HK1178087A - Methods for performing a coronary artery bypass graft procedure - Google Patents

Abstract

The invention provides methods of treating an obstructive coronary artery disease in a mammalian subject. The methods comprise administering to the subject an effective amount of an aromatic-cationic peptide to subjects in need thereof, and performing a coronary artery bypass graft procedure on the subject.

Description

Method for performing coronary artery bypass graft surgery

Cross Reference to Related Applications

Priority of the present application claims priority of united states provisional patent application No. 61/291699 filed on 12/31/2009, united states provisional patent application No. 61/363138 filed on 7/9/2010, and united states provisional patent application No. 61/406713 filed on 10/26/2010, which are incorporated herein by reference in their entirety.

Technical Field

The present invention relates generally to compositions and methods for the treatment of obstructive coronary artery disease using Coronary Artery Bypass Graft (CABG) surgery. In particular, the methods involve administering an effective amount of an aromatic-cationic peptide prior to CABG surgery, during CABG surgery, and/or after CABG surgery.

Background

The following description is provided to facilitate the understanding of the reader. None of the information provided or references cited is admitted to be prior art to the present invention.

Coronary Artery Bypass Graft (CABG) surgery is effective in alleviating angina and in improving survival and quality of life in patients with obstructive coronary artery disease. CABG surgery is one of the most common procedures performed in the world and results in more resources being spent in cardiovascular medicine than any other single procedure. In fact, nearly 500000 hospitalized patients underwent CABG surgery in 2006 in the United states. However, during perioperative and post-operative periods, myocardial infarction, ventricular failure, life-threatening arrhythmias, renal insufficiency, nerve damage, and death may occur. Patients in need of CABG belong to an older and older population and are characterized by co-morbid conditions (including advanced atherosclerosis), and in such patients, the incidence of such adverse events is expected to increase.

Left Ventricular (LV) function is an important predictor of early and late mortality following coronary surgery. Left ventricular function is associated with increased perioperative and distant mortality risks in patients undergoing coronary artery bypass surgery compared to patients with normal LV function. Low Ejection Fraction (EF) and clinical heart failure indicate higher surgical mortality in CABG surgery. It has recently been reported that post-operative N-terminal brain natriuretic peptide precursor (NT-proBNP) concentrations are associated with a higher mortality in hospitalization and a prolonged Intensive Care Unit (ICU) stay (> 4 days) after CABG surgery.

Any increase in creatine kinase isoenzyme fragment (CK-MB) following CABG surgery suggests myocardial cell necrosis, and higher CK-MB concentrations may be associated with worse outcomes. A linear relationship between postoperative CK-MB elevation and mortality has been reported as follows: post-operative CK-MB peaks of less than 5-fold, 5-fold to less than 10-fold, 10-fold to less than 20-fold, and greater than 20-fold normal upper limit were associated with six-month mortality of 3.4%, 5.8%, 7.8%, and 20.2%, respectively. A recent consensus document suggests defining post-CABG myocardial infarction based on elevated CK-MB levels of at least 5-fold above the normal upper limit during the first 72 hours after CABG surgery in association with the emergence of new pathological Q-wave or left bundle branch block, imaging evidence of new graft or autologous coronary artery occlusion recorded by angiography, or new depletion of viable myocardium.

Finally, if it is intended to improve the efficacy of CABG patients, it would be important to develop better methods for preventing myocardial ischemia reperfusion injury, an important mechanism leading to increased cardiovascular morbidity and mortality. Furthermore, the development of new adjunctive therapies and targeting them to reduce or minimize damage to other fragile terminal organs (e.g., kidney and brain) remains important for improving the efficacy of patients undergoing CABG.

Disclosure of Invention

The present invention relates generally to the treatment of obstructive coronary artery disease in a mammal by administering a therapeutically effective amount of an aromatic-cationic peptide to a subject in need thereof. In one aspect, the present invention provides a method for treating obstructive coronary artery disease, the method comprising: (a) a therapeutically effective amount of the peptide D-Arg-2 '6' -Dmt-Lys-Phe-NH₂Or a pharmaceutically acceptable salt thereof, to a mammalian subject in need thereof; and (b) performing a coronary artery bypass graft surgery on the subject. In another aspect, the present invention provides a method for preventing renal or cerebral complications during Coronary Artery Bypass Graft (CABG) surgery, the method comprising: (a) a therapeutically effective amount of the peptide D-Arg-2 '6' -Dmt-Lys-Phe-NH₂Or a pharmaceutically acceptable salt thereof, to a mammalian subject; and (b) performing coronary artery bypass graft surgery (CABG) on the subject.

In some embodiments, the aromatic-cationic peptide is a peptide having the following characteristics:

at least one net positive charge;

a minimum of four amino acids;

up to about 20 amino acids;

minimum number of net positive charges (p)_m) And the total number of amino acid residues (r) is: 3p of_mIs the maximum number less than or equal to r + 1; and aromatic hydrocarbonThe minimum number of aromatic groups (a) and the total number of net positive charges (p)_t) The relationship between them is: except that when a is 1, p_tMay be other than 1, 2a is p or less_tMaximum of +1. In one embodiment, 2p_mIs the maximum number less than or equal to r +1, and a may be equal to p_t. The aromatic-cationic peptide can be a water-soluble peptide having a minimum of two positive charges or a minimum of three positive charges.

In one embodiment, the peptide includes one or more non-naturally occurring amino acids, such as one or more D-amino acids. In some embodiments, the C-terminal carboxyl group at the C-terminus of the amino acid is amidated. In certain embodiments, the peptide has a minimum of 4 amino acids. The peptide may have up to about 6, up to about 9, or up to about 12 amino acids.

In one embodiment, the peptide includes a tyrosine residue or a 2 ', 6' -dimethyltyrosine (Dmt) residue at the N-terminus. For example, the peptide may have the formula Tyr-D-Arg-Phe-Lys-NH₂Or2 ', 6' -Dmt-D-Arg-Phe-Lys-NH₂. In another embodiment, the peptide comprises a phenylalanine residue or a 2 ', 6' -dimethylphenylalanine residue at the N-terminus. For example, the peptide may have the formula Phe-D-Arg-Phe-Lys-NH₂Or2 ', 6' -Dmp-D-Arg-Phe-Lys-NH₂. In certain embodiments, the aromatic-cationic peptide has the formula D-Arg-2 ', 6' -Dmt-Lys-Phe-NH₂(also referred to as SS-31).

In one embodiment, the peptide is defined by the following formula I:

wherein R is¹And R²Each independently selected from:

(i) hydrogen;

(ii) linear or branched C₁-C₆Alkyl groups of (a);

（iii）

wherein m =1-3;

（iv）

（v）

R³and R⁴Each independently selected from:

(i) hydrogen;

(ii) linear or branched C₁-C₆Alkyl groups of (a);

（iii）C₁-C₆alkoxy group of (a);

(iv) an amino group;

（v）C₁-C₄an alkylamino group of (a);

（vi）C₁-C₄a dialkylamino group of (a);

(vii) a nitro group;

(viii) a hydroxyl group;

(ix) halogen, wherein "halogen" includes chlorine, fluorine, bromine and iodine;

R⁵、R⁶、R⁷、R⁸、R⁹each independently selected from:

(i) hydrogen;

(ii) linear or branched C₁-C₆Alkyl groups of (a);

（iii）C₁-C₆alkoxy group of (a);

(iv) an amino group;

（v）C₁-C₄an alkylamino group of (a);

（vi）C₁-C₄a dialkylamino group of (a);

(vii) a nitro group;

(viii) a hydroxyl group;

(ix) halogen, wherein "halogen" includes chlorine, fluorine, bromine and iodine; and is

n is an integer of 1 to 5.

In certain embodiments, R¹And R²Is hydrogen; r³And R⁴Is methyl; r⁵、R⁶、R⁷、R⁸、R⁹Are all hydrogen; and n is 4.

In one embodiment, the peptide is defined by the following formula II:

wherein R is¹And R²Each independently selected from:

(i) hydrogen;

(ii) linear or branched C₁-C₆Alkyl groups of (a);

（iii）

wherein m =1-3;

（iv）

（v）

R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹and R¹²Each independently selected from:

(i) hydrogen;

(ii) linear or branched C₁-C₆Alkyl groups of (a);

（iii）C₁-C₆alkoxy group of (a);

(iv) an amino group;

（v）C₁-C₄an alkylamino group of (a);

（vi）C₁-C₄a dialkylamino group of (a);

(vii) a nitro group;

(viii) a hydroxyl group;

(ix) halogen, wherein "halogen" includes chlorine, fluorine, bromine and iodine; and is

n is an integer of 1 to 5

In certain embodiments, R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹And R¹²Are all hydrogen; and n is 4. In another embodiment, R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹And R¹¹Are all hydrogen; r⁸And R¹²Is methyl; r¹⁰Is a hydroxyl group; and n is 4.

The aromatic-cationic peptide can be administered in various ways. In some embodiments, the peptide can be administered orally, topically, intranasally, intraperitoneally, intravenously, subcutaneously, or transdermally (e.g., by iontophoresis).

In one embodiment, the peptide is administered to the subject prior to ischemia. In one embodiment, the peptide is administered to the subject prior to reperfusion of the ischemic tissue. In one embodiment, the peptide is administered to the subject at about the time of reperfusion of the ischemic tissue. In one embodiment, the peptide is administered to the subject after reperfusion of the ischemic tissue.

In one embodiment, the peptide is administered to the subject prior to CABG surgery. In another embodiment, the peptide is administered to the subject after CABG surgery. In another embodiment, the peptide is administered to the subject during and after CABG surgery. In another embodiment, the peptide is administered to the subject sequentially before, during, and after CABG surgery.

In one embodiment, administration of the peptide to the subject begins at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 3 hours, at least 5 hours, at least 8 hours, at least 12 hours, or at least 24 hours prior to CABG. In one embodiment, the peptide is administered to the subject at about 5 minutes to 30 minutes, from about 10 minutes to 60 minutes, from about 10 minutes to 90 minutes, or from about 10 minutes to 120 minutes prior to CABG surgery. In one embodiment, the peptide is administered to the subject until about 5 minutes to 30 minutes, about 10 minutes to 60 minutes, about 10 minutes to 90 minutes, or about 10 minutes to 120 minutes, or about 10 minutes to 180 minutes after CABG surgery.

In one embodiment, the peptide is administered to the subject at least 30 minutes, at least 1 hour, at least 3 hours, at least 5 hours, at least 8 hours, at least 12 hours, or at least 24 hours after CABG surgery. In one embodiment, the duration of administration of the peptide following CABG surgery is about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 8 hours, about 12 hours, or about 24 hours.

In one embodiment, administration of the peptide to the subject as an intravenous infusion begins about 1 minute to 30 minutes before reperfusion of the tissue (i.e., about 5 minutes, about 10 minutes, about 20 minutes, or about 30 minutes before reperfusion) and continues about 1 hour to 24 hours after reperfusion (i.e., about 1 hour, about 2 hours, about 3 hours, or about 4 hours after reperfusion). In one embodiment, the subject receives a bolus intravenous injection prior to reperfusion of the tissue. In one embodiment, the subject continues to receive the peptide chronically after the reperfusion period, i.e., for about 1 day to 7 days, about 1 day to 14 days, about 1 day to 30 days after the reperfusion period. During this period, the peptide may be administered by any route, such as subcutaneously or intravenously.

In one embodiment, the peptide is administered by systemic intravenous infusion initiated at about 5-60 minutes, about 10-45 minutes, or about 30 minutes prior to induction of anesthesia. In one embodiment, the peptide is administered in combination with cardioplegia. In one embodiment, the peptide is administered as a portion of priming solution in a heart-lung machine during cardiopulmonary bypass.

Drawings

Figure 1 shows a study design of animals used in the examples;

figures 2A and 2B show data representing infarct size in rabbits treated with sham surgery (ligatures applied, but not tightened). Fig. 2A is a photograph of a heart section of a sham operated rabbit treated with placebo and a computer generated image highlighting infarct size. FIG. 2B is a photograph of a heart section of a sham-operated rabbit treated with peptide and a computer generated image highlighting infarct size;

fig. 3A and 3B show data representing infarct size in two different control rabbits that induced myocardial ischemia and were treated with placebo. Each figure shows a photograph of a slice of the heart and a computer generated image highlighting the infarct size;

figures 4A, 4B, 4C, 4D, and 4E show data representing infarct sizes in five different rabbits induced myocardial ischemia and treated with exemplary aromatic-cationic peptides. Each figure shows a photograph of a slice of the heart and a computer generated image highlighting the infarct size;

FIG. 5 is a graph showing the ratio of infarct size to left ventricular area for control and test group rabbits;

FIG. 6 is a graph showing the ratio of infarct size to area at risk for control and test group rabbits.

Detailed Description

It is to be understood that certain aspects, modes, embodiments, variations and features of the present invention are described below in varying degrees of detail in order to provide a substantial understanding of the present invention.

The following provides definitions of some terms used in this specification. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like.

As used herein, "administering" a formulation, drug, or peptide to a subject includes any route by which a compound is introduced or administered to the subject to perform its intended function. "administration" may be carried out by any suitable route, including oral, intranasal, parenteral (by intravenous, intramuscular, intraperitoneal or subcutaneous) or topical administration. "administration" includes self-administration and administration by others.

