Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITION AND METHOD FOR PROTECTING
THE HEART DURING REPERFUSION
BACRGRO~ND
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., 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," Pers~ectives in Clinical Nutrition, ed. J.M. Kenney
and P.R. Borum, 1989, Urban & Schwarzenberg, 8altimore-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.
~ nother 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
con~ensurately greater importance in supplying the energy needs of
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the heart than in circumstances of normal blood flow. Rau, E.E.,
Shine, K.I., Gervais, A., Douglas, A.M., and Amos, E.C. III,
"Enhanced Mech~nical Recovery of Anoxic and Ischemic Myocardium by
Amino Acid Perfusion," Am. J. Physiol., 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 relea~e alanine. Bittl, J.A., Shine,
X.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.
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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.,
suDra; Rau, et. al., su~ra.) 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.
EXANPLE
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.
NET~OD
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 (4-7C) cardioplegic solution and the aorta was
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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 37C. 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 H2O filling
pressure), opening the aortic output line (80cm H2O 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.
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NEA8UREMENT~
Cardiac Output (C0) represents the combination of coronary
flow plus aortic flow (C0 = 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 (C0) multiplied by
mean aortic pressure (MAP) multiplied by 0.0136 (LVMW = Co x MAP
x 0.0136 gm/min.) C0 was measured as described above. M~P was
determined by the formula:
MAP = (2 x ~P~ + ASP
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 lC-MP module.
Lactate was measured according to the procedure of Gutmann,
H. and Wahlefeld, MethodQ of Enzvmatic Determinations, 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" Analvt. Biochem. 29:381-392 (1969).
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RB U~T~
TABLE 1
TREATMENTLACTATE ATP % REC.I % REC2 n
CO WORK
nmole/g/dry wt nmole/g/dry wt.
,
CONTROL 25,050 3,674 2.9 0 3
5Mm Alanine5,987* 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** 44** 100** 4
+ 5mM Glutamate
5mM Glutamate *** *** 16 *** 3
* 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.
% 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
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accumulation and decreased ATP levels in 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
co~trol 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 additisn, alanine may act as a
substrate to increase mitochondrial pyruvate which is rapidly
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converted to acetyl CoA. Acetyl CoA is at a rate-limitinq 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 Xrebs 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 treat~ent 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
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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.
