WO2000069427A1

WO2000069427A1 - Composition and method for treating limb ischemia

Info

Publication number: WO2000069427A1
Application number: PCT/US1999/010867
Authority: WO
Inventors: Scott Howell Davis
Original assignee: WALKER PAUL MOORE
Current assignee: WALKER PAUL MOORE
Priority date: 1999-05-17
Filing date: 1999-05-17
Publication date: 2000-11-23
Anticipated expiration: 2001-11-17

Abstract

This invention presents an aqueous formulation and a method for the perfusion of ischemic limbs. The disclosed formulation includes an oncotic agent, electrolytes, a readily oxidizable energy substrate, magnesium ion, a buffer to maintain the formulation at physiological pH, and a free radical scavenger.

Description

COMPOSITION AND METHOD FOR

TREATING LIMB ISCHEMIA BACKGROUND OF THE INVENTION

This invention is directed to a composition and method for the treatment of circulatory occlusions, and specifically occlusions of the limbs. When such vascular occlusions occur, the adequate flow of blood is presumptively prevented from reaching cellular systems. Because all living tissues require oxygen, nutrients, water, and other substances, this decreased flow of essential substances to the cells may cause cellular damage or cellular death.

Circulatory shock is a common life-threatening pathophysiological state that occurs secondary to trauma, hemorrhage, burns, sepsis, allergic reactions, and heart failure. These different types of circulatory shock are characterized by reduced blood pressure, cardiac output, and fluid volume flow. Furthermore, organ ischemia and inflammation (edema) are associated with certain procedures that can result in shock-like microcirculatory abnormalities. Both systemic shock and localized reactions cause a reduction in blood flow and oxygen delivery to vital organs and tissues. This low blood flow condition causes local hypoxia, interruption of nutrient delivery, and can lead to loss of cellular and organ function and even death. Accepted definitive treatment for some types of circulatory shock and useful therapy in all types of shock includes fluid volume infusions. The electrolytes present to facilitate such treatment include the ions of sodium, potassium, calcium, and chloride in concentrations approximating that found in normal blood plasma. In addition, magnesium ion is often used in excess of normal blood concentrations as a counter-measure to the cell membrane calcium channel transport mechanism. One way of obtaining normal electrolyte concentrations is by using Ringer's lactate solution as the source of the needed electrolytes. Additionally, the desired concentration of electrolytes can be achieved directly by dissolving salts of the desired ions in water, preferably distilled water.

Other isotonic fluid replacement solutions have been used, including isotonic crystalloid solutions mixed with macromolecular solutions of plasma proteins or synthesized molecules with oncotic properties (colloids) similar to natural plasma proteins; including albumin, dextran, hetastarch, and polygelatin in 0.9% NaCl solution. One important feature of these colloid systems is the use of volume expanders that facilitate volume loading of the circulatory system without deleterious interstitial and tissue results. Of course whole blood may also be used, but is expensive, often unavailable, carries some risk of viral contamination, and cross matching may delay therapy and may not be entirely curative of the affliction.

Crystalloids and colloids have been used as volume expanders, but generally must be infused in large volumes so as to include other necessary metabolites. Such large volumes may, however, cause peripheral and pulmonary edema and thus the benefits of use are diminished. Additionally, the large volume requirements for infusion of isotonic fluids further requires time delays and logistic difficulties associated with vascular delivery of effective therapy. Hyperosmotic crystalloid and hyperosmotic/hyperoncotic

(crystalloid/colloid) formulations offer some physiological benefits for the treatment of circulatory shock, including improved efficacy for restoration of overall cardiovascular function in animals and man compared to conventional resuscitation. Normalization of circulatory function has been obtained with such solutions. Small volumes of salt and concentrated dextran formulations have been shown to rapidly restore and sustain normalization of circulatory function in situations involving hemorrhagic shock. However, there remain important limitations regarding the use of these formulations for reperfusion of ischemic limbs. One limitation involves the generation or liberation of metabolic toxins from the previously ischemic tissue.

