WO1992004023A1

WO1992004023A1 - Composition and method for protecting the heart during reperfusion

Info

Publication number: WO1992004023A1
Application number: PCT/US1991/006545
Authority: WO
Inventors: Austin L. Shug
Original assignee: Shug Austin L
Priority date: 1990-09-10
Filing date: 1991-09-09
Publication date: 1992-03-19
Also published as: AU8513191A; CA2068333A1; EP0509066A1; EP0509066A4

Abstract

A pharmaceutical composition and method is described for protecting the heart of an animal or human during ischemia and reperfusion. The amino acid alanine when administered in an amount sufficient to increase the blood level of the animal or human to 1.0 to 20.0 mM alanine, will protect the myocardium from damage during ischemia and reperfusion.

Description

COMPOSITION AND METHOD FOR PROTECTING THE HEART DURING REPERFUSION

BACKGROUND

During acute myocardial infarction ("MI") , blood flow to the cardiac muscle becomes severely restricted if not stopped. If flow is not restored, myocardial ischemia and death ultimately result. Three major objectives in treatment of MI are to restore, toward normal, the compromised blood flood and to protect the heart muscle until such blood flow is restored, and to prevent further damage during reperfusion.

Myocardial ischemia leads to a number of well documented sequelae, one of which is the breakdown of myocardial protein. Several references have reported success in protecting heart muscle during ischemia by administering branched chain amino acids ("BCAA") , leucine, valine and metabolites of leucine. Rubinstein, A., Gur, Y., "Branched Chain Amino Acids in the Protection of the Myocardium from Ischemic Damage," Israel J. Med. Sci. _f Vol. 25:1, 1989; Schwalb, H.J., Freund, H.R. and Uretsky, G. , "Role of Amino Acids Acids in Myocardial Protection During Ischemia and Reperfusion," Perspectives in Clinical Nutrition, ed. J.M. Kenney and P.R. Borum, 1989, Urban & Schwarzenberg, Baltimore-Munich, pages 57-67. One explanation for the observed protective effect has been that such preparations may stimulate protein synthesis and reduce the degradation of heart proteins. Id.

Another sequelae of developing ischemia is the disturbance in energy metabolism which one would expect if the heart is deprived of oxygen. In this regard, it has been suggested that during developing ischemia anaerobic metabolic pathways may assume a commensurately greater importance in supplying the energy needs of the heart than in circumstances of normal blood flow. Rau, E.E., Shine, K.I., Gervais, A., Douglas, A.M., and Amos, E.C. Ill, "Enhanced Mechanical Recovery of Anoxic and Ischemic Myocardium by Amino Acid Perfusion," Am. J. Phvsiol.. 236(6) :H873-H879, 1979. An enhanced anaerobic metabolism has been offered by some investigators to explain the cardiotonic effect of glutamate in the ischemic heart. Support for this hypothesis is said to be found in the observation that stressed or paced heart preparations appear to take up glutamate and release alanine. Bittl, J.A. , Shine, K.I., "Protection of Ischemic Rabbit Myocardium by Glutamic Acid," Am. J. Phvsiol.. 245 (Heart Circ. Physiol. 14): H406-H412, 1983.

Although protein degradation is ordinarily viewed by most authorities as an adverse consequence of developing myocardial ischemia, it may, in fact, have protective aspects - at least in the early stages of ischemia. During the research that led to the present invention, applicant observed that during the early stages of ischemia, heart mitochondria exhibited an increase, rather than the expected decrease, in respiration. In searching for an explanation to this unexpected finding, applicant noted that liver mitochondrial studies have shown that conditions which result in protein breakdown, such as acute exercise or gluconeogenic hormones cause a lasting stimulation of liver mitochondrial respiration. Bobyleva-Guarriero, V., Battelli, D. , Bellei, M. and Lardy, A., "Sources of Intramitochondrial Malate," FASEB J.. 3:2208-2211; 1989. This reference demonstrated that the cause of the stimulation in liver mitochondrial respiration was an increased level of malate in the mitochondria. This, in turn, was shown to be related to hepatic blood increases of alanine and glutamate - products of protein catabolism. Id. The experiments of the applicant were conducted to test whether the products of protein catabolism, namely, alanine and glutamate are protective of the heart during ischemia and reperfusion, and whether this protective effect is mediated by an increase in respiration in myocardial mitochondria. While others have reported the protective effects of glutamate (Bittl, et. al., supra; Rau, et. al., supra.) on the ischemic heart, to applicant's knowledge, no one has previously studied the protective effect of alanine, either alone or in combination with glutamate. To applicant's surprise, alanine alone has significant protective effect on the ischemic and reperfused heart. A further unexpected finding was that the combination of alanine and glutamate produced a synergistic protective effect. This latter finding is significant as it permits one to limit the amount of glutamate, a known neurotoxin (Finkbeiner, S., Stevens, C.F., "Applications of Quanitative Measurements for Assessing Glutamate Neurotoxicity." Proc. Nat. Acad. Sci.. USA, Vol. 85, 4071-4074, June 1988) and still get an extraordinary protective effect.

