WO1993010784A1

WO1993010784A1 - Therapeutic composition and method for preventing reperfusion injury

Info

Publication number: WO1993010784A1
Application number: PCT/US1992/010116
Authority: WO
Inventors: Donald E. Hultquist; Marshal Shlafer
Original assignee: University of Michigan Ann Arbor
Current assignee: University of Michigan Ann Arbor
Priority date: 1991-11-25
Filing date: 1992-11-24
Publication date: 1993-06-10
Anticipated expiration: 1994-05-25
Also published as: AU3222893A

Abstract

A pharmacologic intervention for protecting human cells, tissues, and organs from reperfusion injury following ischemia, or mitigating against the harmful effects thereof, comprises the introduction of a therapeutic composition to blood and/or oxygen-deprived cells, tissues or organs preferably prior to, or concurrent with, reintroduction of oxygen. In a practical embodiment, the therapeutic composition is a sterile, isotonic solution comprising about 1 to 200 micromoles of an antioxidant compound, such as riboflavin or a riboflavin analog. In alternative embodiments, the therapeutic composition is a sterile, isotonic solution comprising about 0.1 to 200 micromoles of the antioxidant compound, pyrroloquinoline quinone. In preferred embodiments, about 5 to 10 millimoles of a cellular nutrient, e.g., glucose, is coadministered with the antioxidant compound.

Description

THERAPEUTICCOMPOSITIONANDMETHODFOR PREVENTINGREPERFUSIONINJURY

BACKGROUND OF THE INVENΗON

This invention relates generally to a therapeutic composition and method for protecting human cells, tissues, and organs from reperfusion injury following ischemia, or mitigating against the harmful effects thereof, and more particularly, to a safe and effective pharmacologic intervention procedure comprising administration of a therapeutic composition containing an antioxidant compound, .such as riboflavin or a riboflavin analog, or in alterna¬ tive embodiments, pyrroloquinoline quinone.

Ischemia, the result of reduction of blood flow, and hence, of oxygen to living tissues, is a common clinical event. It is often associated with morbidity and mortali¬ ty. Ischemia occurs spontaneously, for example, during cardiac arrest, myocardial infarction, stroke, shock, suffocation, drowning, and acute lung injury. In organ (e.g., heart, liver, kidney, lung) transplant procedures, ischemia is induced in the transplanted organ in the period of time between harvesting from a donor and implantation in the recipient. In all of the foregoing situations, the ultimate goal is to restore blood flow to the organs. However, an additional component of damage to the organs, called reperfusion injury, occurs when oxygen is restored after deprivation of oxygen and blood flow. Reperfusion injury is also attendant to a variety of surgical proce¬ dures such as cardiac or vascular surgery.

In view of the prevalence of ischemia, and the resultant health consequences, a pharmacologic intervention that could reduce or prevent the resulting damage is greatly needed. Such an intervention would find applica¬ bility in routine health care, i.e., for the prevention of anticipated ischemic organ damage in high risk patients, or in emergency transplantation situations.

Pharmacologic interventions have been proposed and tested. These include drugs that are called calcium channel blockers, agents that allegedly inhibit the breakdown of metabolic fuels, and redpx enzymes such as superoxide dismutase. The aforementioned drugs disadvanta- geously require complex and/or expensive technology for their manufacture, such as special chemical synthesis or recombinant DNA technology. Further, these drugs must be administered in relatively high doses which lead to side effects, toxicity, or, in the case of proteins (enzymes) , potentially serious antigen-antibody reactions. In some cases, the efficacy of treatment is limited only to certain organs or tissues, or depends upon administration of the agent before ischemia has occurred.

There is thus a .need in the art for a pharmacologic intervention which is easily and inexpensively manufac¬ tured, which is efficacious at low doses, which is non- toxic and lacks significant side effects and/or antigenic potential, which is not organ or tissue specific, and which can be administered after ischemia occurs to mitigate the - -effects of reperfusion injury.

It is, therefore, an object of this invention to provide a pharmacologic intervention to prevent and/or mitigate reperfusion injury to cells, tissues, and organs, after ischemia.

It is another object of this invention to provide a safe and effective pharmacologic intervention to prevent and/or mitigate reperfusion injury using a compound which occurs naturally in human tissues.