Small volume resuscitation of hypovolemic hemorrhage shock using

7.5% NaCl solution and a combination of 7.5% NaCl and 6% Dextran-70 has been extensively studied. These studies have shown that, when administered in volumes of 4-6 ml/kg to animals, treatment results in a rapid improvement of blood pressure and near normalization of cardiac output, vital organ perfusion, and O₂ delivery. However, in patients with internal injury, pre-hospital resuscitation before surgical intervention has led to increased bleeding secondary to a rapid rise in blood pressure with concomitant reopening of clotted and tamponaded vascular injuries. This phenomenon in uncontrolled hemorrhage has been demonstrated in different animal models in which mortality was increased subsequent to resuscitating or reperfusion treatment.

Although hypertonic saline rapidly improves both blood pressure and cardiac output, these beneficial effects may be overshadowed by deleterious effects from increased blood pressure. Uncontrolled internal bleeding in trauma patients is aggravated by increased pressure, leading to increased internal bleeding. Return of normal blood pressure, resulting in increased bleeding due to an increase of arterial pressure, may lead to increased mortality as compared to no treatment.

Hypertonic saline infusions in shocked animals and patients have been shown to cause an initial acidosis and hypokalemia. Circulatory shock is often associated with an acidosis and thus increased acidotic insult may be deleterious. Treatment with hypertonic saline can also lead to a hyperchloremic acidosis, possibly due to excessive chloride ion load. Some isotonic Ringers solutions and mildly hypertonic formulations mimic sodium and chloride concentration ratios found in plasma and are thought to decrease the likelihood of acidosis. U.S. Patent No. 5,130,230 discloses a blood substitute that comprises an aqueous solution of electrolytes at physiological concentration, a macromolecular oncotic agent, a biological buffer having a buffering capacity in the range of physiological pH, simple nutritive sugar or sugars, magnesium ion in a concentration sufficient to substitute for the flux of calcium ion across cell membranes, and an anticoagulant. The blood substitute also includes a cardioplegic agent, such as potassium ion, in a concentration sufficient to prevent or arrest cardiac fibrillation.

Absent, however, in prior publications is the observation that revascularization creates potentially profound consequences both locally and systemically. Local complications include reperfusion injury of muscle resulting in swelling, development of compartment syndrome, and compressive muscle necrosis. Systemic complications include development of renal dysfunction due to myoglobinuria, myocardial arrhythmias, ventilatory embarrassment, and fatal cardia-pulmonary failure.

Limb ischemia as a consequence of local mural thrombus or distal embolus which occludes the arterial supply of the leg results in both local tissue injury and a systemic inflammatory response. The local injury can result in muscle death with release of sequestered intramuscular products which are deleterious to the patient including hyperkalemia, lactic acidosis and myoglobinuria. The hyperkalemia and lactic acidosis can result in cardiac arrythmias and cardiac arrest, while the myoglobinuria can cause acute tubular necrosis and renal failure. The applicants have delineated the time course of muscle necrosis following ischemia and the relationship between intramuscular adenine nucleotide depletion and tissue survival. Harris, K., Walker, P., Mickle,

D., Harding R., Gatley, R., Wilson, G., Kuzon, B., McKee, N. and, Romaschin,

A., "Metabolic Response of Skeletal Muscle to Ischemia, " American J. of

Physiology 250(19): H213-H220, 1986. Labbe, R., Lindsay, T., Walker, P., "The Extent and Distribution of Skeletal Muscle Necrosis Following Graded

Periods of Normothermic Ischemia, " J. Vascular Surgery 6(s): 152-157, 1987.

These studies have defined the major variables which affect the extent of muscle necrosis including muscle temperature, collateral flow and duration of ischemia. Petrasek, P., Homes-Vanniasinkam, S., Walker, P., "Determinants of Ischemic

Injury to Skeletal Muscle, " J. of Vascular Surgery 19(4): 623-631, 1994.

Previous studies by the applicants have identified products of oxidant mediated membrane injury in skeletal muscle during ischemia but particularly following reperfusion. Lindsay, T., Walker, P., Mickle, D., Romaschin, A., "Measurement of Hydroxy-Conjugated Dienes After Ischemia/Reperfusion in

Canine Skeletal Muscle, " American J. of Physiology 254(3): H578, 1988.

Alternation of oxidants with free radical scavengers (superoxide diomatase and catalase) resulted in significant muscle salvage (> 40% compared to untreated control muscle). Walker, P., Lindsay, T., Labbe, R., Mickle, D., and Romaschin, A., "Salvaging Skeletal Muscle with Free Radical Scavengers, " J. of Vascular Surgery 5(1): 68, 1987. Other strategies to minimize reperfusion induced injury employing controlled re-oxygenation with a stroma free crystalloid solution containing hydroxy-ethyl starch have been successful.