The term "amino acid" as used herein includes naturally occurring amino acids and synthetic amino acids as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code and those amino acids which are later modified, such as hydroxyproline, γ -carboxyglutamic acid and O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha-carbon, a carboxyl group, an amino group, and an R group that are bound to a hydrogen, such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by commonly known three-letter symbols or one-letter symbols as recommended by the IUPAC-IUB Biochemical nomenclature Commission.

The term "effective amount" as used herein refers to an amount sufficient to obtain a desired therapeutic and/or prophylactic effect, e.g., an amount that results in the prevention or alleviation of cardiac ischemia-reperfusion injury or one or more conditions associated with cardiac ischemia-reperfusion injury. In the context of therapeutic or prophylactic use, the amount of the composition administered to a subject will depend on the type and severity of the disease and the nature of the individual, such as general health, age, sex, body weight and tolerance to drugs. The amount also depends on the extent, severity and type of the disease. One skilled in the art will be able to determine the appropriate dosage based on these and other factors. The composition may also be administered in combination with one or more other therapeutic compounds. In the methods described herein, the aromatic, cationic peptide can be administered to a subject having one or more symptoms or signs of vascular occlusion. In other embodiments, the mammal has one or more symptoms or signs of myocardial infarction, such as chest pain described as pressure sensation, fullness, or compression in the middle of the chest; chest pain radiating to the chin or teeth, shoulders, arms and/or back; dyspnea or breathlessness; upper abdominal discomfort with or without nausea and vomiting; and sweating or sweating. For example, a "therapeutically effective amount" of an aromatic-cationic peptide refers to an average level that minimally reduces the physiological effects of cardiac ischemia-reperfusion injury during CABG.

The term "ischemia reperfusion injury" as used herein refers to damage caused by: blood supply to a tissue is first restricted, followed by a sudden resupply of blood with concomitant production of free radicals. Ischemia is the reduction of blood supply to a tissue and reperfusion, a sudden influx of oxygen into the ischemic tissue, following ischemia.

An "isolated" or "purified" polypeptide or peptide is substantially free of cellular material or other contaminated polypeptide derived from a source of cells or tissue from which the agent was derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, an isolated aromatic cationic peptide will not have materials that would interfere with diagnostic or therapeutic uses of the agent. Such interfering materials may include enzymes, hormones, and other proteinaceous and non-proteinaceous solutes.

The terms "polypeptide", "peptide" and "protein" as used herein are interchangeable herein to refer to a polymer comprising two or more amino acids linked to each other by peptide bonds or modified peptide bonds, such as peptide isosteres. Polypeptides refer to short chains (often referred to as peptides, glycopeptides, or oligomers) as well as longer chains (often referred to as proteins). The polypeptide may include amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences that are modified by natural processes such as post-translational processing or chemical modification techniques well known in the art.

The term "simultaneous" therapeutic application as used herein refers to the administration of at least two active ingredients by the same route and at the same time or substantially at the same time.

The term "separate" therapeutic application as used herein refers to the administration of at least two active ingredients by different routes at the same time or at substantially the same time.

The term "sequential" therapeutic application as used herein refers to the administration of at least two active ingredients at different times, the routes of administration being the same or different. More specifically, sequential application refers to the complete administration of one of the active ingredients before the other active ingredients begin. Thus, it is possible to administer one active ingredient several minutes, hours or days before the other active ingredient. In this case no concurrent treatment was performed.

The terms "treatment" or "alleviating" as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the target condition or disorder. A subject is successfully "treated" for vasoocclusive injury if, upon receiving a therapeutic amount of an aromatic cationic peptide according to the methods described herein, the subject exhibits an observable and/or measurable reduction and disappearance of one or more symptoms and signs of the vasoocclusive injury, e.g., a reduction in infarct size. It will also be understood that the various modes of treating or preventing a medical condition described herein are intended to mean "significant," which includes complete treatment or prevention as well as less than complete treatment or prevention, in which some biologically or medically relevant result is achieved.

As used herein, "preventing" a disorder or condition refers to a compound that reduces the occurrence of the disorder or condition in a treated sample relative to an untreated control sample, or delays the occurrence or reduces the severity of one or more symptoms of the disorder or condition relative to an untreated control sample. As used herein, preventing renal or brain complications of CABG includes preventing or reducing, on a statistical sample, damage to the brain or kidney in a patient undergoing CABG. Prevention does not mean that the subject no longer develops a condition later in life, but merely that there is a reduced likelihood of occurrence.

Method for performing CABG surgery using aromatic cationic peptides

The present invention relates to the treatment or prevention of obstructive coronary artery disease by administering certain aromatic-cationic peptides in combination with CABG surgery. A method for treating or preventing cardiac ischemia reperfusion injury is also provided. In one aspect, the invention relates to a method of coronary revascularization comprising administering to a mammalian subject a therapeutically effective amount of an aromatic-cationic peptide and subjecting the subject to Coronary Artery Bypass Graft (CABG) surgery.

Certain aromatic-cationic peptides have been shown (including D-Arg-2 ', 6' -Dmt-Lys-Phe-NH)₂) Effective in a number of in vivo animal models with myocardial Ischemia Reperfusion (IR) injury. In the acute myocardial IR model, rabbits underwent 30 minutes of ischemia followed by 180 minutes of reperfusion. Infusion of D-Arg-2 ', 6' -Dmt-Lys-Phe-NH was initiated 20 min before reperfusion₂Or vehicle and the infusion continued throughout the experiment. Measurement by calculating the amount of LV at risk of infarction (measured by the amount of area of histologically non-absorbing Evans Blue dye)And) to infarct size (measured from the amount of the area not stained with triphenyltetrazolium chloride nitrogen).

In one aspect, the present invention relates to methods for using aromatic-cationic peptides as multi-organ protectants when administered preoperatively, intraoperatively, and immediately post-operatively in patients undergoing CABG surgery. Cardiopulmonary bypass is known to induce oxidative stress. During ischemic myocardial reoxidation and reperfusion, oxygen-derived free radicals (superoxide anion, hydroxyl anion, and hydrogen peroxide) are produced and the normal endogenous mechanisms for scavenging these free radicals are reduced. In most cases, these oxygen radicals are byproducts of cellular metabolism and are cleared or inactivated enzymatically by superoxide dismutase, catalase, and peroxidase, and by antioxidant receptors such as glutathione, vitamin E, and hemoglobin. The excessive production of Reactive Oxygen Species (ROS) during reperfusion of the myocardium causes damage to cell membranes and leakage of enzymes into the interstitial tissue, leading to the consumption of superoxide dismutase and catalase.

Mitochondria are the major intracellular source of ROS. Functionally, mitochondria are the initiator of oxidative stress and are the first target of oxidative stress. Mitochondrial damage can lead to cell death. This reflects the important role played by mitochondria in energy metabolism and calcium homeostasis, as well as their ability to release pro-apoptotic factors (such as cytochrome C) and apoptosis-inducing factors. Mitochondria are very sensitive to ischemia. Indeed, mitochondrial damage and dysfunction may occur even after periods of mildly reduced myocardial blood flow without immediate changes in the concentration of ATP or phosphocreatine.

Reperfusion injury is related at least in part to the problem of mitochondrial permeability transition. Ultimately, this results in the production of low ATP concentrations and altered ion homeostasis, leading to rupture of the cell membrane and cell death. Post-reperfusion arrhythmias are also associated with mitochondrial dysfunction. Previous attempts to use antioxidant therapy, calcium channel blockers, sodium-hydrogen exchange inhibitors, and anti-inflammatory drugs to address known mediators of ischemia reperfusion injury in patients alone were largely disappointing. This has led to the notion that: there is a need for a multi-objective mechanical approach for ischemia reperfusion injury that successfully translates experimental intervention into prevention of clinical manifestations of reperfusion injury, including: reperfusion arrhythmias, myocardial stunning, and muscle cell death and infarction.

This general approach to myocardial rescue at the cellular level in CABG surgery must involve therapy to prevent ischemia reperfusion injury while maintaining blood flow throughout the myocardial microcirculation. Ideally, this is most ideally achieved by combining the technically perfect CABG procedure with a therapeutic agent that achieves the dual goals of opening the major ductal artery with tight timing requirements and maintaining an open microvasculature. Unfortunately, because of the conflict between pathophysiological disorders in many hearts and CABG surgery, finding an effective therapy to reduce or prevent CABG-associated myocardial ischemia-reperfusion injury has proven difficult.

Certain aromatic-cationic peptides can target mitochondria. Uptake studies showed that: the peptide D-Arg-2 ', 6' -Dmt-Lys-Phe-NH₂Is six times higher than the intracellular concentration in the extracellular fluid, and the concentration of the drug in the mitochondrial particle is about 5000 times higher than the intracellular concentration in the extracellular fluid. Thus, the peptide is selectively absorbed by mitochondria. In conjunction with this aggregation in mitochondria, the peptide has been shown to have a number of unique characteristics, including: scavenging Reactive Oxygen Species (ROS), promoting electron transfer within the mitochondrial electron transport chain, maintaining mitochondrial respiration (oxygen consumption), maintaining Adenosine Triphosphate (ATP) levels, preventing the disappearance of mitochondrial membrane potential, preventing cytochrome c release, and preventing mitochondrial swelling consistent with inhibiting opening of the mitochondrial permeability transition pore (mPTP).

In some embodiments, the aromatic-cationic peptide is administered prior to, during, and/or immediately after coronary artery bypass graft surgery to prevent or treat renal complications of CABG surgery. In post-operative CABG patients, even minor increases in serum creatinine above baseline values are associated with adverse outcomes, and any degree of renal insufficiency, however small, even in the absence of complete loss of function, has significant clinical consequences. Perioperative injury, including ischemia reperfusion injury, can lead to the development of renal injury manifested by decreased Glomerular Filtration Rate (GFR) and increased serum creatinine concentration. Despite advances in coronary artery bypass graft technology, intensive care and hemodialysis over the past decade, morbidity and mortality associated with postoperative renal dysfunction have not changed significantly. Although different intra-operative strategies have been developed to provide renal protection to patients undergoing cardiovascular surgery, these strategies have focused primarily on the use of drugs such as dopamine, mannitol, and furosemide. However, pharmaceutical intervention has been shown to be unprotected to the kidney.

The peptide D-Arg-2 ', 6' -Dmt-Lys-Phe-NH has been shown₂Is effective in reducing ARI caused by ischemia-reperfusion. See U.S. patent publication No. 20090221514. In particular, the peptides are effective in reducing interstitial fibrosis, tubular apoptosis, macrophage infiltration, and tubular cell proliferation in animal models with ARI. The peptide significantly improved histopathological scores from 45 min ischemia and 24 hr reperfusion, and also significantly increased ATP production rate after reperfusion.

Risk factors associated with renal dysfunction in post-operative CABG patients can be divided into patient-related and surgery-related criteria. Patient-related factors include diabetes, hypertension, left ventricular dysfunction, and preexisting renal disease. For example, patients with preoperative renal serum creatinine (SCr) ≧ 1.5mg/dL are at greater risk for acute deterioration of postoperative renal function, prolonged mechanical ventilation, increased intensive care and hospitalization, and greater short-term and long-term mortality than subjects with lower preoperative renal serum creatinine values. Perioperative mortality is gradually increased and can reach 33 percent for non-dialysis patients with preoperative SCR of more than or equal to 1.7mg/dL and less than 2.5 mg/dL. Overall, patients with a reduced number of properly functioning nephrons before surgery are more sensitive to maldistributed and reduced renal blood flow, increased renovascular resistance and reduced glomerular filtration rates perioperatively and post-operatively.

The existing kidney disease greatly increases the risk of perioperative complications. Renal function declines with age, and renal insufficiency is the result of several common cardiovascular risk factors (e.g., hypertension and diabetes). Impaired renal function worsens the effects of these conditions and is associated with a number of other non-well-defined risk factors, including increased acute phase proteins, decreased antioxidants, and disturbances of calcium/phosphate metabolism. Renal insufficiency is also a common consequence of reduced left ventricular systolic function and heart failure. In addition, chronic kidney disease is itself a risk factor for left ventricular hypertrophy, left ventricular dilation, and left ventricular insufficiency.

The work in Acute Kidney Injury (AKI) defines the need for a sudden (within 48 hours) decline in renal function defined as either an absolute increase in serum creatinine levels of > 26.4 μmol/l (0.3 mg/dl) or a percent increase in serum creatinine levels of > 50% (1.5 fold above baseline) or a decrease in urine output (recorded oliguria < 0.5ml/kg/h for > 6 hours). It is assumed that these criteria apply in the context of clinical presentation and subsequent adequate fluid resuscitation when applicable. Overall, the Acute Kidney Injury Network (AKIN) proposed three grades describing an increase in serum creatinine relative to baseline and a decrease in post-operative urine output.

A number of common pathophysiological processes are thought to contribute to AKI associated with CABG surgery. Ischemic reperfusion injury, oxidative stress and inflammation are included in this list. In particular, these factors appear to act in an interrelated and possibly synergistic manner. Typically, renal perfusion is self-regulated in order to maintain glomerular filtration rate until mean arterial blood pressure drops below 80mm hg. Mean arterial blood pressure during cardiac surgery is often at a lower limit or below the limit of self-regulation, especially during hemodynamic instability. In addition, many cardiac surgery patients have impaired self-regulation due to the presence of co-morbidities (e.g., advanced age, atherosclerosis, chronic hypertension, or chronic kidney disease), administration of drugs that impair renal self-regulation (e.g., non-steroidal anti-inflammatory drugs, ACE inhibitors, angiotensin receptor blockers, and radiocontrast agents), or pro-inflammatory states. In patients with impaired self-regulation, renal function can deteriorate even when mean arterial blood pressure is within the normal range.