It is also an object of this invention to provide a pharmacologic intervention to prevent and/or mitigate reperfusion injury which is non-toxic, non-antigenic, does not produce contractile or hemodynamic effects, etc. It is a further object of this invention to provide a pharmacologic intervention to prevent and/or mitigate reperfusion injury which is effective at very low dosages (e.g., micromolar range). It is additionally an object of this invention to provide a pharmacologic intervention to prevent and/or mitigate reperfusion injury which is inexpensive to manufacture and administer.

SUMMARY OF THE INVENTION The foregoing and other objects are addressed by this invention which provides a composition for treating blood and oxygen-deprived cells, tissues or organs of a living being to prevent reperfusion injury upon reintroduction of blood flow and/or oxygen. The composition comprises an antioxidant compound, preferably in a solution suitable for introduction into living tissues, cells, organs, or organisms, in a therapeutically effective amount. As used herein, the term "antioxidant compound" refers to a compound which, in a biological environment, counteracts the deleterious effects of reactive oxygen species. In some embodiments of the invention, the antioxidant compound is riboflavin or a riboflavin analog. In other embodiments of the invention, the antioxidant compound is pyrroloquino- line quinone (PQQ) . The therapeutically effective amount is that amount of antioxidant compound which produces the desired effect, i.e., prevention or mitigation of reperfusion injury. In certain preferred embodiments, about l to 200 micromoles riboflavin in solution has been found to be effective. The term "riboflavin" includes riboflavin analogs such as flavin mononucleotide, flavin adenine dinucleotide, riboflavin tetraacetate, riboflavin tetrabutyrate, deriva¬ tives of ribitol, and isoalloxazine derivatives, such as proflavin, lumiflavin, lumiflavin-3-acetate, and lumi- chrome.

In other embodiments of the invention, about 0.1 to 200 micromoles pyrroloquinoline quinone in solution has been found to be effective.

In particularly preferred embodiments of the inven¬ tion, the composition further contains a cellular nutrient. This cellular nutrient may advantageously be glucose. The presence of glucose has been found to reduce the amount of antioxidant compound required for therapeutic effective¬ ness. In preferred embodiments, about 5 to 10 millimoles of glucose in the solution has been efficacious.

In a specific illustrative embodiment of the inven¬ tion, the composition may comprise a sterile, isotonic solution (such as a parenteral fluid) containing about 1 to 200 micromoles of riboflavin and about 5 to 10 millimoles of glucose.

In another specific illustrative embodiment of the invention, the composition may comprise a sterile, isotonic solution containing about 0.1 to 200 micromoles of pyrrolo¬ quinoline quinone and about 5 to 10 millimoles of glucose. In a method aspect of the invention, a living being is treated to prevent reperfusion injury upon reintroduction of oxygen to blood and oxygen-deprived cells, tissues or organs by introducing to the oxygen-deprived cells, tissues or organs of the living being a therapeutically effective amount of an antioxidant compound. The therapeutically effective amount of the antioxidant compound is preferably in the range of about 0.1 to 200 micromoles. In particularly preferred embodiments, a cellular nutrient is co-introduced with the antioxidant compound. The cellular nutrient is preferably glucose which may be present in the range of about 5 to 20 millimolar.

Although the antioxidant compound may be introduced post-reperfusion, it is particularly preferred to introduce the antioxidant compound and/or cellular nutrient, prior to or concurrent with reintroduction of oxygen to the oxygen- deprived cells, tissues or organs of the living being.

In certain embodiments of the invention, the antioxi- dant compound and/or glucose can be provided in solution in a therapeutically effective amount and administered by perfusing the cell, tissue, or organ with the solution. In an alternative embodiment, a sterile, isotonic solution can be administered systemically to the living being, such as by intravenous injection.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the principles of the invention, an

- antioxidant compound, such as riboflavin or a riboflavin analog or PQQ is administered to a living being preferably prior to, or concurrent with, reintroduction of oxygen and blood flow to ischemic or anaerobic cells or tissues in order to protect the cells, tissues, and organs from reperfusion injury. In preferred embodiments, the antioxi¬ dant compound is coadministered with a cellular nutrient, glucose.