Rubin, B., Titley, Jr., Chang, G., Smith, A., Liauw, K., Romaschin., A., and Walker, P., "A Clinically Applicable Method for Long Term Salvage of Post

Ischemic Skeletal Muscle, " J. of Vascular Surgery 13(1): 58-68 1991. In these studies, removal of plasma proteins and white cells during the first 20 minutes of reperfusion following 5 hours of ischemia resulted in a significant muscle salvage. These studies demonstrated that plasma proteins such as complement and polymorphonuclear white cells play a key role in initiating reperfusion injury and that a brief period of controlled reperfusion excluding these components had a major effect on local muscle salvage. Further studies by the applicants have demonstrated that blood plasma effluent from ischemia muscle contains mediators which activate neutrophils. Petrasek, P., Lindsay, T., Romaschin, A., Walker, P., "Plasma Activation of Neutrophil CD 18 After Skeletal Muscle Ischemia - a Potential Mechanism for Late Systemic Injury, " American J. of Physiology 39(5): H1515-H1520, 1996. These mediators likely play a major role in the systemic dissemination of injury beyond the ischemic/reperfused muscle and may contribute to the surprisingly high and unexplained mortality (15-20%) associated with a relatively simple surgical procedure such as embolectory/thrombectomy. The combined use of anti- neutrophil adhesion protein antibody with fasciotomy has also been used by the applications to attemiate muscle necrosis. Petrasek, P., Liauw, S., Romaschin, A., Walker, P., "Salvage of Post-Ischemia Skeletal Muscle by Monoclonal Antibody Blockade of Neutrophil Adhesion Molecule CD 18, " J. of Surgical Research 56: 5-12, 1994.

SUMMARY AND OBJECTS OF THE INVENTION Accordingly, it is an object of this invention to provide a perfusate composition and method that alleviate the adverse effects associated with the previously known reperfusion compositions and methods.

Specifically, it is an object of this invention to provide a perfusate composition that minimizes free-radical-mediated injury, edema, and subsequent local muscle loss.

It is a further object of this invention to provide a pH-buffered perfusate composition that is electrolytically and osmotically balanced to biological conditions, provides reperfusion without causing edema, and includes biologically essential nutrients. It is a yet another object of this invention to provide a method whereby an ischemic limb is reperfused with a suitable amount of a perfusate that minimizes free radical damage to the affected tissue and reduces edema, muscle necrosis, and subsequent free-radical-induced systemic cascades that may result in failure of the heart, lungs, and kidneys.

These and other objects are addressed by providing a composition of matter comprising: (a) an amount of an oncotic agent sufficient to adjust colloid osmotic pressure of the composition to about 28 mm Hg;

(b) about 1-100 mM of an easily oxidizable energy substrate;

(c) about 5-200 mM of a free radical scavenger;

(d) about 0.1-10 mM of magnesium; (e) a sufficient amount of a biologically compatible buffer wherein the pH of the composition is in the range of physiological pH; and

(f) a mixture of electrolytes comprising about 115-175 mM sodium, 0.5-4 mM potassium, 90-160 mM chloride, and 0.25-0.45 mM calcium.

Preferably, the oncotic agent is a member selected from the group consisting of serum albumin and glucan polymers and mixtures thereof.

Preferred glucan polymers are selected from the group consisting of low molecular weight starches and waxy starches comprising hydroxyethyl ether groups introduced into alpha (1-4) linked glucose units and mixtures thereof. A preferred amount of the oncotic agent is about 5-10% (w/v). It is also preferred that the easily oxidizable energy substrate is a member selected from the group consisting of simple sugars and amino acids and mixtures thereof. Preferred simple sugars are selected from the group consisting of glucose, fructose, and sucrose and mixtures thereof, and preferred amino acids are selected from the group consisting of glutamate and aspartate and mixtures thereof.