In CABG patients, these factors can lead to cellular ischemia with damage and activation of tubular epithelial and vascular endothelial cells. In addition, blockages of the microvasculature and tubules can occur, resulting in damage and a cytopenic vicious cycle. During maintenance, damage is stabilized when cell repair, cell division and cell redifferentiation occur, and the maintenance phase transitions to the recovery phase; or the sustained release of harmful mediators drives cellular responses toward inappropriate proliferation and fibrosis. Eventually, nucleoside depletion culminates in the accumulation of hypoxanthine and contributes to the production of reactive oxygen molecules. Small tubular oxidative stress is evident even in non-extracorporeal circulatory heart surgery and is exacerbated by cardiopulmonary bypass. Existing evidence suggests that apoptosis is the primary mechanism of early tubular cell death in AKI. The key step in apoptosis is the activation of caspases (cysteine aspartate-specific proteases) that are extremely functional during programmed cell death. Caspase activation occurs through pathways that control mitochondrial membrane permeability, which induce pores in the mitochondria to egress cytochrome c into the cytoplasm, and then activate the caspase cascade.

Cardiac surgery can also contribute to ischemic kidney injury by eliciting a strong systemic inflammatory response. Proinflammatory events during cardiac surgery include surgical trauma, contact of blood components with artificial surfaces of the CPB circuit, ischemia reperfusion injury, and endotoxemia. This systemic inflammatory response can lead to dysfunction of various end organs including the kidney, lungs, heart and brain.

The recognition that the AKI associated with CABG is a complex interaction between ischemia, endothelial dysfunction and tubular injury has led to the search for alternative methods of renal protection. The ability of certain aromatic cations to target all of these processes and to favorably affect multiple sites upstream, and in the termination of tubular and vascular injury, has led to the discovery that this molecule is a nephroprotective agent.

In some embodiments, the aromatic-cationic peptide is administered prior to, during, and/or immediately after CABG surgery to prevent or treat a brain complication of CABG surgery. Post-operative neurodegeneration has been reported in patients undergoing CABG surgery, particularly when CPB is used. See Terrando et al, Tumor neocross factor-alpha triggers a cytokine cassette and induced porous chemical definition, Proc Natl Acad Sci USA 107(47): 20518-. Despite many advances in cardiac surgery, perioperative and post-operative brain damage remains a problem. Reduced perfusion pressure during CPB and embolization of air or particulate matter during aortic cannulation or during CPB shutdown can produce neurological damage and complications of neuropsychiatric disease.

The peptide D-Arg-2 ', 6' -Dmt-Lys-Phe-NH has been shown₂Mice were protected from cerebral ischemia. See U.S. patent publication No. 0070129306. Treatment of wild type mice with the peptide at 0, 6, 24 and 48 hours after 30 minutes of occlusion of the middle cerebral artery resulted in a significant reduction in infarct volume and hemispherical swelling compared to saline-treated controls.

It has been demonstrated that: CPB can elicit a systemic inflammatory response that can contribute to the development of nerve damage in patients undergoing CABG surgery. This systemic inflammatory response can be mediated by trauma, exposure of blood to collateral circulation in vitro, and lung reperfusion injury following CPB discontinuation. The CPB-associated systemic inflammatory response is related to the concentrations of serum C-reactive protein, IL-6, IL-8, and cortisol. Attenuation of this systemic inflammatory response is an important therapeutic goal and is associated with increased efficacy.

In addition, local brain events can be detrimental to patients during cardiac surgery. The presence of cerebral ischemia itself induces a complex series of molecular pathways including signaling mechanisms, gene transcription and protein formation. Within seconds to minutes after the reduction of blood flow to the brain region, an ischemic cascade is initiated leading to a series of biochemical events that ultimately lead to the breakdown of cell membranes and neuronal death at the center/core of the infarction. Associated with these events are a combination of pathogenic effectors including oxidative stress, ATP depletion, excitotoxicity, inflammation, apoptosis, microvascular occlusion and disruption of the blood-brain barrier of the brain. Oxidative stress leads to ischemic cell death involving multiple injury mechanisms (including mitochondrial inhibition, Ca)²⁺Overload and ischemia reperfusion injury) to form ROS/reactive nitrogen. Local inflammation from ischemic injury is caused by activated microglia and infiltrating inflammatory cells that further release proinflammatory cytokines and ROS at the site of injury. These processes threaten the survival of ischemic brain tissue, since brain tissue is not well equipped with antioxidant and anti-inflammatory defenses. Cerebral ischemia reperfusion causes a significant shift towards pro-oxidative states in the brain. For this reason, the peptide D-Arg-2 ', 6' -Dmt-Lys-Phe-NH₂Can be used as a neuroprotective agent during CABG surgery.

In one embodiment, the peptide is administered to the subject during and after CABG surgery. In another embodiment, the peptide is administered to the subject sequentially before, during, and after CABG surgery. In one embodiment, administration of the peptide to the subject is initiated at least 10 minutes, at least 30 minutes, at least 1 hour, at least 3 hours, at least 5 hours, at least 8 hours, at least 12 hours, or at least 24 hours prior to CABG surgery. In one embodiment, the peptide is administered to the subject at least 3 hours, at least 5 hours, at least 8 hours, at least 12 hours, or at least 24 hours after CABG surgery. In one embodiment, administration of the peptide to the subject begins at least 8 hours, at least 4 hours, at least 2 hours, at least 1 hour, or at least 30 minutes prior to CABG surgery. In one embodiment, the peptide is administered to the subject at least one week, at least one month, or at least one year after CABG surgery.

The aromatic-cationic peptide is water-soluble and has strong polarity. Despite these properties, the peptide can still readily penetrate cell membranes. The aromatic-cationic peptide typically comprises at least 3 amino acids or at least 4 amino acids covalently linked by peptide bonds. The maximum number of amino acids present in the aromatic-cationic peptide is about 20 amino acids covalently linked by peptide bonds. Suitably, the maximum number of amino acids is about 12, more preferably about 9, and most preferably about 6.

The amino acid of the aromatic-cationic peptide can be any amino acid. The term "amino acid" as used herein is intended to mean any organic molecule comprising at least one amino group and at least one carboxyl group. Typically, at least one amino group is in the alpha position relative to the carboxyl group. The amino acids may be naturally occurring. For example, naturally occurring amino acids include the twenty most common L-amino acids such as those typically found in mammalian proteins, namely alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Other naturally occurring amino acids include amino acids synthesized in metabolic processes not related to protein synthesis, for example. For example, the amino acids ornithine and citrulline are synthesized during urea production in mammalian metabolism. Another example of a naturally occurring amino acid includes hydroxyproline (Hyp).

The peptide optionally includes one or more non-naturally occurring amino acids. Suitably, the peptide does not have naturally occurring amino acids. The non-naturally occurring amino acid can be a levorotatory (L-) amino acid, a dextrorotatory (D-) amino acid, or a mixture thereof. Non-naturally occurring amino acids are those amino acids: are not normally synthesized in the natural metabolic processes of an organism and occur non-naturally in proteins. Furthermore, non-naturally occurring amino acids are suitably not recognized by common proteases. Non-naturally occurring amino acids can be present in any position of the peptide. For example, the non-naturally occurring amino acid can be N-terminal, C-terminal, or anywhere between the N-terminal and C-terminal.

For example, an unnatural amino acid can include an alkyl, aryl, or alkylaryl group that is not present in a natural amino acid. Some examples of unnatural alkyl amino acids include α -aminobutyric acid, β -aminobutyric acid, γ -aminobutyric acid, δ -aminopentanoic acid, and ε -aminocaproic acid. Some examples of unnatural aryl amino acids include anthranilic acid, m-aminobenzoic acid, and p-aminobenzoic acid. Some examples of unnatural alkylaryl amino acids include o-, m-, and p-aminophenylacetic acids, and γ -phenyl- β -aminobutyric acid. Non-naturally occurring amino acids include derivatives of naturally occurring amino acids. Derivatives of the naturally occurring amino acids can include, for example, the addition of one or more chemical groups to the naturally occurring amino acid.

For example, one or more chemical groups may be added to the phenylalanine residue or to the 2 ', 3 ', 4 ', 5 ' or 6 ' position of the aromatic ring of the tyrosine residue or to the 4 ', 5 ', 6 ' or 7 ' position of the benzo ring of the tryptophan residue. The group may be any chemical group that can be added to an aromatic ring. Some examples of such groups include branched or unbranched C₁-C₄Alkyl groups of (a), such as methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, or tert-butyl; c₁-C₄Alkoxy group of(i.e., alkoxy); an amino group; c₁-C₄Alkylamino and C₁-C₄Dialkylamino groups of (e.g., methylamino, dimethylamino); a nitro group; a hydroxyl group; halogen (i.e., fluorine, chlorine, bromine, or iodine). Some specific examples of non-naturally occurring derivatives of naturally occurring amino acids include norvaline (Nva) and norleucine (Nle).

Another example of modification of amino acids in a peptide is derivatization of the carboxyl group of an aspartic acid or glutamic acid residue in the peptide. One example of derivatization is amidation with ammonia or a primary or secondary amine (e.g., methylamine, ethylamine, dimethylamine, or diethylamine). Another example of derivatization includes esterification with, for example, methanol or ethanol. Another such modification includes derivatization of the amino group of lysine, arginine or histidine residues. For example, these amino groups may be acylated. For example, some suitable acyl groups include benzoyl or include C as mentioned above₁-C₄An alkanoyl group of any one of the alkyl groups of (1), such as acetyl or propionyl.

Non-naturally occurring amino acids are suitably resistant to common proteases, and/or are insensitive. Examples of non-naturally occurring amino acids that are resistant or insensitive to proteases include the dextrorotatory (D-) form of any of the naturally occurring levorotatory (L-) amino acids mentioned above, as well as the levorotatory and/or dextrorotatory non-naturally occurring amino acids. Although dextrorotatory amino acids are present in certain peptide antibiotics synthesized by methods different from the conventional ribosomal protein synthesis methods of cells, dextrorotatory amino acids are not generally present in proteins. Dextrorotatory amino acids as used herein are considered to be non-naturally occurring amino acids.

To minimize protease sensitivity, the peptide should have less than 5, less than 4, less than 3, or less than 2 consecutive l-amino acids recognized by common proteases, regardless of whether the amino acids are naturally occurring. Preferably, the peptide has only dextrorotatory amino acids and no levorotatory amino acids. If the peptide has a protease-sensitive amino acid sequence, at least one of the amino acids is preferably a non-naturally occurring dextrorotatory amino acid, thereby providing protease resistance. Examples of protease sensitive sequences include two or more consecutive basic amino acids that can be easily cleaved by common proteases such as endopeptidases and trypsin. Examples of basic amino acids include arginine, lysine and histidine.

Aromatic-cationic peptides should have a minimum number of net positive charges at physiological pH compared to the total number of amino acid residues in the peptide. The minimum number of net positive charges at physiological pH will be referred to hereinafter as (p)_m). The total number of amino acid residues in a peptide will be referred to hereinafter as (r). The minimum number of net positive charges discussed below are all at physiological pH. The term "physiological pH" as used herein refers to the normal pH in cells of tissues and organs of the mammalian body. For example, the physiological pH of humans is typically about 7.4, while the normal physiological pH in mammals can be any pH from about 7.0 to about 7.8.

As used herein, "net charge" refers to the balanced remainder between the number of positive charges and the number of negative charges carried by the amino acids present in the peptide. In the present specification, it is understood that the net charge is measured at physiological pH. Naturally occurring amino acids that are positively charged at physiological pH include l-lysine, l-arginine, and l-histidine. Naturally occurring amino acids that are negatively charged at physiological pH include l-aspartic acid and l-glutamic acid.

Typically, peptides have a positively charged N-terminal amino group and a negatively charged C-terminal carboxyl group. The charges cancel each other out at physiological pH. As an example of calculating the net charge, the peptide Tyr-Arg-Phe-Lys-Glu-His-Trp-D-Arg has one negatively charged amino acid (i.e., Glu) and four positively charged amino acids (i.e., two Arg residues, one Lys and one His). Thus, the above peptides have 3 net positive charges.

In one embodiment, the aromatic-cationic peptide has a minimum net positive charge at physiological pHNumber (p)_m) And the total number of amino acid residues (r) has the following relationship: wherein, 3p_mIs the maximum number less than or equal to r +1. In this embodiment, the minimum number of net positive charges (p)_m) And the total number of amino acid residues (r) as follows:

TABLE 1 number of amino acids and net positive charge (3 p)_m≤p+1）

（r）	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20
																			（pm）	1	1	2	2	2	3	3	3	4	4	4	5	5	5	6	6	6	7

In another embodiment, the minimum number of net positive charges (p) of the aromatic-cationic peptide_m) And the total number of amino acid residues (r) has the following relationship: wherein, 2p_mIs the maximum number less than or equal to r +1. In this embodiment, the minimum number of net positive charges (p)_m) And the total number of amino acid residues (r) as follows:

TABLE 2 amino acid number and Net Positive Charge (2 p)_m≤p+1）

（r）	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20
																			（pm）	2	2	3	3	4	4	5	5	6	6	7	7	8	8	9	9	10	10

In one embodiment, the minimum number of net positive charges (p)_m) And amino acid residuesAre equal. In another embodiment, the peptide has three or four amino acid residues with a minimum number of net positive charges of 1, suitably a minimum number of net positive charges of 2 and more preferably a minimum number of net positive charges of 3.