Riboflavin, also known as vitamin B₂, is a small molecule which is essential to human nutrition, and hence is naturally present in human tissues in both the intracel- lular and extracellular spaces. Thus, riboflavin would not be antigenic. Moreover, riboflavin is non-toxic and does not appear to have contractile or hemodynamic effects, when administered at low doses. Further, riboflavin passes easily into the cells when administered systemically or directly to the vasculature of an isolated (i.e., trans- plantable) organ.

The term "riboflavin" as used herein refers to riboflavin, riboflavin derivatives (including flavin mononucleotide, flavin adenine dinucleotide, riboflavin tetraacetate, riboflavin tetrabutyrate, and derivatives in which various hydroxyl groups of the ribitol have been derivatized) , and other isoalloxazine derivatives (includ¬ ing proflavin, lumiflavin, lumiflavin-3-acetate, lumi- chrome, and derivatives in which chemical groups are substituted on the various carbon and nitrogen atoms of the isoalloxazine ring) . Riboflavin can be obtained commer¬ cially from Sigma Chemical Co., St. Louis, MO.

In a preferred embodiment of the invention, the riboflavin is provided in a sterile, preferably isotonic, solution where the concentration of riboflavin in solution would range from about 1 to 200 micromolar. The components of the riboflavin-containing solution should be compatible with living tissue and suitable for administration to living tissue or to a living being. Riboflavin, especially at the concentration herein stated to be efficacious, is compatible with most parenter- al fluids^"in which it can be administered. These fluids include typical, well-known intravenous electrolyte- containing and nutrient-containing media, as well as modified solutions, such as those commonly used at the present time by cardiac surgeons in attempts to minimize (with incomplete success) heart damage due to ischemia and reperfusion.

In particularly preferred embodiments, the sterile solution also contains a cellular nutrient. In a specific preferred embodiment the cellular nutrient is glucose in a concentration of approximately 5 to 10 millimolar. Glucose is advantageously coad inistered during riboflavin treat¬ ment. Although not wishing to be bound by theory, it is possible that the protective effect of riboflavin is a result of the ability of a tissue enzyme to convert it to dihydroriboflavin. Dihydroriboflavin can destroy destruc¬ tive oxidants, or reactive oxygen species, which are ^'generated in cells during reperfusion of ischemic tissues. The enzyme, flavin reductase, catalyzes the reaction between a flavin and NADPH to form dihydroriboflavin and NADP⁺. This enzyme is present in red blood cells, but has recently been discovered in the cystolic fraction of a number of cells other than red blood cells, specifically bovine liver and rat liver, spleen, kidney, heart, lung, brain, ovary, and testis. Highly reactive compounds, specifically hydroxyl radicals and the Fe(IV)0 and Fe(V)0 oxidation states of hemeproteins, are believed to be responsible for much of the damage during reperfusion injury. It has now been demonstrated that dihydroribo- -flavin efficiently destroys hydroxyl radicals and the higher oxidation states of hemeproteins.

Therefore, the _.. administration of riboflavin is believed to permit the ischemic or anaerobic cells to generate greater than normal quantities of dihydroribofla¬ vin by the action of cellular flavin reductase on flavin. The dihydroriboflavin would then destroy the destructive agents and protect the cell from reperfusion injury. It is believed that the coadministration of a cellular nutrient, such as glucose, generates NADPH which is required to reduce riboflavin to dihydroribo lavin.

In the alternative embodiment, PQQ, or 4,5-dihydro- 4,5-dioxo-lH-pyrrolo[2,3-f]quinolone-2,7,9-tricarboxylic acid, also known as methoxatin, is a naturally-occurring compound that serves as a coenzyme for certain enzymes. PQQ has been shown to be present in a variety of living cells, including mammalian tissues and fluids. We have discovered that PQQ is a high-affinity substrate for the intracellular enzyme flavin reductase. Kinetic studies of this enzyme have revealed that the Michaelis constant, !_„,, for PQQ is much lower than the _„, for riboflavin. There¬ fore, even with minute quantities of PQQ, the enzyme flavin reductase can function more efficiently in cells than with riboflavin. The reduced form of PQQ has been observed to reduce readily the ferric forms of hemeproteins, namely hemoglobin, cytochrome c, and myoglobin. The reduced form of PQQ also reduces the Fe(IV)0, or ferryl form, of myoglobin. PQQ operates in a manner analogous to ribofla- vin to reduce destructive Fe(IV)0 forms of hemeproteins and other reactive oxygen species.