EXAMPLE

The effect on ischemic myocardium during reperfusion of alanine and alanine in combination with glutamate was compared with glutamate in the isolated perfused working rat heart.

METHOD

The studies were conducted using hearts removed from adult male Sprague-Dawley rats weighing 250-350 grams. Each removed heart was prepared in the following manner: The heart was placed in a cold (4-7°C) cardioplegic solution and the aorta was dissected. The heart was cannulated onto a modified, isolated rat heart perfusion apparatus within 60 seconds of excision. Temperature in the perfusion chamber throughout the experimental procedure was maintained at 37°C. Non-recirculating retrograde (Lagendorff) perfusion in oxygenated Krebs Henseleit bicarbonated saline solution, containing 11 mM glucose_, (KBH) proceeded for 10-12 minutes during which the left atrial appendage - pulmonary vein main trunk was cannulated. The heart was then perfused in a recirculating anterograde "working" mode by switching perfusate input source to the left atrial pre-load reservoir (15cm H₂0 filling pressure) , opening the aortic output line (80cm H₂0 after-load pressure) , and pacing at 315 beats per minute by atrial anodal stimulus for a 30 minute stabilization period at which time baseline measurements were made of various parameters including cardiac output (CO) .

At the end of the stabilization period, no flow ischemia ("NFI") was induced by terminating pacing stimulus and clamping the left atrial input and aortic output lines. NFI was maintained for 21.5 minutes.

At the conclusion of the 21.5 minute period of NFI, reperfusion was started by reversing the sequence in NFI. Reperfusion was continued in each case for slightly more than 40 minutes. In some cases resuscitation manipulations were employed until recovery. The measurements reported below were taken at the end of 40 minutes of reperfusion. In the control experiments, the reperfusion solution was KBH. In the experimental solutions, alanine, or alanine plus glutamate or glutamate were added to the KBH solution in the concentrations indicated in Table 1. MEASUREMENTS

Cardiac Output (CO) represents the combination of coronary flow plus aortic flow (CO = CF + AF) . This was determined by timed collection of both the coronary flow and the aortic flow. Aortic flow was determined in a graduated cylinder for 20 seconds. This number was multiplied by three to yield aortic flow per minute.

Coronary flow was determined in a analogous fashion.

Cardiac work was determined by measuring left ventricular minute work (LVMW) . LVMW equals cardiac output (CO) multiplied by mean aortic pressure (MAP) multiplied by 0.0136 (LVMW *■=• CO x MAP x 0.0136 gm/min.) CO was measured as described above. MAP was determined by the formula:

MAP = (2 x ADP) + ASP 3 where ADP represents aortic diastolic pressure and ASP represents aortic systolic pressure. Aortic pressures were measured using a spectromed strain gauge transducer, P23XL, with a Gilson 5/6 polygraph outfitted with a 1C-MP module.

Lactate was measured according to the procedure of Gutmann, H. and Wahlefeld, Methods of Enzymatic Determinations_f Bergmeyer, H. (ed.) Vol. 3, Academic Press, New York, N.Y. (1974), "Lactate Determination" pp. 1464-1468.

Adenosine triphosphate (ATP) was measured according to the method of Stanley, P.E. and Williams, S.G., "Use of the Liquid Scintillation Spectrometer for Determining Adenosine triphosphate by the Luciferase Enzyme" Analyt. Biochem. 29:381-392 (1969). RESULTS

TABLE 1

TREATMENT LACTATE ATP % REC .¹ % REC² n

CO WORK nmole/g/dry w nmole/g/dry t.

CONTROL 25,050 3,674 2.9 0 3

5Mm Alanine 5,987* 5,679* 15 63 6 lOmM Alanine 4,527* 5,977* 17 75 3

20mM Alanine 6,768* 6,018* 24 100 4

20mM Alanine 5,517* 8,386** 4 ** 100** 4 + 5mM Glutamate

5mM Glutamate *** *** 16 ***

* p=< 0.05 when compared with control value ** p=< 0.01 when compared with control value *** no measurement taken Statistical significance was calculated with the use of the students T test and significance was placed at p=< 0.05.

¹ % REC. CO. represents the % of recovery of cardiac output as compared to the control.

² % REC. WORK represents the % of recovery of cardiac work as compared with the control.

The data displayed in Table 1 indicate that treatment with alanine, or alanine plus glutamate, or glutamate greatly enhances the recovery of cardiac output and cardiac work during reperfusion. While alanine or glutamate when administered alone show an enhancement of CO, a significantly greater enhancement is found when the two amino acids are given in combination with one another. It should be appreciated that while recovery of CO is not back to control, the experiment model used presents an extreme stress to the rat myocardial as evidenced by the fact that in the control rats recovery of CO is negligible. Also, a large lactate accumulation and decreased ATP levels jn untreated ischemic animals (control) indicates serious ischemic damage. Braunwald, E. and Sobel, B.E., "Coronary Blood Flow and Myocardial Ischemia," In Braunwald, E. (Ed.), Heart Disease , 2nd ed. W.B. Saunders Co., Philadelphia, pages 1235-1261, 1984.