Also important is the total number of aromatic-cationic peptides (p) compared to the net positive charge_t) With a minimum number of aromatic groups. The minimum number of aromatic groups will be referred to as (a) hereinafter. Naturally occurring amino acids having an aromatic group include the amino acids histidine, tryptophan, tyrosine and phenylalanine. For example, the hexapeptide Lys-Gln-Tyr-D-Arg-Phe-Trp has a net positive charge of 2 (provided by a lysine residue and an arginine residue) and three aromatic groups (provided by a tyrosine residue, a phenylalanine residue, and a tryptophan residue).

The aromatic-cationic peptide should also have a minimum number of aromatic groups (a) and a total number of net positive charges (p) at physiological pH conditions_t) Have the following relationship between: wherein, except when p_tWhen the number is 1, a may be other than 1, and 3a is p or less_tMaximum of +1. In this embodiment, the minimum number of aromatic groups (a) and the total number of net positive charges (p)_t) The relationship between them is as follows:

TABLE 3 aromatic groups and net positive charge (3 a. ltoreq. p)_t+1 or a = p_t=1）

（pt）	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20
																					（a）	1	1	1	1	2	2	2	3	3	3	4	4	4	5	5	5	6	6	6	7

In another embodiment, the aromatic-cationic peptide has a minimum number of aromatic groups (a) and a total number of net positive charges (p)_t) Have the following relationship between: wherein 2a is less than or equal to p_tMaximum of +1. In this embodiment, the minimum number of aromatic amino acid residues (a) and the total number of net positive charges (p)_t) The relationship between them is as follows:

TABLE 4 aromatic groups and net positive charge (2 a. ltoreq. p)_t+1 or a = p_t=1）

（pt）	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20
																					（a）	1	1	2	2	3	3	4	4	5	5	6	6	7	7	8	8	9	9	10	10

In another embodiment, the number of aromatic groups (a) and the total number of net positive charges (p)_t) Are equal.

The carboxyl group, and especially the terminal carboxyl group of the C-terminal amino acid, is suitably amidated with, for example, ammonia to form a C-terminal amide. Alternatively, the terminal carboxyl group of the C-terminal amino acid may be amidated with either a primary or secondary amine. The primary or secondary amine may be, for example, an alkyl group, especially a branched or unbranched C₁-C₄Alkyl of (2)A base or an aromatic amine. Thus, the amino acid at the C-terminal end of the peptide may be converted into an amide group, an N-methylamide group, an N-ethylamide group, an N, N-dimethylamide group, an N, N-diethylamide group, an N-methyl-N-ethylamide group, an N-phenylamide group, or an N-phenyl-N-ethylamide group. Free carboxylic acid groups of asparagine residues, glutamine residues, and aspartic acid and glutamic acid residues not present at the C-terminus of the aromatic cationic peptide can also be amidated, whether or not they are present within the peptide. Amidation of these internal positions can be with ammonia or any of the primary or secondary amines described above.

In one embodiment, the aromatic-cationic peptide is a tripeptide having two net positive charges and at least one aromatic amino acid. In a particular embodiment, the aromatic-cationic peptide is a tripeptide having two net positive charges and two aromatic amino acids.

Aromatic-cationic peptides include, but are not limited to, the following examples of peptides:

Lys-D-Arg-Tyr-NH₂

Phe-D-Arg-His

D-Tyr-Trp-Lys-NH₂

Trp-D-Lys-Tyr-Arg-NH₂

Tyr-His-D-Gly-Met

Phe-Arg-D-His-Asp

Tyr-D-Arg-Phe-Lys-Glu-NH₂

Met-Tyr-D-Lys-Phe-Arg

D-His-Glu-Lys-Tyr-D-Phe-Arg

Lys-D-Gln-Tyr-Arg-D-Phe-Trp-NH₂

Phe-D-Arg-Lys-Trp-Tyr-D-Arg-His

Gly-D-Phe-Lys-Tyr-His-D-Arg-Tyr-NH₂

Val-D-Lys-His-Tyr-D-Phe-Ser-Tyr-Arg-NH₂

Trp-Lys-Phe-D-Asp-Arg-Tyr-D-His-Lys

Lys-Trp-D-Tyr-Arg-Asn-Phe-Tyr-D-His-NH₂

Thr-Gly-Tyr-Arg-D-His-Phe-Trp-D-His-Lys

Asp-D-Trp-Lys-Tyr-D-His-Phe-Arg-D-Gly-Lys-NH₂

D-His-Lys-Tyr-D-Phe-Glu-D-Asp-D-His-D-Lys-Arg-Trp-NH₂

Ala-D-Phe-D-Arg-Tyr-Lys-D-Trp-His-D-Tyr-Gly-Phe

Tyr-D-His-Phe-D-Arg-Asp-Lys-D-Arg-His-Trp-D-His-Phe

Phe-Phe-D-Tyr-Arg-Glu-Asp-D-Lys-Arg-D-Arg-His-Phe-NH₂

Phe-Try-Lys-D-Arg-Trp-His-D-Lys-D-Lys-Glu-Arg-D-Tyr-Thr

Tyr-Asp-D-Lys-Tyr-Phe-D-Lys-D-Arg-Phe-Pro-D-Tyr-His-Lys

Glu-Arg-D-Lys-Tyr-D-Val-Phe-D-His-Trp-Arg-D-Gly-Tyr-Arg-D-Met-NH₂

Arg-D-Leu-D-Tyr-Phe-Lys-Glu-D-Lys-Arg-D-Trp-Lys-D-Phe-Tyr-D-Arg-Gly

D-Glu-Asp-Lys-D-Arg-D-His-Phe-Phe-D-Val-Tyr-Arg-Tyr-D-Tyr-Arg-His-Phe-NH₂

Asp-Arg-D-Phe-Cys-Phe-D-Arg-D-Lys-Tyr-Arg-D-Tyr-Trp-D-His-Tyr-D-Phe-Lys-Phe

His-Tyr-D-Arg-Trp-Lys-Phe-D-Asp-Ala-Arg-Cys-D-Tyr-His-Phe-D-Lys-Tyr-His-Ser-NH₂

Gly-Ala-Lys-Phe-D-Lys-Glu-Arg-Tyr-His-D-Arg-D-Arg-Asp-Tyr-Trp-D-His-Trp-His-D-Lys-Asp

Thr-Tyr-Arg-D-Lys-Trp-Tyr-Glu-Asp-D-Lys-D-Arg-His-Phe-D-Tyr-Gly-Val-Ile-D-His-Arg-Tyr-Lys-NH₂

in one embodiment, the peptide has mu-opioid (mu-opioid) receptor agonist activity (i.e., the peptide activates the mu-opioid receptor). Mu-opioid activity can be assessed by radioligands linked to cloned mu-opioid receptors or by bioassay using guinea pig ileum (Schiller et al, Eur J Med Chem,35: 895-. Activation of mu opioid receptors often causes analgesic effects. In some cases, aromatic-cationic peptides with mu-type opioid receptor agonist activity are preferred. For example, during short-term treatment, such as acute diseases or disorders, it may be advantageous to use aromatic-cationic peptides that activate the mu-opioid receptor. Such acute diseases and conditions are often associated with moderate or severe pain. In these cases, the analgesic effect of the aromatic-cationic peptide is advantageous in a therapeutic regimen for a human patient or other mammal. However, aromatic-cationic peptides that do not activate mu-opioid receptors may also be used with or without analgesics, depending on the clinical needs.

Alternatively, in other cases, aromatic-cationic peptides that do not have mu-type opioid receptor agonist activity are preferred. For example, in long-term treatments, such as chronic disease conditions or disorders, it may be undesirable to use aromatic-cationic peptides that activate mu-opioid receptors. In these cases, the potentially adverse or addictive effects of the aromatic-cationic peptide may preclude the use of an aromatic-cationic peptide that activates the mu-opioid receptor in a therapeutic regimen for a human patient or other mammal. Potentially adverse effects may include sedation, constipation, and respiratory depression. In such cases, aromatic-cationic peptides that do not activate the mu-type opioid receptors may be suitable treatments.

Peptides having mu-opioid receptor agonist activity are typically those having a tyrosine residue at the N-terminus (i.e., at the first amino acid position) or having a tyrosine derivative. Suitable derivatives of tyrosine include 2 ' -methyltyrosine (Mmt), 2 ', 6 ' -dimethyltyrosine (2 ' 6 ' -Dmt), 3 ', 5 ' -dimethyltyrosine (3 ' 5 ' -Dmt), N,2 ', 6 ' -trimethyltyrosine (Tmt) and 2 ' -hydroxy-6 ' -methyltyrosine (Hmt).

In one embodiment, the peptide having mu-type opioid receptor agonist activity has the formula Tyr-D-Arg-Phe-Lys-NH₂. The peptide has three net positive charges contributed by the amino acids tyrosine, arginine and lysine, and two aromatic groups contributed by the amino acids phenylalanine and tyrosine. The tyrosine may be a modified derivative of tyrosine, for example 2 ', 6' -dimethyltyrosine, to prepare compounds having the formula 2 ', 6' -Dmt-D-Arg-Phe-Lys-NH₂The compound of (1). The peptide has a molecular weight of 640 and carries three net positive charges at physiological pH. The peptide readily crosses the plasma membrane of several mammalian cell types in an energy independent manner (ZHao et al, J Pharmacol Exp ther.,304:425-432, 2003).

Peptides that do not have mu-opioid receptor agonist activity typically do not have a tyrosine residue or tyrosine derivative at the N-terminus (i.e., amino acid position 1). The amino acid at the N-terminus can be any naturally occurring or non-naturally occurring amino acid other than tyrosine. In one embodiment, the amino acid at the N-terminus is phenylalanine or a derivative of phenylalanine. Exemplary derivatives of phenylalanine include 2 ' -methyl phenylalanine (Mmp), 2 ', 6 ' -dimethyl phenylalanine (2 ', 6 ' -Dmp), N,2 ', 6 ' -trimethyl phenylalanine (Tmp), and 2 ' -hydroxy-6 ' -methyl phenylalanine (Hmp).

An example of an aromatic-cationic peptide not having mu-type opioid receptor agonist activity has the formula Phe-D-Arg-Phe-Lys-NH₂. Alternatively, the N-terminal phenylalanine may be a derivative of phenylalanine, such as 2 ', 6' -dimethylphenylalanine (2 '6' -Dmp). In one embodiment, the peptide having 2 ', 6' -dimethylphenylalanine at amino acid position 1 has the formula 2 ', 6' -Dmp-D-Arg-Phe-Lys-NH₂. In one embodiment, the amino acid sequence is rearrangedSo that Dmt is not at the N-terminus. An example of such aromatic-cationic peptides not having mu-opioid receptor agonist activity has the formula D-Arg-2 '6' -Dmt-Lys-Phe-NH₂。

The peptides and their derivatives mentioned herein may also include functional analogs. A peptide is considered to be a functional analog if the analog has the same function as the recited peptide. For example, the analog can be a substituted variant of a peptide in which one or more amino acids are substituted with another amino acid. Suitable substitution variants of the peptides include conservative amino acid substitutions. Amino acids can be grouped according to their physicochemical properties as follows:

(a) non-polar amino acids: ala (A) Ser (S) Thr (T) Pro (P) Gly (G) Cys (C);

(b) acidic amino acids: asn (N) Asp (D) Glu (E) Gln (Q);

(c) basic amino acids: his (H) Arg (R) Lys (K);

(d) hydrophobic amino acids: met (M) Leu (L) Ile (I) Val (V); and

(e) aromatic amino acids: phe (F) Tyr (Y) Trp (W) His (H).

Substitution of an amino acid in a peptide with another amino acid in the same group is called conservative substitution, and can retain the physicochemical properties of the original peptide. Conversely, substitution of an amino acid in a peptide with another amino acid in a different group is generally more likely to alter the physicochemical properties of the original peptide.

In some embodiments, one or more naturally occurring amino acids in the aromatic-cationic peptide are substituted with an amino acid analog. Examples of analogs that activate the mu opioid receptor include, but are not limited to, the aromatic-cationic peptides shown in table 5.