PQQ can be purchased as a highly purified compound from Sigma Chemical Company, St. Louis, MO. PQQ is soluble in water and can be provided in a sterile, preferably isotonic, solution where the concentration of PQQ in solution would range from about 0.1 to 200 micromolar.

In a method embodiment, the procedure comprises introducing to the tissue, organ, or systemic circulation, a sufficient amount of antioxidant compound in a sterile, isotonic solution, such that the concentration of the riboflavin in the solution which reaches the ischemic tissues will be in the range of 1 to 200 micromolar, or 0.1 to 200 micromolar for PQQ. In preferred embodiments, the introduction of the antioxidant compound-containing solution is initiated prior to introduction of oxygen. However, in emergency situations, such as cardiac arrest, the treatment might begin concurrently with introduction of oxygen.

Specific methods of administering the therapeutic composition of the present invention include, without limitation, (a) perfusing the tissue, or organ, in situ ; (b) perfusing an isolated organ or tissue being replaced or transplanted prior to transplantation; or (c) intravenous injection into the patient as part of first line medical intervention, such as during cardiopulmonary resuscitation and advanced cardiac life support techniques. The treat¬ ment preferably should continue for a period of time following introduction of oxygen to the ischemic tissues, illustratively from 20 minutes to 60 minutes. In riboflavin embodiments where an isolated organ for transplant is being perfused, it is advantageous to use riboflavin rather than an analog of riboflavin, such as riboflavin mononucleotide, which must be metabolized to the flavin.

Experimental Section:

The technique of the present invention is applicable to the protection of tissues in general, however, the most common tissues, or organs, subjected to reperfusion injury are: heart, lung, liver, kidney, intestine, and brain. In order to demonstrate the efficacy of the invention, the riboflavin treatment has been tested on ischemic isolated organs.

Specifically, experiments have been performed on isolated rabbit hearts using leakage of intracellular lactate dehydrogenase as a measure of reperfusion injury and using ventricular pressure, heart rate, and coronary flow as measures of heart function. The rabbit hearts were made hypoxic for 55 minutes in the presence of glucose. The administration of 20 micromolar riboflavin and 10 millimolar glucose for 5 minutes prior to the onset of re- oxygenation and during re-oxygenation significantly reduced the reperfusion injury. Rabbit and rat hearts which had been perfused in this manner were found to contain flavin reductase.

More particularly, rabbit hearts were perfused with a physiologic saline solution, the composition of which mimics the cell-free and protein-free components of blood with respect to glucose and salts. The hearts were instrumented in such a way that their function (e.g., ventricular pressure development, heart rates, and perfus- ate flows through the vasculature) could be monitored, and the effluent coming out of the heart could be collected and assayed for chemical markers of cell damage. After allowing the hearts to stabilize, with the perfusing solution being well oxygenated, the hearts were deprived of adequate oxygen for 55 minutes. Some hearts were perfused for an additional 5 minutes with the same oxygen- depriving solution, but containing 20 micromolar ribofla¬ vin. These hearts were then perfused with the same riboflavin-containing solution that was well oxygenated. Control hearts were treated identically, but were never exposed to riboflavin. A hallmark of reoxygenation/reper- fusion injury is a sudden release of intracellular enzymes from damaged cells into the perfusate. One enzyme marker that is often studied is lactate dehydrogenase (LDH) . Hearts that were reoxygenated in the absence of riboflavin treatment released LDH at a rate of 0.8 ± 0.2 Unit per minute per gram of heart dry weight during reoxygenation. In contrast, riboflavin-treated hearts released signifi¬ cantly less LDH (a rate of 0.4 ± 0.1 Unit/minute/gram.)