A preferred free radical scavenger is a member selected from the group consisting of vitamin E, vitamin C, b-carotene, lipoic acid, and N-acetyl cysteine and mixtures thereof. A method of use of a composition of matter for treating ischemia comprising:

(a) inserting an arterial balloon cannula in an artery that supplies blood to the ischemic organ; (b) inserting a venous balloon cannula in a vein that conducts blood from the ischemic organ;

(c) administering a composition of matter through the arterial balloon cannula and perfusing the ischemic organ therewith, wherein the composition comprises (i) an amount of an oncotic agent sufficient to adjust colloid osmotic pressure of the composition to about 28 mm Hg;

(ii) about 1-100 mM of an easily oxidizable energy substrate; (iii) about 5-200 mM of a free radical scavenger; (iv) about 0.1-10 mM of magnesium; (v) a sufficient amount of a biologically compatible buffer wherein the pH of the composition is in the range of physiological pH; and

(vi) a mixture of electrolytes comprising about 115-175 mM sodium, 0.5-4 mM potassium, 90-160 mM chloride, and 0.25-0.45 mM calcium; and

(d) collecting venous effluent through the venous balloon cannula.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Before the present composition and method for treating ischemia of the limb are disclosed and described, it is to be understood that this invention is not limited to the particular configurations, process steps, and materials disclosed herein as such configurations, process steps, and materials may vary somewhat. It is also to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof. It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a composition containing "a buffer" includes a mixture of two or more of such buffers, reference to "an oncotic agent" includes reference to one or more of such oncotic agents, and reference to "a free radical scavenger" includes reference to a mixture of two or more free radical scavengers.

In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

As used herein, a "biologically acceptable" or "biologically compatible" component is one that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio.

Also, as it will be understood by those skilled in the art, the composition of the present invention is useful for treatment of different ischemic organs, including limbs.

The pH of the presently claimed composition or perfusate should be maintained in the range of physiological pH, generally between about pH 6.8 and 7.9. More preferably, the pH is maintained in the range of about pH 7.4 to 7.8, and most preferably about pH 7.6. The pH is maintained by the use of biologically acceptable or compatible buffers. Such buffers have buffering capacities in the range of physiological pH between about 6.8 and 7.9, but may have also have a buffering capacity outside the range of physiological pH. Many such biologically compatible buffers are well known to a person of ordinary skill in the art. One buffer suitable for use in the perfusate according to the invention is N-2-hydroxyethylpiperazine-N-2-hydroxypropanesulfonic acid (HEPES) buffer, which has a useful pH range between 6.8 and 8.2. Other buffers, such as 3-(N-morpholino) propanesulfonic acid (MOPS, pH range 6.5- 7.9); N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid; 2-([2-hydroxyl- l,l-bis(hydroxymethyl)ethyl] amino) ethanesulfonic acid (TES, pH range 6.8 - 8.2); 3-[N-tris(hydroxy- methyl) methylamino]-2-hydroxypropanesulfonic acid (TAPSO, pH range 7.2 - 8.2); 4-[2-hydroxyethyl]-l-piperazinepropanesulfonic acid (EPPS, pH range 7.3 - 8.7); tris(hydroxymethyl)aminomethane (THAM®, TRIS), sodium phosphate; and potassium phosphate, may be used. Such buffers will generally be used in sufficient amounts to maintain the pH of the presently claimed composition or perfusate in the range of physiological pH. Examplary concentrations of biologically compatible buffers may range from about 10 mM to 200 mM, and more preferably from 25 mM to 100 mM. Mixtures of buffers may also be used.

The perfusate, according to the present invention, also includes a concentration of divalent metal ions of a type and in an amount sufficient to displace or block the effects of calcium ion at the cellular membrane. Magnesium ion is preferred in this regard, and it is preferable to supply the magnesium ion by the addition of a chloride salt of magnesium. The additional magnesium ion in the perfusate is believed to displace calcium ion in the so- called cellular membrane calcium channel. The magnesium ion preferably should be present in an amount in the range of about 0.1-10 mM. In one preferred embodiment of the invention, the perfusate contains about 0.5-2.0 mM Mg ^" in the form of MgCl₂

It is also preferable that the formulation contain additional electrolytes. Examplary preferred formulations and ranges of such electrolytes approximately are as follows: 115-175 mM sodium; 0.5-4 mM potassium, and more preferably 2-3 mM potassium; 90-160 mM chloride; and 0.25-0.45 mM calcium. Reasonable variations of the preferred ranges of electrolytes as understood by those skilled in the art are also within the scope of the present invention.