TABLE 5 peptide analogs with mu opioid activity

Amino acid position 1	Amino acid position 2	Amino acid position 3	Amino acid position 4	C-terminal modification
					Tyr	D-Arg	Phe	Lys	NH2
Tyr	D-Arg	Phe	Orn	NH2
					Tyr	D-Arg	Phe	Dab	NH2
Tyr	D-Arg	Phe	Dap	NH2
					2’6’Dmt	D-Arg	Phe	Lys	NH2
2’6’Dmt	D-Arg	Phe	Lys-NH(CH2)2-NH-dns	NH2
					2’6’Dmt	D-Arg	Phe	Lys-NH(CH2)2-NH-atn	NH2
2’6’Dmt	D-Arg	Phe	dnsLys	NH2
					2’6’Dmt	D-Cit	Phe	Lys	NH2
2’6’Dmt	D-Cit	Phe	Ahp	NH2
					2’6’Dmt	D-Arg	Phe	Orn	NH2
2’6’Dmt	D-Arg	Phe	Dab	NH2
					2’6’Dmt	D-Arg	Phe	Dap	NH2
2’6’Dmt	D-Arg	Phe	Ahp (2-aminoheptanoic acid)	NH2
					Bio-2’6’Dmt	D-Arg	Phe	Lys	NH2
3’5’Dmt	D-Arg	Phe	Lys	NH2
					3’5’Dmt	D-Arg	Phe	Orn	NH2
3’5’Dmt	D-Arg	Phe	Dab	NH2
					3’5’Dmt	D-Arg	Phe	Dap	NH2
Tyr	D-Arg	Tyr	Lys	NH2

Tyr	D-Arg	Tyr	Orn	NH2
					Tyr	D-Arg	Tyr	Dab	NH2
Tyr	D-Arg	Tyr	Dap	NH2
					2’6’Dmt	D-Arg	Tyr	Lys	NH2
2’6’Dmt	D-Arg	Tyr	Orn	NH2
					2’6’Dmt	D-Arg	Tyr	Dab	NH2
2’6’Dmt	D-Arg	Tyr	Dap	NH2
					2’6’Dmt	D-Arg	2’6’Dmt	Lys	NH2
2’6’Dmt	D-Arg	2’6’Dmt	Orn	NH2
					2’6’Dmt	D-Arg	2’6’Dmt	Dab	NH2
2’6’Dmt	D-Arg	2’6’Dmt	Dap	NH2
					3’5’Dmt	D-Arg	3’5’Dmt	Arg	NH2
3’5’Dmt	D-Arg	3’5’Dmt	Lys	NH2
					3’5’Dmt	D-Arg	3’5’Dmt	Orn	NH2
3’5’Dmt	D-Arg	3’5’Dmt	Dab	NH2
					Tyr	D-Lys	Phe	Dap	NH2
Tyr	D-Lys	Phe	Arg	NH2
					Tyr	D-Lys	Phe	Lys	NH2
Tyr	D-Lys	Phe	Orn	NH2
					2’6’Dmt	D-Lys	Phe	Dab	NH2
2’6’Dmt	D-Lys	Phe	Dap	NH2
					2’6’Dmt	D-Lys	Phe	Arg	NH2
2’6’Dmt	D-Lys	Phe	Lys	NH2
					3’5’Dmt	D-Lys	Phe	Orn	NH2
3’5’Dmt	D-Lys	Phe	Dab	NH2
					3’5’Dmt	D-Lys	Phe	Dap	NH2
3’5’Dmt	D-Lys	Phe	Arg	NH2
					Tyr	D-Lys	Tyr	Lys	NH2
Tyr	D-Lys	Tyr	Orn	NH2
					Tyr	D-Lys	Tyr	Dab	NH2
Tyr	D-Lys	Tyr	Dap	NH2
					2’6’Dmt	D-Lys	Tyr	Lys	NH2
2’6’Dmt	D-Lys	Tyr	Orn	NH2
					2’6’Dmt	D-Lys	Tyr	Dab	NH2
2’6’Dmt	D-Lys	Tyr	Dap	NH2
					2’6’Dmt	D-Lys	2’6’Dmt	Lys	NH2
2’6’Dmt	D-Lys	2’6’Dmt	Orn	NH2
					2’6’Dmt	D-Lys	2’6’Dmt	Dab	NH2
2’6’Dmt	D-Lys	2’6’Dmt	Dap	NH2
					2’6’Dmt	D-Arg	Phe	dnsDap	NH2
2’6’Dmt	D-Arg	Phe	atnDap	NH2
					3’5’Dmt	D-Lys	3’5’Dmt	Lys	NH2

3’5’Dmt	D-Lys	3’5’Dmt	Orn	NH2
					3’5’Dmt	D-Lys	3’5’Dmt	Dab	NH2
3’5’Dmt	D-Lys	3’5’Dmt	Dap	NH2
					Tyr	D-Lys	Phe	Arg	NH2
Tyr	D-Orn	Phe	Arg	NH2
					Tyr	D-Dab	Phe	Arg	NH2
Tyr	D-Dap	Phe	Arg	NH2
					2’6’Dmt	D-Arg	Phe	Arg	NH2
2’6’Dmt	D-Lys	Phe	Arg	NH2
					2’6’Dmt	D-Orn	Phe	Arg	NH2
2’6’Dmt	D-Dab	Phe	Arg	NH2
					3’5’Dmt	D-Dap	Phe	Arg	NH2
3’5’Dmt	D-Arg	Phe	Arg	NH2
					3’5’Dmt	D-Lys	Phe	Arg	NH2
3’5’Dmt	D-Orn	Phe	Arg	NH2
					Tyr	D-Lys	Tyr	Arg	NH2
Tyr	D-Orn	Tyr	Arg	NH2
					Tyr	D-Dab	Tyr	Arg	NH2
Tyr	D-Dap	Tyr	Arg	NH2
					2’6’Dmt	D-Arg	2’6’Dmt	Arg	NH2
2’6’Dmt	D-Lys	2’6’Dmt	Arg	NH2
					2’6’Dmt	D-Orn	2’6’Dmt	Arg	NH2
2’6’Dmt	D-Dab	2’6’Dmt	Arg	NH2
					3’5’Dmt	D-Dap	3’5’Dmt	Arg	NH2
3’5’Dmt	D-Arg	3’5’Dmt	Arg	NH2
					3’5’Dmt	D-Lys	3’5’Dmt	Arg	NH2
3’5’Dmt	D-Orn	3’5’Dmt	Arg	NH2
					Mmt	D-Arg	Phe	Lys	NH2
Mmt	D-Arg	Phe	Orn	NH2
					Mmt	D-Arg	Phe	Dab	NH2
Mmt	D-Arg	Phe	Dap	NH2
					Tmt	D-Arg	Phe	Lys	NH2
Tmt	D-Arg	Phe	Orn	NH2
					Tmt	D-Arg	Phe	Dab	NH2
Tmt	D-Arg	Phe	Dap	NH2
					Hmt	D-Arg	Phe	Lys	NH2
Hmt	D-Arg	Phe	Orn	NH2
					Hmt	D-Arg	Phe	Dab	NH2
Hmt	D-Arg	Phe	Dap	NH2
					Mmt	D-Lys	Phe	Lys	NH2
Mmt	D-Lys	Phe	Orn	NH2
					Mmt	D-Lys	Phe	Dab	NH2

Mmt	D-Lys	Phe	Dap	NH2
					Mmt	D-Lys	Phe	Arg	NH2
Tmt	D-Lys	Phe	Lys	NH2
					Tmt	D-Lys	Phe	Orn	NH2
Tmt	D-Lys	Phe	Dab	NH2
					Tmt	D-Lys	Phe	Dap	NH2
Tmt	D-Lys	Phe	Arg	NH2
					Hmt	D-Lys	Phe	Lys	NH2
Hmt	D-Lys	Phe	Orn	NH2
					Hmt	D-Lys	Phe	Dab	NH2
Hmt	D-Lys	Phe	Dap	NH2
					Hmt	D-Lys	Phe	Arg	NH2
Mmt	D-Lys	Phe	Arg	NH2
					Mmt	D-Orn	Phe	Arg	NH2
Mmt	D-Dab	Phe	Arg	NH2
					Mmt	D-Dap	Phe	Arg	NH2
Mmt	D-Arg	Phe	Arg	NH2
					Tmt	D-Lys	Phe	Arg	NH2
Tmt	D-Orn	Phe	Arg	NH2
					Tmt	D-Dab	Phe	Arg	NH2
Tmt	D-Dap	Phe	Arg	NH2
					Tmt	D-Arg	Phe	Arg	NH2
Hmt	D-Lys	Phe	Arg	NH2
					Hmt	D-Orn	Phe	Arg	NH2
Hmt	D-Dab	Phe	Arg	NH2
					Hmt	D-Dap	Phe	Arg	NH2
Hmt	D-Arg	Phe	Arg	NH2

Dab = diaminobutyl

Dap = diaminopropionic acid

Dmt = dimethyltyrosine

Mmt = 2' -methyl tyrosine

Tmt = N,2 ', 6' -trimethyltyrosine

Hmt =2 '-hydroxy-6' -methyl tyrosine

dnsDap = beta-dansyl-L-alpha, beta-diaminopropionic acid

atnDap = beta-anthranilic acid-L-alpha, beta-diaminopropionic acid

Bio = biotin

Examples of analogs that do not activate mu opioid receptors include, but are not limited to, the aromatic-cationic peptides shown in table 6.

TABLE 6 peptide analogs lacking mu-opioid activity

Amino acid position 1	Amino acid position 2	Amino acid position 3	Amino acid position 4	C-terminal modification
					D-Arg	Dmt	Lys	Phe	NH2
D-Arg	Dmt	Phe	Lys	NH2
					D-Arg	Phe	Lys	Dmt	NH2
D-Arg	Phe	Dmt	Lys	NH2
					D-Arg	Lys	Dmt	Phe	NH2
D-Arg	Lys	Phe	Dmt	NH2
					Phe	Lys	Dmt	D-Arg	NH2
Phe	Lys	D-Arg	Dmt	NH2
					Phe	D-Arg	Phe	Lys	NH2
Phe	D-Arg	Dmt	Lys	NH2
					Phe	D-Arg	Lys	Dmt	NH2
Phe	Dmt	D-Arg	Lys	NH2
					Phe	Dmt	Lys	D-Arg	NH2
Lys	Phe	D-Arg	Dmt	NH2
					Lys	Phe	Dmt	D-Arg	NH2
Lys	Dmt	D-Arg	Phe	NH2
					Lys	Dmt	Phe	D-Arg	NH2
Lys	D-Arg	Phe	Dmt	NH2
					Lys	D-Arg	Dmt	Phe	NH2
D-Arg	Dmt	D-Arg	Phe	NH2
					D-Arg	Dmt	D-Arg	Dmt	NH2
D-Arg	Dmt	D-Arg	Tyr	NH2
					D-Arg	Dmt	D-Arg	Trp	NH2
Trp	D-Arg	Phe	Lys	NH2
					Trp	D-Arg	Tyr	Lys	NH2
Trp	D-Arg	Trp	Lys	NH2
					Trp	D-Arg	Dmt	Lys	NH2
D-Arg	Trp	Lys	Phe	NH2
					D-Arg	Trp	Phe	Lys	NH2
D-Arg	Trp	Lys	Dmt	NH2
					D-Arg	Trp	Dmt	Lys	NH2
D-Arg	Lys	Trp	Phe	NH2
					D-Arg	Lys	Trp	Dmt	NH2
Cha	D-Arg	Phe	Lys	NH2
					Ala	D-Arg	Phe	Lys	NH2

Cha = cyclohexyl alanine

The amino acids of the peptides shown in tables 5 and 6 may be in a levorotatory structure or a dextrorotatory structure.

Peptides may also be synthesized by any method well known in the art. For example, suitable methods for chemically synthesizing peptides include methods such as those described by Stuart and Young in: solid Phasepeptide Synthesis, second edition, Pierce Chemical Company (1984); and MethodsEnzymol.289, Academic Press, New York (1997).

Prophylactic and therapeutic uses of aromatic-cationic peptides

Overview. The aromatic-cationic peptides described herein are useful for preventing or treating diseases. In particular, the invention provides prophylactic and therapeutic methods for treating a subject at risk of (or susceptible to) a vaso-occlusive injury or cardiac ischemia-reperfusion injury. Thus, the present methods provide for preventing and/or treating vaso-occlusive or cardiac ischemia-reperfusion injury in a subject by administering an effective amount of an aromatic-cationic peptide to a subject in need thereof and performing CABG surgery.

Determination of the biological effect of aromatic-cationic peptide-based therapeutics. In various embodiments, suitable in vitro or in vivo assays are performed to determine the effect of a particular aromatic-cationic peptide-based therapy and whether the administration of the aromatic-cationic peptide is suitable for treatment. In various embodiments, in vitro assays can be performed using representative animal models to determine whether a given aromatic-cationic peptide-based therapy has the desired effect in preventing or treating ischemia-reperfusion injury. Prior to testing in a human subject, the compounds used in treatment may be tested in suitable animal model systems including, but not limited to, rat, mouse, chicken, pig, cow, monkey, rabbit, and the like. Similarly, for in vivo testing, any animal model system known in the art may be used prior to administration to a human subject.

A method of prevention. In one aspect, the invention provides a method of preventing vasoocclusive injury in a subject by administering to the subject an aromatic-cationic peptide that prevents the onset or progression of vasoocclusive injury. For example, a subject at risk for a vessel occlusion injury may be determined by the diagnostic or prognostic analysis described herein. In prophylactic applications, pharmaceutical compositions or medicaments of aromatic-cationic peptides are administered to a subject susceptible to or at risk of a disease or condition in an amount effective to eliminate or reduce the risk of the disease, reduce the severity of the disease, or delay the onset of the disease, including biochemical, histological, and/or behavioral disorders of the disease, complications of the disease, and intermediate pathological phenotypes present during the development of the disease. Administration of a prophylactic aromatic-cationic peptide can occur before the abnormal condition characteristic manifests itself, such that the disease or disorder is prevented, or alternatively its development is delayed. Suitable compounds may be determined based on the screening assays described above. In some embodiments, the peptide is administered in an amount sufficient to prevent renal complications or brain complications from CABG.