By contrast, treatment with the amino acids of the present invention results in a significantly lower lactate concentration when compared with the control. This finding alone is evidence of the protective effect of the treatment of the invention. Moreover, the treated hearts show a significance increase from control in ATP concentrations which is evidence of a stimulation of mitochondrial respiration and oxidative phosphorylation.

The data presented above indicate the usefulness of alanine or the combination of alanine plus glutamate to protect the heart in humans and other mammals during developing ischemia and reperfusion. Medical situations where such treatment would be useful are acute MI, chronic low flow caused by coronary artery disease and open heart surgery where the heart is stopped and needs to be resuscitated. In accordance with this invention, alanine with a combination of alanine plus glutamate is administered to a patient, either alone or in combination with one or more drugs known to be useful in the treatment of MI. While the exact mechanism by which alanine (or alanine plus glutamate) enhances cardiac output and protects the myocardial ischemia requires further delineation, the findings reported here strongly suggest that the amino acids of the invention act by increasing mitochondrial respiration. Both glutamate and alanine may act to increase mitochondrial malate. In addition, alanine may act as a substrate to increase mitochondrial pyruvate which is rapidly converted to acetyl CoA. Acetyl CoA is at a rate-limiting step in the citric acid cycle and is known to decrease markedly in myocardial ischemia. Thus, an increase in Acetyl CoA by conversion from alanine may be a possible mechanism by which alanine so effectively increases heart mitochondrial respiration.

The administration of the amino acids of the present invention can be effected orally, intraperitonally, subcutaneously, intravenously or intramuscularly. Conveniently, the amino acids of the present invention are mixed or dissolved in any innocuous vehicle such as water or sterile saline solution or an electrolyte solution such as Krebs Heneleit Solution or in tablet or powder form containing the usual solid diluents or carriers.

If given as a combination of alanine and glutamate, the glutamate concentration should be sufficient to stimulate respiration but not so great as to risk the development of unwanted neurotoxic side effects. In humans, a dosage sufficient to raise the blood concentration to 1-20 mM alanine plus 0-5 mM glutamate is preferable.

The amino acids of the invention can be co-administered with other amino acids, branched chain amino acids or with drugs that are known to be useful in the treatment of heart disease, such as drugs which display positive iontropic action or drugs with anti- arrhythmic effects. Effective amounts of the amino acids of the invention may also be co-administered with other compounds such as L-Carnitine which are believed to be useful supplements for treating heart disease.

The principals, preferred embodiments and modes of operation of the present invention have been described in the foregoing Specification. The invention which is intended to be protected herein, however, is not to be construed as limited to the particular forms disclosed, since these are to be regarded as illustrative, rather than restrictive. Variations and changes may be made by those skilled in the art without departing from the spirit of the invention.

Claims

CLAIMS: What is claimed:

1. A pharmaceutical composition for protecting the heart of an animal or human, comprising: an effective amount of alanine.

2. A pharmaceutical composition as claimed in claim 1, wherein said effective amount is an amount sufficient to stimulate heart mitochondrial respiration.

3. A pharmaceutical composition as claimed in claim 1, wherein said effective amount is an amount sufficient to cause the concentration of alanine in the blood of said heart to be 1 to 20 mM.

4. A pharmaceutical composition for protecting the heart, comprising: an effective amount of alanine and glutamate.

5. A pharmaceutical composition as claimed in claim 4, wherein said effective amount is an amount sufficient to stimulate heart mitochondrial respiration.

6. A pharmaceutical composition as claimed in claim 4, wherein said effective amount is an amount sufficient to cause the concentration in the blood of said heart to be 1 to 20 mM alanine and 0 to 5 mM glutamate.

7. A method for protecting the heart of an animal or a human comprising the steps of: administering to said animal or human an effective amount of alanine.

8. A method as claimed in claim 7, wherein said effective amount is an amount sufficient to stimulate myocardial mitochondrial respiration.

9. A method as claimed in claim 7, wherein said effective amount is an amount sufficient to cause said animal or human's blood to have a concentration of 1 to 20 mM alanine.

10. A method for protecting the heart of an animal or a human comprising the steps of: administering to said animal or human an effective amount of alanine and glutamate.

11. A method as claimed in claim 10, wherein said effective amount is an amount sufficient to stimulate myocardial mitochondrial respiration.

12. A method as claimed in claim 10, wherein said effective amount is an amount sufficient to cause said animal or human's blood to have a concentration of 1 to 20 mM alanine and 0 to 5 mM glutamate.

13. A pharmaceutical solution for protecting the heart during cardioplegia comprising: an effective amount of alanine.

14. A pharmaceutical solution as claimed in claim 13 wherein said effective amount is an amount sufficient to cause the concentration of alanine in the blood of said heart to be 1 to 20 mM.

15. A method for protecting the heart of an animal or human during cardioplegia comprising the steps of administering to said animal or human an effective amount of alanine.

16. A method as claimed in claim 15 wherein said effective amount is an amount sufficient to cause the concentration of alanine in the blood of said heart to be 1 to 20 mM.