sequence of events ensuing after prolonged ischemia (20 minutes or longer)

followed by reinstitution of blood flow.

While magnesium has been used in treating myocardial infarction, magnesium

gluconate has not. The restoration of blood flow in the infarct-related artery is critical

for miriimizing necrosis and salvaging myocardium. There is always the risk of

possible reperfusion injury. The reperfusion injury may include death of cells that

were still viable at the onset of the reperfusion, the no-reflow phenomenon and loss of vasodilator reserve, myocardial smnning and arrhythmias. In accordance with the

present invention, magnesium gluconate improves myocardial energy production,

inhibits calcium cellular overload, stabilizes injured cell membranes, diminishes free radical induced damage, inhibits intracoronary thrombosis, promotes both coronary

and systemic vasodilation and minimizes the development of sinus tachycardia.

The timing of the treatment with magnesium gluconate modulation of

reperfusion injury with restoration flow is important. Typically, it should be

administered as close to the time of perfusion as feasible. However, it may be

administered within about 4 hours after the time of reperfusion.

The dosage of magnesium gluconate for myocardial infarction is defined as that which will elevate the patient's serum level (usually 1.7 to 2.0 mEq/Liter) two-fold

to 3.4 to 4.0 mEq/Liter during the first hour of reperfusion, followed by 40 to 60

mmol over the ensuing 24 hours. Generally, the magnesium serum level should be elevated by adπiinisteration of magnesium gluconate to about 4 mEq/Liter during the

first hour of reperfusion.

There are other methods relating to the preferred embodiments of the present

invention.

The method of treating stroke wherein the effective amount of intravenous

magnesium gluconate is administered to the stroke victim with the on set of symptoms

with continuous dosage for 24 hours as shown above.

The method of preserving an organ for transplantation comprises adrmnistering

magnesium gluconate to the donar prior to harvest and in the subsequent perfusion

solution in an amount to preserve the organ for storage and transplantation and to prevent preimplantation ischemic and reperfusion injury. The amount of the

magnesium gluconate needed for preservation is about 40 to 60 mmol over 12 to 24 hours. This dose is generally two times normal level.

The use of magnesium gluconate in a cardioplegic solution for use in cardiac

bypass surgery is another embodiment of the present invention. This embodiment

includes an adequate and sufficient amount of magnesium gluconate being added to an

aqueous solution containing minerals, electrolytes and preservation agents to insure

adequate preservation of the heart without reperfusion injury. The amount of

magnesium gluconate added to an aqueous solutions of electrolytes and minerals is

about 40 meq/Liter of magnesium.

The intravenous route of adrninistration is preferred, however, other routes of

administration may be effective, for example, direct infusion into an organ to be preserved for transplantation.

The concentration of the magnesium gluconate should be about 5 to about 10

mmol in sterile water in a 20 ml to 50 ml vial. Such product is commercially

available from Sigma Chemical Company, St. Louis, Missorri.

The following examples are to illustrate the invention, and are not intended to limit the invention.

Example I

Effects of magnesium salts on " ite-specific" Fe-mediated deoxyribose

oxidation.

This experiment was designed to assess whether or not each of three

magnesium salts, magnesium chloride, magnesium sulfate and magnesium gluconate, would affect the "site-specific" Fe-mediated oxidation of the deoxyribose. The procedure was similar to that developed by Gutteridge and Hallivwell (the deoxyribose

assay: an assay both for free hydroxyl radical and for site-specific hydroxyl radical

production. Biochem J. 253: 932-933, 1988). The following ingredients were

combined an assay mixture of 1 ml: deoxyribose (1-2.8mM), FeCl₃ (10-20 uM), H₂O₂ (2 mM) ascorbic acid (0.1 mM) in a 10 mM potassium phosphate buffer pH 7.4 ±

each magnesium salt (0-4 mM). After 30 minutes of incubation at 30 °C the oxidation

product, malondialdehyde, was determined by the thiobarbituric acid med od as

described in Mak & Weglicki, Methods Enzymology 234: 620-630, 1994.