The perfusate should also contain an oncotic agent. By "oncotic agent" is meant substances, generally macromolecules, that are of a size that is unable to leave the circulation by traversing the fenestrations of the capillary bed. Such oncotic agents are exemplified by blood plasma expanders, which are known in general as macromolecules having a size sufficient to prevent their escape from the blood plasma through the circulatory capillary bed into the interstitial spaces of the body. Human serum albumin is one well known plasma protein that is used to expand plasma volume. Other well known blood plasma expanders include dextran, hetastarch, and polygelatin. Polysaccharide blood plasma expanders are generally characterized as glucan polymers. Pentastarch is an artificial colloid derived from a waxy starch composed almost entirely of hydroxyethyl ether groups introduced into the alpha (1-4) linked glucose units.

Other polysaccharide derivatives may be suitable as oncotic agents in the perfusate according to this invention. Among such other polysaccharide derivatives are cross linked hydroxy ethyl starch products. In the perfusate according to this invention it is desirable that the polysaccharide be sufficiently large such that it not escape from the capillary bed of the treated vasculature.

Low molecular weight starches having a molecular weight of between about

200,000 to 300,000 are generally preferred.

The concentration of the oncotic agent in the perfusate is sufficient to achieve, when taken together with electrolytes and simple sugar discussed below, a colloid osmotic pressure approximating that of normal human serum, about 28 mm Hg. In particular, when Dextran 40 is used, about 6% dextran 40

(w/v) or about 60 grams (g) per liter (1) of water is used. By way of example, in currently preferred embodiments oncotic agents are generally present in amounts ranging from about 5.0 to 10.0% (w/v).

The perfusate according to the invention also includes a readily oxidizable energy substrate, such as simple sugars, amino acids, and the like. The concentration of the readily oxidizable substrate will generally be in a range of from about 1 mM to about 100 mM. Simple sugars include sucrose, fructose, and glucose or dextrose (alpha-D-glucose); most preferred is dextrose. Glucose at a concentration of about 3.6 g per liter or about 20 mM dextrose is preferred. Additionally the perfusate of the present invention may include organic compounds that are easily oxidizable under biological conditions. Such easily oxidizable organic compounds would be glycolized and simultaneously enter the mitochondria (such as glutamate and aspartate). The presence of these easily metabolized compounds provides an accessible energy source for the damaged cells, further facilitating cellular recovery. Depending upon the specific purpose for which the perfusate is to be used the concentration of the nutritive sugar may be further varied in a range between about 1 mM and 1 M. Thus, if the blood substitute is to be used to maintain a subject during a surgical procedure the lower concentration of dextrose of about 10 to 20 mM is used. To be included in a preferred embodiment in the perfusate is an osmolality adjusting agent. A preferred osmolality adjusting agent is mannitol. The inclusion of mannitol is beneficial for adjusting the final osmolality of the perfusate without the need to reduce or augment the concentrations of other components. One of skill in the art will readily perceive alterations in concentration that would further the utility of the perfusate of this invention. Osmolality of the perfusate according to the invention will be in a range of about 300 to 450 milliosmoles with an osmolality of about 405 to 415 being preferred. In addition to the above ingredients, it is advantageous to add certain specific antioxidants or free radical scavengers including b-carotene, vitamin E (d-alpha-tocopherol), N-acetyl cysteine, lipoic acid, and vitamin C (ascorbic acid). Lipophilic (e.g. vitamin E) or hydrophilic (e.g. N-acetyl cysteine, vitamin C) antioxidants or a mixture thereof can be used in the formulation. These substances provide a protective effect against free radicals and oxidative damage that can occur in an ischemic limb. These ingredients can be added in the following amounts: vitamin E, 0-100 mM; b-carotene, 0-100 mM; N-acetyl cysteine, 5-100 mM; vitamin C, 1-50 mM; lipoic acid, 0-50 mM. The concentration of free radical scavengers in the formulation should generally be in the range of about 5-200 mM, and preferably about 20-100 mM.