The ability of PQQ to reduce reperfusion injury was assessed in the isolated rabbit heart model in a similar manner. The administration of 20 micromolar PQQ and 10 millimolar glucose for 5 minutes prior to the onset of re¬ oxygenation and during re-oxygenation significantly reduced the reperfusion injury. Hearts that were reoxygenated in the absence of the antioxidant treatment released LDH, summed over a 30 minute re-oxygenation period, in the amount of 22 ± 4 Unit per gram of heart dry weight. In contrast, riboflavin-treated hearts released 10 ± 4 Unit/gram LDH and PQQ-treated hearts released 4.2 ± 1 Unit/gram. This represents 81% protection by PQQ and 55% protection by riboflavin.

Although the invention has been described in terms of specific embodiments and applications, persons skilled in the art can, in light of this teaching, generate additional embodiments^'without exceeding the scope or departing from the spirit of the claimed invention. Accordingly, it is to be understood that the drawing and description in this disclosure are proffered to facilitate comprehension of the invention, and should not be construed to limit the scope thereof.

Claims

WHAT IS CLAIMED IS:

1. A composition for treating blood and oxygen- deprived cells, tissues or organs of the living being to prevent reperfusion injury comprising: a solution of an antioxidant compound in a therapeuti¬ cally effective amount to prevent reperfusion injury upon reintroduction of oxygen to the blood and oxygen-deprived cells, tissues or organs of the living being.

2. The composition of claim 1 wherein the antioxi- dant compound is a riboflavin.

3. The composition of claim 2 wherein the riboflavin is selected from the group consisting of riboflavin, flavin mononucleotide, flavin adenine dinucleotide, riboflavin tetraacetate, riboflavin tetrabutyrate, derivatives of ribitol, and isoalloxazine derivatives.

4. The composition of claim 3 wherein the isoalloxa¬ zine derivatives are selected from the group consisting of proflavin, lumiflavin, lumiflavin-3-acetate, and lumi- chrome.

5. The composition of claim 1 wherein the therapeu¬ tically effective amount of riboflavin is in the range of about l to 200 micromolar.

6. The composition of claim 1 wherein the antioxi¬ dant compound is pyrroloquinoline quinone.

7. The composition of claim 6 wherein the therapeu¬ tically effective amount of pyrroloquinoline quinone is in the range of about 0.1 to 200 micromolar.

8. The composition of claim 1 further comprising a cellular nutrient.

9. The composition of claim 8 wherein the cellular nutrient is glucose, the glucose being present in a range of about 5 to 10 millimoles.

10. A sterile, isotonic solution comprising about 1 to 200 micromoles of riboflavin and about 5 to lO milli- moles of glucose.

11. A sterile, isotonic solution comprising about 0.1 to 200 micromoles of pyrroloquinoline quinone and about 5 to 10 millimoles of glucose.

12. A method of treating a living being to prevent reperfusion injury upon reintroduction of oxygen to blood and oxygen-deprived cells, tissues or organs of the living being comprising: introducing to the oxygen-deprived cells, tissues or organs of the living being a therapeutically effective amount of an antioxidant compound.

13. The method of claim 12 wherein the antioxidant 'compound is riboflavin.

14. The method of claim 13 wherein the therapeutical¬ ly effective amount is in the range of about 1 to 200 micromolar.

15. The method of claim 12 wherein the antioxidant compound is pyrroloquinoline quinone.

16. The method of claim 15 wherein the therapeutical¬ ly effective amount is in the range of about 0.1 to 200 micromolar.

17. The method of claim 12 wherein a cellular nutrient is introduced with the antioxidant compound.

18. The method of claim 17 wherein the cellular nutrient is glucose.

19. The method of claim 18 wherein the amount of glucose is in the range of about 5 to 20 millimolar.

20. The method of claim 12 wherein the introducing step is performed prior to or concurrent with reintroduc¬ tion of oxygen to the oxygen-deprived cells, tissues or organs of the living being.

21. The method of claim 12 wherein the step of introducing comprises: perfusing the cell, tissue, or organ tissue with a solution of a therapeutically effective amount of an antioxidant compound.

22. The method of claim 12 wherein the step of introducing comprises: administeringsystemicallyatherapeuticallyeffective amount of antioxidant compound to the living being.

23. The method of claim 22 wherein the step of administering comprises intravenous injection of a sterile, isotonic solution of a therapeutically effective amount of an antioxidant compound.