In this assay, iron binds weakly to deoxyribose; in the presence of hydrogen

peroxide, hydroxyl radical is generated site-specifically and which oxidizes the

deoxyribose. Oxidation of deoxyribose is determined by the accumulation of the

degradation product which reacts with thiobarbituric acid to form colored products

with absorbance of 532 mM. Results are seen in Figure 1. The higher the absorbents

represented by the Y-axis, the more extensive the oxidation of deoxyribose. Any

agent with metal binding capacity would be able to withdraw the deoxyribose-bound

iron and inhibit the reaction.

As seen in Figure 1, of me three magnesium salts, it appears that magnesium

gluconate produced the most prominent concentration-dependent inhibition of the

deoxyribose oxidation. At 4 mM, magnesium gluconate displayed a 55% inhibition whereas either magnesium chloride or magnesium sulfate at the same concentration

only afforded approximately 15% inhibition, magnesium gluconate can function as

a more superior "iron-chelator' than MgCl₂ or MgS0₄. Example II

Effects of Magnesium Salts on Fe₂+ -mediated membrane lipid peroxidation

This experiment was designed to see if magnesium gluconate would protect

against iron-mediated membrane lipid peroxidation. The complete assay procedure

in which the microsomal membrane lipid peroxidation was induced by ferrous iron is

described in a procedure published by Mak and Weglicki, JCI 75: 58-65, 1985. The

assay mixture (500 ul) consisted of rat liver microsomal membranes (0.2 mg/ml), ±

each magnesium-salt, the chloride, the sulfate, and the gluconate, (0-4mM), in the 10

mM potassium phosphate buffer, pH 7.4. The lipid peroxidation reaction was initiated by the final addition of 100 uM ferrous sulfate (FeSO₄. 7H₂O). After 20 minutes of

reaction at 30 °C, the membrane lipid peroxidation was determined by the MDA-TBA

method as described in Mak and Weglicki, Methods of Enzymology 234: 620-630,

1994. Membrane lipid peroxidation was monitored by malondialdehyde (MDA) formation on the Y-axis. When magnesium gluconate was introduced into the reaction

mixture, lipid peroxidation was inhibited to a varying degrees depending upon the

magnesium salt concentration. At 4 mM, magensium gluconate inhibited MDA

formation by about 45%, whereas MgCl₂ for the entire concentration range did not

inhibit more than 10% . The inhibitory effect of Mg-gluconate was primarily due to

significant iron-chelating activity which protected the membrane against irion-mediated

lipid peroxidation.

Claims

What is claimed is:

1. A method of treating ischemia/reperfusion injury due to oxidative

stress in a patent in need thereof comprising administering

intravenously to me patient an amount of magnesium gluconate

effective to substantially reduce an excess free radical surge.

2. The method of Claim 1 wherein the magnesium gluconate is

administered to the patient in need thereof in amounts ranging from about 40 mmol to about 60 mmol over 24 hours.

3. The method of Claim 1 wherein the ischemia/reperfusion injury is

selected from the group consisting of myocardial infarction and stroke.

4. The method of Claim 3 wherein the intravenous administration of the magnesium gluconate is administered as close to the time of perfusion

as feasible.

5. The method of Claim 3 within the intravenous administration of magnesium gluconate is within about 4 hours after the time of

reperfusion.

6. A method of treating acute myocardial infarction which comprises

intravenous administration to a subject suffering from myocardial

infarction and in need of treatment, a dose of magnesium gluconate

effective to alleviate some or all of the manifestations of the myocardial

infarction.

7. The method of Claim 6 where the magnesium gluconate is adrninistered

wid in about 4 hours after the myocardial infarction.

8. A method of preserving an organ for transplantation comprising

administering an amount of magnesium gluconate to the organ prior to

harvest that is sufficient to preserve the organ for transplantation and

prevent ischemia/reperfusion injury upon transplantation.

9. A cardioplegia solution comprising an effective amount of magnesium

gluconate, and minerals and electrolytes.