Optional ingredients that can be added to the formulation include stroma free hemoglobin (e.g. "HEMASOL"), acetyl-carnitine, desferal (as an iron chelator), and SCR1 (a complement regulatory protein for preventing edema). Carnitine, (3-carboxy-2-hydroxypropyl)trimethylammonium hydroxide, inner salt; (CAR), one of the nutrients supplied mainly from meat and dairy products, is an essential cofactor for many metabolic interactions in the body. The function of CAR to excrete an excess amount of organic acids upon their precipitation in the body has been clearly demonstrated in acidemia such as propionic acidemia and methylmalonic aciduria. Organic acids precipitated in the mitochondria form acyl-coenzyme A (CoA), which may be harmful because of the disturbance in the balance of the ratio between acetyl and acyl-CoA in the mitochondria. Carnitine, in this situation, accepts organic acid from the corresponding acyl CoA. Carnitine facilitates the excretion of organic acids from the mitochondria by the reverse sequence of the reaction responsible for the influx of fatty acids into the mitochondria. Each alternative additive could be included in a concentration commensurate with envisioned purpose in ranges of about 0 to 25 mM. EXAMPLE 1 By way of example and not limitation, the following formulae represent specific embodiments of the invention. They may be prepared by dissolving solid ingredients in sterile distilled water and/or mixing solutions of such ingredients in proper proportions. Formulation I

1.5% (w/v) low molecular weight starch (Pentaspan; oncotic agent)

25 mM sodium phosphate/citrate buffer, pH 7.65

20 mM N-acetyl cysteine 5 mM vitamin E

5 mM vitamin C

25 mM glucose

2.5 mM glutamate

2.5 mM aspartate 130 mM sodium chloride

3 mM potassium chloride

0.45 mM calcium chloride

0.5 mM magnesium chloride

Formulation II

6% (w/v) dextran 40 (oncotic agent)

50 mM Tris buffer, pH 7.6

25 mM N-acetyl cysteine

10 mM vitamin C 30 mM glucose

5 mM glutamate mannitol (adjust to 350 mosmol/kg) stroma free hemoglobin 10 mM

115 mM sodium chloride 2 mM potassium chloride

0.3 mM calcium chloride

1.0 mM magnesium chloride

Formulation III 7.5% PENTASTARCH 20 mM dextrose

50 mM Tris buffer + 1.0 mM dibasic sodium phosphate, pH 7.6 140 mM sodium chloride 2.8 mM potassium chloride 2.5 mM glutamate 2.5 mM aspartate 0.4 mM calcium chloride 1.0 mM magnesium chloride 25 mM N-acetyl cysteine 5 mM ascorbic acid

Formulation IV 10% PENTASPAN 30 mM glucose 50 mM TES buffer, pH 7.5 120 mM sodium chloride 3.0 mM potassium chloride 5 mM aspartate

0.4 mM calcium chloride 0.8 mM magnesium chloride 10 mM N-acetyl cysteine 5 mM vitamin C 2.3 mM b-carotene 5 mM acetyl-carnitine

Formulation V

8% (w/v) PENTASTARCH 33 mM glucose

75 mM Tris buffer, pH 7.8 130 mM sodium chloride 2.5 mM potassium chloride

3 mM glutamate 2 mM aspartate

0.4 mM calcium chloride 1.2 mM magnesium chloride 25 mM N-acetyl cysteine

4 mM vitamin C 1 mM lipoic acid

1 mM desferal 1 mM SCR1 Formulation VI

5% (w/v) dextran 40 25 mM glucose 25 mM fructose 100 mM MOPS buffer, pH 7.6 150 mM sodium chloride 2.2 mM potassium chloride 0.25 mM calcium chloride 2 mM magnesium chloride 25 mM N-acetyl cysteine 10 mM ascorbic acid 10 mM acetyl carnitine

Formulation VII 7.5% (w/v) PENTASPAN 50 mM glucose 50 mM Tris buffer, pH 7.4 140 mM sodium chloride 2 mM potassium chloride 5 mM glutamate

0.3 mM calcium chloride 0.5 mM magnesium chloride 100 mM N-acetyl cysteine 50 mM vitamin C 1 mM desferal

Formulation VIII

7.5% (w/v) PENTASPAN

50 mM sucrose 100 mM THAM buffer, pH 7.7

150 mM sodium chloride

3 mM potassium chloride

2.5 mM glutamate

2.5 mM aspartate 0.4 mM calcium chloride

1 mM magnesium chloride 25 mM N-acetyl cysteine

2 mM vitamin C

1 mM stroma free hemoglobin Formulation IX pH may be up to 8.0 at room temperature - for example, pH 7.6 at 37°

7.5% (w/v) Hydroxyethyl starch (molecular weight 200,000) 20 mM dextrose

50 mM Tris buffer

115 mM sodium chloride

1.0 mM sodium phosphate (di-basic)