A therapeutic method. Another aspect of the invention includes a method of treating a vasoocclusive lesion in a subject for therapeutic purposes. In therapeutic applications, an amount of the composition or drug is administered to a subject suspected of having such a disease or a subject already having such a disease to substantially eliminate or at least partially inhibit a condition of the disease, including its complications and intermediate pathological manifestations in the development of the disease. Accordingly, the present invention provides methods of treating a subject suffering from cardiac ischemia reperfusion injury by administering an effective amount of an aromatic-cationic peptide and performing CABG surgery.

Mode of administration and effective dose

Any method known to those skilled in the art may be used to contact the cells, organs or tissues with the peptide. Effective amounts can be determined during preclinical testing and clinical trials using methods familiar to physicians and clinicians. An effective amount of a peptide useful in the method can be administered to a mammal in need thereof by any of a variety of known methods for administering pharmaceutical compositions. The peptide may be administered systemically or locally.

The peptide may be expressed as a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salt" means a salt made from a base or an acid that is acceptable for administration to a patient (e.g., a mammal) (e.g., a salt that is acceptably safe in the mammal for a given dosing regimen). It will be understood, however, that a salt need not be a pharmaceutically acceptable salt, such as a salt of an intermediate compound that is not intended for administration to a patient. Pharmaceutically acceptable salts can be obtained from pharmaceutically acceptable inorganic or organic bases, and from pharmaceutically acceptable inorganic or organic acids. Furthermore, when a peptide contains a basic moiety, such as an amine, pyrimidine, or imidazole, and an acidic moiety, such as a carboxylic acid or tetrazole, zwitterions may be formed and are included within the term "salt(s)" as used herein. Salts derived from pharmaceutically acceptable inorganic bases include ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, and zinc salts, and the like. Salts derived from pharmaceutically acceptable organic bases include primary, secondary and tertiary amine salts, including substituted amines, cyclic amines, naturally occurring amines, and the like, such as arginine, betaine, caffeine, choline, N' -dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, reduced glucamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine (pipradine), polyamine esters, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. Salts derived from pharmaceutically acceptable inorganic acids include borates, carbonates, hydrohalites (hydrobromides, hydrochlorides, hydrofluorides or hydroiodides), nitrates, phosphates, sulfamates and sulfates. Salts derived from pharmaceutically acceptable organic acids include aliphatic hydroxy acid salts (e.g., citrate, gluconate, glycolate, lactate, lactobionate, malate, and tartrate), aliphatic monocarboxylic acid salts (e.g., acetate, butyrate, formate, propionate, and trifluoroacetate), amino acid salts (e.g., aspartate and glutamate), aromatic carboxylic acid salts (e.g., benzoate, p-chlorobenzoate, diphenylacetate, gentisate, hippurate, and triphenylacetate), aromatic hydroxy acids (e.g., o-hydroxybenzoate, p-hydroxybenzoate, 1-hydroxynaphthalene-2-carboxylate, and 3-hydroxynaphthalene-2-carboxylate), ascorbate salts, dicarboxylate salts (e.g., fumarate, maleate, oxalate, and succinate), and pharmaceutically acceptable salts thereof, Glucuronate, mandelate, mucate, nicotinate, orotate, pamoate, pantothenate, sulfonates (benzenesulfonate, camphorsulfonate, edisylate (edisylic), ethanesulfonate, isethionate, methanesulfonate, naphthalenesulfonate, naphthalene-1, 5-disulfonate, naphthalene-2, 6-disulfonate, and p-toluenesulfonate), xinafoic acid (xinafoic) salt, and the like.

The aromatic-cationic peptides described herein can be added to pharmaceutical compositions, alone or in combination, for administration to a subject to treat or prevent a disease described herein. The compositions generally include an active agent and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein includes physiological saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with the administration of a drug. Auxiliary active compounds may also be added to the composition.

Pharmaceutical compositions are generally formulated to conform to their intended route of administration. Examples of routes of administration include injection (e.g., intravenous, intradermal, intraperitoneal, or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoretic, and transmucosal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following ingredients: sterile diluents such as water for injection, physiological saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl benzoate preservatives; antioxidants such as ascorbic acid or sodium bisulfite; complexing agents, such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate and phosphate; and a solvent for adjusting osmotic pressure, such as sodium chloride or levulose. The pH can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. The intravenous formulation may be contained in glass or plastic ampoules, disposable syringes or multi-dose vials. For the convenience of the patient or treating physician, dosage formulations may be provided in the form of kits comprising all the equipment (e.g., vials, bottles of diluent, syringes and needles) required for the course of treatment (e.g., 7 days of treatment).

Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (where water soluble) or dispersions or sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous injection, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL^TM(BASF, pasiboni, n.j.) or Phosphate Buffered Saline (PBS). In all cases, compositions for parenteral administration must be sterile and should be a liquid present to the extent that easy injection is achieved. Compositions for parenteral administration must be stable under the conditions of manufacture and storage and should be preserved while preventing the contaminating action of microorganisms such as bacteria and fungi).

The aromatic-cationic peptide composition can include a carrier, which can be a solvent or dispersion medium including, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable combinations thereof. Proper fluidity can be maintained, for example, by: coatings using, for example, lecithin; in the case of dispersoids, by maintaining the desired particle size; and by using a surfactant. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Glutathione and other antioxidants may be included to prevent oxidation. In many cases, it will be preferred to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or a pharmaceutically acceptable gum.

Sterile solutions for injection can be prepared by adding the required amount of the active compound to the appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile solutions for injection, typical methods of preparation include vacuum drying and freeze-drying, which yields a powder of the active ingredient plus any other desired ingredient in the previously sterile-filtered solution.

Oral compositions typically include an inert diluent or an edible carrier. For oral therapeutic administration, the active compounds can be mixed with excipients and used in the form of tablets, dragees or capsules (e.g., gelatin capsules). Oral compositions can also be prepared using liquid carriers for use as mouth washes. Pharmaceutically compatible binders and/or auxiliary materials may be included as part of the composition. The tablets, pills, capsules, lozenges, or the like can include any of the following ingredients or compounds with similar properties: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; excipients, such as starch or lactose; a disintegrating agent such as alginic acid, sodium carboxymethyl starch, or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds may be delivered in the form of an aerosol spray from a pressurized container or dispenser or a nebulizer containing a suitable propellant (e.g., a gas such as carbon dioxide). Such methods include those described in U.S. patent No. 6468798.

Systemic administration of the therapeutic compounds described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, or fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays. For transdermal administration, the active compounds are formulated into ointments, salves, gels, lotions, which are generally known in the art. In one embodiment, transdermal administration may be by iontophoresis.

The therapeutic protein or peptide may be formulated in a carrier system. The support may be a colloidal system. The colloidal system may be a liposome, phospholipid bilayer vehicle. In one embodiment, the therapeutic peptide is encapsulated in liposomes while maintaining peptide integrity. One skilled in the art understands that there are various methods to prepare liposomes. (see: Methods biochem. anal.,33:337-462(1988) by Lichtenberg et al; Liposome Technology, CRC Press (1993) by Anselem et al). Liposomal formulations can delay drainage and enhance cellular uptake (see Reddy, Ann. Pharmacother,34(7-8):915-923 (2000)). The active agent may also be loaded into particles prepared from medically acceptable ingredients, including but not limited to soluble, insoluble, permeable, impermeable, biodegradable, or gastro-retentive polymers or liposomes. Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles, and viral vector systems.

The carrier may also be a polymer, such as a biodegradable, biocompatible polymer matrix. In one embodiment, the therapeutic peptide may be embedded in a polymer matrix while maintaining protein integrity. The polymer may be natural, such as a polypeptide, protein or polysaccharide; or may be synthetic, such as poly alpha hydroxy acids. Examples include carriers made of, for example: collagen, fibronectin, elastin, cellulose acetate, nitrocellulose, polysaccharides, fibrin, gelatin, and combinations thereof. In one embodiment, the polymer is polylactic acid (PLA) or lactic glycolic acid copolymer (PGLA). The polymer matrix, including microspheres and nanospheres, can be prepared and isolated in a variety of forms and sizes. The polymer formulation may cause an extension of the duration of the therapeutic effect (see: Reddy, Ann. Pharmacother.,34(7-8):915-923 (2000)). Polymer formulations for human growth hormone (hGH) have been used in clinical trials (see: Kozarrich and Rich, Chemical Biology,2: 548-.

Examples of polymeric microsphere sustained release agents are described in PCT publication WO 99/15154 (Tracy et al), U.S. Pat. No. 5674534, and U.S. Pat. No. 5716644 (all of Zale et al), PCT publication WO96/40073 (Zale et al), and PCT publication WO00/38651 (Shah et al). United states patent No. 5674534 and united states patent No. 5716644 and PCT publication WO96/40073 describe a polymer matrix comprising particles containing erythropoietin stabilized against aggregation with a salt.

In some embodiments, the therapeutic compound is prepared with a carrier that will protect the therapeutic compound from rapid expulsion from the body, such as a controlled release agent, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Such formulations may be prepared using known techniques. Such materials are also commercially available, for example, liposome suspensions (including liposomes directed to specific cells having a monoclonal antibody to a cell-specific antigen) available from Alza Corporation and Novapharmaceuticals, Inc. can also be used as medically acceptable carriers. The colloidal suspensions may be prepared using methods known to those skilled in the art, for example, as described in U.S. patent No. 4522811.

The therapeutic compound may also be formulated to enhance intracellular delivery. For example, liposomal delivery systems are known in the art, see, for example: "Recent Advances in Liposome Drug Delivery Systems" by Chonn and Cullis, Current Opinion in Biotechnology 6: 698-. The following references describe the delivery of proteins to cells in vivo or in vitro using fusogenic liposomes: cancer Lett.100:63-69(1996) of Mizguchi et al.

The amount of therapeutic agent, toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining LD50 (the lethal dose for 50% of total) and ED50 (the therapeutically effective dose in 50% of total). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED 50. Compounds exhibiting high therapeutic indices are preferred. While compounds with toxic side effects may be used, it is contemplated to design a delivery system that targets such compounds to the affected site of the tissue to minimize possible damage to uninfected cells and thereby reduce side effects.

Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that include the ED50, which is less or non-toxic. The dosage may vary within this range depending upon the type of dosage employed and the route of administration utilized. For any compound used in the method, a therapeutically effective dose may be initially calculated from cell culture assays. A dose can be formulated in animal models to achieve a circulating blood concentration range that includes IC50 (i.e., the concentration of the test compound that achieves half the maximal inhibitory disorder) as determined in bacterial culture. Such formulations can be used to more accurately determine useful dosages in humans. Blood drug levels can be determined, for example, by high performance liquid chromatography.

Typically, an effective amount of the aromatic-cationic peptide sufficient to obtain a therapeutic or prophylactic effect ranges from about 0.000001 mg/kg body weight/day to about 10000 mg/kg body weight/day. Suitably, the dosage ranges from about 0.0001 mg/kg body weight/day to about 100 mg/kg body weight/day. For example, the dose may be 1mg/kg body weight or 10mg/kg body weight daily, every second or third day, or in the range of 1mg/kg body weight to 10mg/kg body weight weekly, biweekly or triweekly. In one embodiment, a single dose of the peptide ranges from 0.1mg/kg body weight to 10000 mg/kg body weight. In one embodiment, the concentration of the aromatic-cationic peptide in the carrier ranges from 0.2 mg/ml to 2000 mg/ml. Exemplary methods of treatment provide for dosing once a day or week. In therapeutic applications, relatively high doses are sometimes required in relatively short time intervals until the progression of the disease is slowed or stopped, and preferably until the subject exhibits a condition that partially or completely ameliorates the disease. Thereafter, prophylactic administration to the patient may be carried out.

In an exemplary embodiment, the peptide is administered to the subject by intravenous infusion at about 0.001mg/kg/h to about 1mg/kg/h, i.e., about 0.005mg/kg/h, about 0.01mg/kg/h, about 0.025mg/kg/h, about 0.05mg/kg/h, about 0.10mg/kg/h, about 0.25mg/kg/h, or about 0.5 mg/kg/h. The intravenous infusion may be initiated prior to or after tissue reperfusion.

In some embodiments, a therapeutically effective amount of an aromatic-cationic peptide can be defined as a concentration of 10 peptides in a target tissue^-12Mole to 10^-6Mols, e.g. about 10^-7And (3) mol. The foregoing concentrations may be administered in a systemic dose of 0.01mg/kg to 100 mg/kg or an equivalent dose of body surface area. The schedule of dosages is optimized to maintain a therapeutic concentration in the target tissue, most preferably by daily or weekly administration, but also includes continuous administration (e.g., infusion or transdermal administration).

In some embodiments, the dose of the aromatic-cationic peptide is provided at a "low", "medium", or "high" dose level. In one embodiment, the low dose is provided from about 0.001mg/kg/h to about 0.5mg/kg/h, suitably from about 0.01mg/kg/h to about 0.1 mg/kg/h. In one embodiment, the medium dose is provided from about 0.1mg/kg/h to about 1.0mg/kg/h, suitably from about 0.1mg/kg/h to about 0.5 mg/kg/h. In one embodiment, the high dose is provided from about 0.5mg/kg/h to about 10mg/kg/h, suitably from about 0.5mg/kg/h to about 2 mg/kg/h. The intravenous infusion may be initiated prior to or after tissue reperfusion. In some embodiments, the subject may receive a bolus intravenous injection prior to tissue reperfusion. In one embodiment, the peptide is administered in conjunction with cardioplegic fluid. In one embodiment, the peptide is administered as a portion of the priming solution of a heart-lung machine during cardiopulmonary bypass.