2.8 mM potassium chloride 5.0 mM glutamic acid

5.0 mM aspartic acid

0.4 mM calcium chloride

1.0 mM magnesium chloride

5.0 mM ascorbic acid (Vitamin C)

N-Acetyl-Cysteine (Mucomyst or Parvolex) - 25 mM added immediately prior to perfusion (60cc of 1.225 M commercial prepartion 3L perfusate)

The perfusate of the present invention can be administered through the use of a specialized arterial cannula composed of two main segments. This cannula provides simultaneous occlusion and perfusion capabilities in the arterial circulation vasculature. The artery is first occluded by the occlusion segment thereby facilitating the incorporation of the pure perfusate into the vasculature. The perfusate solution is next introduced into the circulatory system in the region to be treated through the same occlusion-perfusion cannula.

The perfusate infused into the muscle tissue is drained out through a retrograde venous cannula composed of an occlusive balloon at the distal end of the cannula and fenestrations proximal to the balloon through which the perfusate enters the cannula. In this manner the perfusate is introduced into the region of the limb in need thereof and subsequently drained from the limb. The composition is infused at a pO₂ not to exceed about 150 mm Hg. EXAMPLE 2 In this example, the ischemic lower extremity of a patient in need of treatment therefor is perfused with a composition according to the present invention to control edema, minimize free radical mediated injury, and provide a fluid that is hypocalcemic, alkalotic, and contains substrates that can be glycolized and simultaneously enter the mitochondria.

An arterial balloon catheter is inserted into the common femoral artery through an arteriotomy performed by standard anesthesia and vascular surgery procedures. A venous balloon cannula is placed in the common femoral vein proximal to the deep femoral vein via a small venotomy using a purse string suture to secure the cannula. A blood collection bag to collect venous perfusate is connected to the femoral venous balloon cannula. Using standard perfusion procedures, the ischemic organ, for example, limb such as an arm or a leg, is perfused for 15-20 minutes with a perfusate composition according to Example 1 at a rate of 100-200 ml/min at a pressure of 80-150 mmHg via a Sarns single chamber perfusion pump or rigid pressure bag attached to the femoral arterial balloon cannula. The femoral venous effluent is collected at the same rate.

Claims

What is claimed is:

1. A composition of matter comprising:

(a) an amount of an oncotic agent sufficient to adjust colloid osmotic pressure of the composition to about 28 mm Hg; (b) about 1-100 mM of an easily oxidizable energy substrate;

(c) about 5-200 mM of a free radical scavenger;

(d) about 0.1-10 mM of magnesium;

(e) a sufficient amount of a biologically compatible buffer, wherein the pH of the composition is in the range of physiological pH; and

2. The composition of claim 1 wherein said oncotic agent is a member selected from the group consisting of albumin and glucan polymers and mixtures thereof.

3. The composition of claim 2 wherein said oncotic agent is a glucan polymer selected from the group consisting of low molecular weight starches and waxy starches comprising hydroxyethyl ether groups introduced into alpha (1-4) linked glucose units and mixtures thereof.

4. The composition of claim 1 wherein amount of oncotic agent is about 5-10% (w/v).

5. The composition of claim 1 wherein said easily oxidizable energy substrate is a member selected from the group consisting of simple sugars and amino acids and mixtures thereof.

6. The composition of claim 5 wherein said simple sugars are selected from the group consisting of glucose, fructose, and sucrose and mixtures thereof, and said amino acids are selected from the group consisting of glutamate and aspartate and mixtures thereof.

7. The composition of claim 1 wherein said free radical scavenger is a member selected from the group consisting of vitamin E, vitamin C, b- carotene, lipoic acid, and N-acetyl cysteine and mixtures thereof.

8. The composition of claim 1 wherein said biologically compatible buffer is a member selected from the group consisting of N-2- hydroxyethylpiperazine-N-2-hydroxypropanesulfonic acid; 3-(N-morpholino) propanesulfonic acid; N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid; 2-([2-hydroxyl-l,l-bis(hydroxymethyl)ethyl]amino) ethanesulfonic acid; 3-[N-tris(hydroxy- methyl) methylamino]-2-hydroxypropanesulfonic acid; 4-[2- hydroxyethyl]- 1-piperazinepropanesulfonic acid; tris(hydroxymethyl)aminomethane, sodium phosphate; and potassium phosphate and mixtures thereof.

9. The composition of claim 1 wherein amount of said biologically compatible buffer is about 10-200 mM.

10. The composition of claim 1 wherein amount of said biologically compatible buffer is about 25-100 mM.

11. The composition of claim 1 wherein amount of said magnesium is about 0.5-2.0 mM.

12. The composition of claim 1 wherein amount of said potassium is about 2-3 mM.

13. The composition of claim 1 further comprising 0-25 mM of a member selected from the group consisting of stroma free hemoglobin, desferal, acetyl carnitine, and SCR1 and mixtures thereof.

14. The composition of claim 1 further comprising an amount of an osmolality adjusting agent sufficient to adjust osmolality of the composition from 300-420 milliosmoles.

15. The composition of claim 14 wherein the osmolality of the composition is about 405-415 milliosmoles.

16. The composition of claim 14 wherein the osmolality adjusting agent is mannitol.

17. A medical kit comprising the composition according to claim 1, an arterial cannula for administering said composition, and a venous cannula for collecting venous effluent containing said composition.

18. A method of use of a composition of matter for treating ischemia comprising:

(c) administering a composition of matter through the arterial balloon cannula and perfusing the ischemic organ therewith, wherein the composition comprises: (i) an amount of an oncotic agent sufficient to adjust colloid osmotic pressure of the composition to about 28 mm Hg;

(ii) about 1-100 mM of an easily oxidizable energy substrate; (iii) about 5-200 mM of a free radical scavenger;

(iv) about 0.1 - 10 mM of magnesium; (v) a sufficient amount of a biologically compatible buffer, wherein the pH of the composition is in the range of physiological pH; and (vi) a mixture of electrolytes comprising about 115-

175 mM sodium, 0.5-4 mM potassium, 90-160 mM chloride, and 0.25-0.45 mM calcium; and (d) collecting venous effluent through the venous balloon cannula.

19. The method of claim 18 wherein said oncotic agent is a member selected from the group consisting of serum albumin and glucan polymers and mixtures thereof.

20. The method of claim 19 wherein said oncotic agent is a glucan polymer selected from the group consisting of low molecular weight starches and waxy starches comprising hydroxyethyl ether groups introduced into alpha (1-4) linked glucose units and mixtures thereof.

21. The method of claim 18 wherein amount of oncotic agent is about 5-10% (w/v).

22. The method of claim 18 wherein said easily oxidizable energy substrate is a member selected from the group consisting of simple sugars and amino acids and mixtures thereof.

23. The method of claim 22 wherein said simple sugars are selected from the group consisting of glucose, fructose, and sucrose and mixtures thereof, and said amino acids are selected from the group consisting of glutamate and aspartate and mixtures thereof.

24. The method of claim 18 wherein said free radical scavenger is a member selected from the group consisting of vitamin E, vitamin C, b-carotene, lipoic acid, and N-acetyl cysteine and mixtures thereof.

25. The method of claim 18 wherein said biologically compatible buffer is a member selected from the group consisting of N-2- hydroxyethylpiperazine-N-2-hydroxypropanesulfonic acid; 3-(N-morpholino) propanesulfomc acid; N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid; 2-([2-hydroxyl-l,l-bis(hydroxymethyl)ethyl]amino) ethanesulfonic acid; 3-[N-tris(hydroxy- methyl) methylamino]-2-hydroxypropanesulfonic acid; 4-[2- hydroxy ethyl]- 1 -piperazinepropanesulfonic acid; tris(hydroxymethyl)aminomethane, sodium phosphate; and potassium phosphate and mixtures thereof.

26. The method of claim 18 further comprising 0-25 mM of a member selected from the group consisting of stroma free hemoglobin, desferal, acetyl carnitine, and SCR1 and mixtures thereof.

27. The method of claim 18 further comprising an amount of an osmolality adjusting agent sufficient to adjust osmolality of the composition to about 300-420 milliosmoles.

28. The method of claim 27 wherein the osmolality of the composition is about 405-415 milliosmoles.

29. The method of claim 27 wherein the osmolality adjusting agent is mannitol.

30. The method of claim 18 wherein the ischemic organ is a limb.

31. The method of claim 30 wherein the limb is a leg.

32. The method of claim 30 wherein the limb is an arm.