One skilled in the art will recognize that certain factors may affect the dosage and timing effective to treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the health of the subject and/or the age of the subject, as well as other diseases present. Moreover, treating a subject with a therapeutically effective amount of a therapeutic composition described herein can include a single treatment or a series of treatments.

The mammal treated according to the methods of the invention can be any animal, for example, including farm animals such as sheep, pigs, cattle and horses; pet animals, such as dogs and cats; laboratory animals such as rats, mice and rabbits. In a preferred embodiment, the mammal is a human.

Examples

The invention is further illustrated by the following examples, which should not be construed as limiting the invention in any way.

Example 1 Effect of aromatic-cationic peptides on protection of Rabbit model from vascular occlusion injury

The role of aromatic-cationic peptides in protecting rabbit models from vasoocclusive injury was studied. The peptide D-Arg-2 '6' -Dm was confirmed by this examplet-Lys-Phe-NH₂The myocardial preservation effect of (1).

Experimental methods

New Zealand white rabbits were used in this study. The rabbits were male and were more than 10 weeks old. Environmental control of the animal room is set up to maintain a temperature of 61 ° F to 72 ° F and a relative humidity of between 30% and 70%. The room temperature and humidity were recorded hourly and monitored daily. The animal room was ventilated about 10-15 times per hour. The photoperiod was 12 hours light/12 hours dark (with fluorescent lighting) except for the necessary adjustment dose and data collection period. Routine daily observations were made. Approximately 180 grams of rabbit food, the certified food Harlan Teklad (2030C), was provided daily from the time of arrival at the laboratory. In addition, fresh fruits and vegetables were provided to rabbits three times a week.

The peptide D-Arg-2 '6' -Dmt-Lys-Phe-NH₂(sterile lyophilized powder) was used as the test drug. Dosing solutions are formulated to be no more than 1mg/ml and delivered by continuous Infusion (IV) at a constant rate (e.g., 50 μ L/Kg/min). Physiological saline (0.9% NaCl) was used as a control.

The test drug/excipient drug was provided intravenously under general anesthesia to mimic the desired route of administration in the clinical setting of AMI and PTCA. Intravenous infusion was performed by peripheral vein using a Kd Scientific infusion pump (Holliston, MA 01746) in constant amounts (e.g., 50 μ L/kg/min).

The study was followed by a scheduled placebo and sham-control design. Briefly, 10-20 healthy, domesticated male rabbits were assigned to one of three study groups (approximately 2-10 animals per group). Group A (n =10, CTRL/PLAC) included vehicle-treated animals (vehicle; VEH, IV); group B (n =10, treated) included animals treated with peptides; group C (n =2, SHAM operation (SHAM)) included SHAM-operated time controls treated with vehicle (vehicle; VEH, IV) or peptide.

TABLE 7 study design

In all cases, treatment was initiated at approximately 10 minutes after the start of 30 minutes of ischemic injury (coronary occlusion) and continued until 3 hours after reperfusion. In all cases, cardiovascular function was monitored before and during ischemia, and up to 180 minutes (3 hours) after reperfusion. The experiment was terminated 3 hours after reperfusion (study end); irreversible myocardial damage (infarct size obtained by histomorphometry) at this time point was assessed and was the primary endpoint of the study. The study design is summarized in table 7 and fig. 1.

Anesthesia/surgical preparation. General anesthesia was induced by intramuscular Injection of (IM) ketamine (-35 mg/kg-50 mg/kg)/xylazine (-5 mg/kg-10 mg/kg) mixture. An intravenous catheter is placed in a peripheral vein (e.g., ear) to administer the anesthetic. To preserve autonomic function, anesthesia was maintained by continuous infusion of propofol (-8 mg/kg/h-30 mg/kg/h) and ketamine (-1.2 mg/kg/h-2.4 mg/kg/h). A balloon endotracheal tube is placed through a tracheotomy (lower abdominal median incision) and is used to mechanically ventilate the lungs with 95% O through a volume-switched animal ventilator (-40 breaths/min, breath volume-12.5 ml/kg)₂Per 5% CO₂To convert PaCO to₂Values were maintained primarily within the physiological range.

When the surgical plane of anesthesia is reached, the formation of transthoracic or needle-like electrodes of two standard Electrocardiogram (ECG) leads (e.g., lead II, aVF, V2) is prevented. Dissection of the neck exposes the carotid artery, which is separated from, isolated from, and inserted with a dual sensor high fidelity micro manometer catheter (Millar Instruments); the tip of the catheter is pushed retrograde through the aortic valve into the Left Ventricle (LV) to simultaneously determine the pressure of the aorta (root, proximal sensor) and left ventricle (distal sensor). The incision of the neck also exposes the jugular vein, which is inserted with an empty injection catheter (for blood sampling). Finally, additional intravenous catheters are placed in peripheral veins (e.g., ears) to administer excipient/test drugs.

Subsequently, the animal was placed in a right-side lying position and the heart was exposed by midline thoracotomy and pericardiotomy. The heart was rested on a pericardial stent to expose the Left Circumflex (LCX) and Left Anterior Descending (LAD) coronary arteries. The ligature wire is loosely placed around the proximal LAD (using a tapered-tipped needle) and, depending on the coronary artery of each animal, around one or more branches of the LCX limbal coronary artery, if necessary. Tightening these snares (through a short section of polyethylene tubing) caused a temporary ischemia of a portion of the left ventricular myocardium.

Once the instrumental tests were completed, hemodynamic stability for at least 30 minutes and appropriate depth of anesthesia were verified/ensured. Subsequently, animals were paralyzed using atracurium (-0.1 mg/kg/h to 0.2mg/kg/h, i.v.) to promote hemodynamic/respiratory stabilization. Following atracurium administration, the symptoms of idiopathic hyperkinetic and/or changes in BIS values are used to assess the depth of anesthesia and/or to titrate intravenous anesthetics.

Protocol/cardiovascular data collection. Immediately after surgical preparation, animals were heparinized (100 units heparin/kg/h, bolus intravenous injection) and after hemodynamic stabilization (for about 30 minutes), baseline data were collected, including venous blood for assessment of cardiac myogenases/biomarkers and test drug concentrations.

After hemodynamic stabilization and baseline measurements, the animals were subjected to 30 minutes of acute ischemic injury by tightening the LAD/LCX coronary artery snare. Myocardial ischemia is visually confirmed by a change in color (i.e., cyanosis) of the distal distribution of LAD/LCX and by the onset of an electrocardiographic change. After approximately 10 minutes of ischemia, the animals receive a continuous perfusion vehicle (saline) or peptide; after starting the treatment, ischemia was continued for another 20 minutes (i.e., a total of 30 minutes). Subsequently (i.e. after 30 minutes of ischemia, during which 30 minutes, the last 20 minutes were treated simultaneously), the coronary snare was released and the previously ischemic myocardium was left to be reperfused for 3 hours. Treatment with excipients or peptides was continued throughout the reperfusion period. It should be noted that in sham-operated animals, the vessel snares are manipulated at the beginning of ischemia/reperfusion, but are not tightened or loosened.

Cardiovascular data collection was performed at 11 predetermined time points: after instrument testing/stabilization (i.e., baseline), after 10 minutes of ischemia, and after 30 minutes of ischemia, and at 5 minutes after reperfusion, at 15 minutes after reperfusion, at 30 minutes after reperfusion, at 60 minutes after reperfusion, at 120 minutes after reperfusion, and at 180 minutes after reperfusion. Throughout the experiment, the analog signals were continuously digitally sampled (1000 Hz) and recorded using a data acquisition system (IOX; EMKA Technologies), and the following parameters were determined at the above-mentioned time points: (1) from bipolar transthoracic electrocardiogram (e.g., lead II, aVF): cardiac rhythm (arrhythmia quantification/grading), RR, PQ, QRS, QT, QTc, short-term QT instability and QT: TQ (restitution); (2) from solid state pressure gauges (milliar) in the aorta: arterial/aortic pressure (AoP); and (3) from a solid state pressure gauge in the LV (Millar): left ventricular pressure (ESP, EDP) and derivative indices (dP/dtmax, dP/dtmin, Vmax, and tau). In addition, cardiac biomarkers and infarct size were evaluated in order to determine/quantify the extent of irreversible myocardial damage (i.e., infarct) caused by I/R injury with and without peptide therapy.

A blood sample. Venous whole blood samples (< 3 ml) were collected for Pharmacokinetic (PK) analysis and for assessment of myocardial damage by cardiac biomarker analysis at the following six data collection time points: baseline, at 30 minutes ischemia, at 30 minutes post-reperfusion, at 60 minutes post-reperfusion, at 120 minutes post-reperfusion, and at 180 minutes post-reperfusion. In addition, whole blood samples (-0.5 ml) of three arteries were collected at baseline, 60 minutes of ischemia, and 60 minutes after reperfusion and 180 minutes after reperfusion to determine blood gas, the arterial samples were collected into a blood gas syringe, and the arterial samples were used to measure blood gas by an I-Stat analyzer/cassette (CG4 +).

Histopathology/histomorphometry. After completion of the protocol, irreversible myocardial damage (i.e., infarction) resulting from I/R injury is assessed. Briefly, the coronary artery snare was retightened and Evan's blue dye (1 ml/kg, Sigma, st. louis, MO) was injected intravenously to trace the myocardial Area (AR) at risk during ischemia. After approximately 5 minutes, the heart was stopped (by injecting potassium chloride into the left atrium), and the heart was freshly excised. The left ventricle was cut into 3mm thick slices in a direction perpendicular to its longitudinal axis (from apex to base). Subsequently, the sections were incubated in 2% triphenyltetrazolium (TTC) at 37 ℃ for 20 minutes and fixed in 10% non-buffered formalin solution (NBF).

After fixation, infarcts and areas at risk are digitally traced/measured. For this purpose, the thickness of each slice is measured with a digital micrometer and then photographed/scanned. All photographs were input to an Image analysis program (Image J; National Institutes of Health), and computer-assisted leveling methods were performed to determine the overall size of the infarct (I) area and the area At Risk (AR). For each section, AR (i.e., not stained blue) is expressed as a percentage of LV area, and infarct size (I, unstained tissue) is expressed as a percentage of AR (I/AR). In all cases, quantitative histomorphometric measurements were performed by those not knowledgeable in the assignment/study design.

And (5) observing the animals. Data are acquired on the IOX system of EMKA using Electrocardiogram (ECG) automated software for analysis (EMKA technique). The measurement of all physiological parameters is done manually or automatically from a (digital) oscillogram. Using the average of the data from 60s from each target time point (if possible); however, as described above, the signal/oscillogram is recorded continuously throughout the experiment to allow for more accurate/detailed temporal data analysis (by correction), if necessary. Additional calculations were performed using Microsoft Excel. Data are expressed as mean with standard error.

Results

Figures 2 to 6 show the infarct size from hearts subjected to 30 minutes of ischemia and 3 hours of reperfusion. Figures 2A and 2B show data representing infarct size in rabbits used for sham surgery (ligatures applied, but not tightened) with placebo or peptide treatment. The LV is sliced into 3mm thick slices perpendicular to the longitudinal axis of the LV (from top to bottom). Fig. 2A is a photograph of a heart section of a sham operated rabbit treated with placebo and a computer generated image highlighting infarct size. Fig. 2B is a photograph of a heart section of a sham operated rabbit treated with peptide and a computer generated image highlighting infarct size.

Fig. 3A and 3B show data representing infarct size in two different control rabbits that induced myocardial ischemia and were treated with placebo. Each figure shows a photograph of a slice of the heart and a computer generated image highlighting the infarct size.

Fig. 4A, 4B, 4C, 4D and 4E show data representing infarct sizes in five different rabbits induced with myocardial ischemia and treated with the peptide. Administration of the peptide resulted in a reduction in infarct size compared to the control. Table 8 shows data of the ratio of the area at risk to the area of the left ventricle, the ratio of the infarct area to the area of the left ventricle, and the ratio of the infarct area to the area at risk for each of the animals used in the study. Fig. 5 to 6 show further data representing the ratio of area at risk to the area of the left ventricle, the ratio of infarct area to left ventricle area, and the ratio of infarct area to area at risk in the peptide-treated subjects and control subjects.

TABLE 8 histopathological results of study animals

These results show that in a standard rabbit model of acute myocardial ischemia and reperfusion, administration of peptide by IV continuous infusion starting at 10 minutes up to 30 minutes of the ischemic period and then 180 minutes after reperfusion, myocardial infarction size can be reduced relative to the control group. In rabbits with a determinable response to treatment, the size of the myocardial infarct zone was reduced relative to the size of the infarct noted in the control group. Treatment at less than 3 hours (i.e., 30 minutes) after reperfusion provided similar myocardial rescue (data not shown). These results indicate that peptide treatment prevented the appearance of symptoms of acute myocardial ischemia reperfusion injury. In this regard, the aromatic-cationic peptides are useful in methods of preventing and treating vessel occlusion injury in a mammalian subject.

Example 2 Effect of peptides in protecting humans from vascular occlusion injury

This example will confirm the administration of D-Arg-2 '6' -Dmt-Lys-Phe-NH upon revascularization₂Whether or not to limit infarct size during acute myocardial infarction.

Study group. Males and females 18 years old or older who appeared in the hospital after the onset of chest pain and were treated with revascularization (e.g., PCI or thrombolysis) were eligible for inclusion by clinical decision. Patients may be STEMI (ST elevation myocardial infarction) or non-STEMI. STEMI patients will have the following symptoms: suggesting a cut-off of blood supply to the heart muscle and conditioned on the patient's ECG showing a typical heart attack pattern of ST elevation. Therefore, diagnosis is based only on symptoms, clinical examination, and ECG changes. In the case of a heart attack with non-ST elevation, the symptoms of chest pain may be the same as those of STEMI, but the important difference is that the patient's ECG does not show typical ST elevation changes normally associated with a heart attack. Patients usually have a history of experiencing angina, but the ECG at the time of suspected episode may show no abnormalities. Diagnosis of medical history and symptoms is questionable and confirmed by blood tests showing elevated concentrations of substances known as myocardial enzymes in the blood.

Angiography and revascularization. Prior to revascularization, left ventricular and coronary angiography is performed using standard techniques. Revascularization was performed by PCI using direct stenting. Alternative methods of vascular reconstruction include, but are not limited to, balloon angioplasty, percutaneous transluminal coronary angioplasty, and atherectomy.

Experimental protocol. Patients meeting the inclusion criteria were randomly assigned to the control or peptide groups after coronary angiography was performed and before the stent was implanted. The random grouping is performed using a computer generated random sequence. Patients in the pepset received a bolus of D-Arg-2 '6' -Dmt-Lys-Phe-NH administered intravenously less than 10 minutes prior to direct stenting₂. The peptide was dissolved in saline and injected through a catheter located in the antecubital vein. Patients were randomized in equal amounts into any of the following treatment groups (e.g., 0mg/kg/h, 0.001mg/kg/h, 0.005mg/kg/h, 0.01mg/kg/h, 0.025mg/kg/h, 0.05mg/kg/h, 0.10mg/kg/h, 0.25mg/kg/h, 0.5mg/kg/h, and 1.0 mg/kg/h). Peptides were administered by IV infusion from approximately 10 minutes prior to reperfusion to approximately 3 hours post PCI. After the reperfusion period, the peptide is chronically administered to the subject by any mode of administration (e.g., subcutaneous or intravenous injection).

Infarct size. The primary endpoint was infarct size assessed by measuring cardiac biomarkers. Blood samples were taken at hospitalization and repeated over the following 3 days. Coronary biomarkers were measured in each patient. For example, the area under the curve (AUC) (expressed in arbitrary units) for creatine kinase and troponin I release (Beckman kit) was measured by computerised planimetry in each patient. The primary secondary endpoint was the size of the infarct measured by the region of delayed hyperenhancement technique seen on cardiac Magnetic Resonance Imaging (MRI) assessed on day 5 post-infarct. For the late enhancement analysis, 0.2mmol gadolinium-tetraazacyclododecane tetraacetic acid (DOTA)/kg was injected at a rate of 4ml per second and 15ml of physiological saline was injected. Delayed hyperenhancement was assessed using a three-dimensional inversion recovery gradient echo sequence 10 minutes after gadolinium-DOTA injection. The images were analyzed on short axis slices covering the entire left ventricle.

Myocardial infarction is identified by delayed super-enhancement techniques within the myocardium, defined quantitatively by the intensity of the post-myocardial contrast signal, which is 2SD higher than the signal intensity in the reference region of the distal non-infarcted myocardium within the same sheet. For all sections, the absolute mass of the infarct zone was calculated according to the following formula: infarct mass (in grams of tissue) = ∑ (hyper-enhancement region [ in square centimeters ]) × slice thickness (in centimeters) × myocardial specific gravity (1.05 grams/cubic centimeter).

Biomarkers of the determined risk factors. The concentrations of N-terminal pro-brain natriuretic peptide (NT-proBNP) and glucose were measured, as well as the estimated glomerular filtration rate (eGFR). These biomarkers all significantly predict all-cause mortality by mean follow-up of approximately two and a half years. Calculating a risk score based on these three biomarkers can identify patients at high risk of death during follow-up. It is predicted that peptides will reduce the risk score of these biomarkers in patients undergoing PCI compared to patients not receiving the peptide undergoing PCI. Blood samples can be taken to determine CK-MB and troponin I. The area under the curve (AUC) (expressed in arbitrary units) for CK-MB and troponin I release can be measured by computerized planimetry in each patient.

Other end points. The whole blood concentrations of the peptides were at the time before PCI and at 1 hour, 2 hours, 4 hours, 8 hours, and 12 hours after PCI. Blood pressure and serum concentrations of creatinine and potassium were measured at admission, 24 hours, 48 hours, 72 hours after PCI. Serum concentrations of bilirubin, gamma glutamyltransferase and alkaline phosphatase, as well as white blood cell count were measured at the time of admission and 24 hours after PCI.

The cumulative incidence of major adverse events occurring within the first 48 hours after reperfusion, including death, heart failure, acute myocardial infarction, stroke, ischemic recurrence, need for revascularization, renal or hepatic insufficiency, vascular complications, and hemorrhage were recorded. Adverse events associated with infarction, including heart failure and ventricular fibrillation, were assessed. Furthermore, after 3 months of acute myocardial infarction, cardiac events were recorded and global left ventricular function was assessed by echocardiography (Vivid 7systems; GEVingmed).

It is predicted that, by some measures, administration of the peptide at reperfusion will be associated with a smaller infarct than that observed with placebo.

Example 2 Effect of peptides on organ protection during CABG

This example will confirm the administration of the aromatic-cationic peptide D-Arg-2 '6' -Dmt-Lys-Phe-NH₂Whether the peptide limits the size of damaged myocardium in moderate to high risk patients undergoing non-urgent CABG surgery with planned cardiopulmonary bypass (CPB) and cardioplegia. The effect of the peptides as cardioprotective agents was evaluated using the relative size of the damaged myocardium as measured by the concentration of peak CK-MB enzyme, troponin, or lactate dehydrogenase. The effect of peptide administration on renal and cardiac complications was also assessed.

It is predicted that peptides are superior to placebo in reducing the incidence of cardiac, renal and/or cerebral complications using planned cardiopulmonary bypass and cardioplegic elective CABG surgery in combination with the background of standard of care treatment. The effect of peptides as cardioprotective agents, as measured by the relative size of infarcted myocardium in moderate to high risk patients undergoing phase-selective CABG surgery with planned cardiopulmonary bypass and cardioplegia, was measured by the myocardial enzyme concentration on the day post-surgery (POD) of 4.

Cardiac complications. This study has the following objectives: (1) to evaluate the effect of peptides on a combination of cardiovascular death, non-fatal Myocardial Infarction (MI), or non-fatal stroke from randomized (0 days post-operative) to 4 days post-operative (POD), 30 days post-operative (POD), and 90 days post-operative (POD) in moderate to high risk patients undergoing elective CABG surgery with planned cardiopulmonary bypass and cardioplegia; (2) to evaluate the effect of peptides on individual events of cardiovascular death, non-fatal myocardial infarction or non-fatal stroke from randomized (0 days post-operative) to 4 days post-operative (POD), 30 days post-operative (POD) and 90 days post-operative (POD) in moderate to high risk patients undergoing elective CABG surgery with planned cardiopulmonary bypass and cardioplegia; and (3) to assess the incidence of significant atrial and/or ventricular arrhythmias with a day post-operative (POD) of 2 in moderate to high risk patients undergoing phase-selective CABG surgery with planned cardiopulmonary bypass and cardioplegia.

Renal complications. This study has the following objectives: (1) to evaluate the effect of the peptides as a nephroprotective agent, this was measured by continuous assay of renal function for acute renal failure (AKI) measured by CK-MB concentration 4 days post-surgery in moderate to high risk patients undergoing phase-selective CABG surgery employing planned cardiopulmonary bypass and cardioplegia; and (2) to evaluate the effect of peptides on renal function from randomized (0 days post-operative) to 30 days post-operative (POD) and 90 days post-operative (POD) in moderate to high risk patients undergoing phase-selective CABG surgery with planned cardiopulmonary bypass and cardioplegia.

Brain complications. This study has the following objectives: (1) to evaluate the effect of peptides on acute brain injury, this was assessed by magnetic resonance imaging performed before and not later than 4 (+ 2 days) post-operative days in moderate to high risk patients undergoing phase-selective CABG surgery with planned cardiopulmonary bypass and cardioplegia; (2) to assess the safety and tolerability of peptides administered prior to surgery, administered during surgery, and administered for short periods of time following surgery in moderate to high risk patients undergoing elective CABG surgery with planned cardiopulmonary bypass and cardioplegia; and (3) to evaluate the pharmacokinetics of the peptides in moderate to high risk patients undergoing phase-selective CABG surgery with planned cardiopulmonary bypass and cardioplegia.

Overall study design and planning

The study was a phase II, prospective, randomized, double-blind, placebo-controlled, multicenter dose range study designed to test the hypothesis that: peptides are superior to placebo in reducing cardiac, renal, and cerebral complications in moderate to high risk patients undergoing phase-selective CABG surgery with planned cardiopulmonary bypass (CPB) and cardioplegia, in combination with the background of standard of care therapy. The surgical procedure includes a separate Coronary Artery Bypass Graft (CABG) procedure with or without mitral valve repair for mild to moderate valve dysfunction. The patient is identified as having moderate to high risk for subsequent end organ complications associated with CABG surgery in the patient. The risk analysis of the patient includes at least two of: the age is more than or equal to 65 years old; moderate renal dysfunction defined as an estimated glomerular filtration rate (eGFR) of 31mL/min to 60 mL/min; a history of diabetes requiring treatment rather than a regular diet; and signs of significant left ventricular dysfunction (LV ejection fraction ≦ 0.40) or congestive heart failure in the absence of any type of cardiac pacemaker.

The main exclusion criteria included: acute myocardial infarction occurring less than 48 hours prior to randomization, CABG surgery with intermittent aortic occlusion without cardioplegia, minimally invasive surgery (i.e., without CPB), clinically significant kidney/liver disease, uncontrolled diabetes, a history of old stroke, transient ischemic attack or carotid endarterectomy, and a history of cranial trauma or epilepsy within six months. Prior to the planned CABG procedure, patients will be screened for all inclusion/exclusion criteria. In addition to standard treatments including anticoagulation, eligible patients will also be randomly assigned to receive one of the two peptide dosing regimens or a matching placebo.

The peptide or matched placebo will be administered by three different routes. All patients will take advantage of all three drug delivery modes to receive study drug in the same blinded manner: (1) at about 30 minutes prior to induction of anesthesia and for a total period of time lasting up to about 6 hours, systemic Intravenous (IV) infusion is performed, (2) in combination with cardioplegic fluid, and (3) as part of the priming solution in a heart-lung machine during CPB.

Hospitalization testing will last up to 96 hours. Diffusion weighted magnetic resonance imaging (DW-MRI) assessment of the patient's brain will be performed within 4 days prior to the CABG procedure and within 3 to 6 days after the procedure. At least, clinical follow-up of effects, experimental data, and adverse reactions will be performed daily during the exponential hospitalization period, at 96 hours, 30 days (range, 30-40), and 90 days (range, 76-104) after the exponential CABG surgical procedure. Throughout the course of the experiment, the attendant medical care will be left to the discretion of the cardiac surgeon and/or care physician. Compliance with guideline-based therapies and use of symptom-based drugs (aspirin, beta-blockers, angiotensin converting enzyme inhibitors, angiotensin II receptor antagonists, calcium channel blockers, diuretics, antiarrhythmics, statins, insulin, oral antidiabetics, and coumarin) would be strongly encouraged.

Endpoint and Effect follow-up

The main efficacy endpoints of the study will be: the effect of peptides as cardioprotective agents was evaluated using the relative area of damaged myocardium as measured by peak CK-MB enzyme concentration in 72 hours post-surgery in moderate to high risk patients undergoing non-urgent CABG surgery with planned cardiopulmonary bypass (CPB) and cardioplegia. Secondary endpoints will include the incidence of acute renal failure (AKI) associated with CABG surgery as assessed by continuous measurement of kidney function 72 hours post-surgery and the incidence of new acute brain injury associated with CABG surgery as assessed at POD 3-6 using magnetic resonance imaging of the brain.

A predetermined assessment for cardiovascular death, non-fatal Myocardial Infarction (MI), or non-fatal stroke will be made from the time of randomization (day 0 post-surgery) to POD 90. Diagnosis of MI will be based on clinical information collected from the study site as well as CK-MB laboratory data and ECG experimental data from the core laboratory. Stroke will be defined as a new, focal, non-traumatic neurological deficit that lasts at least 24 hours. An independent, blinded committee of clinical events will adjudicate all suspected myocardial infarction, stroke, and all death causes of death.

TABLE 5 evaluation schedule-first 24 hours

¹= patients not meeting each protocol standard (including pre-PCI and post-PCI TIMI blood flow standards) for any reason will be discontinued toStudy drug was administered and would be excluded from the effective assay. For safety, these patients will be tracked for 72 hours and replaced with new patients in a randomized pattern.

Patient selection and withdrawal

Subjects included male or female patients (45 years old or older) scheduled to undergo non-emergency CABG surgery with planned cardiopulmonary bypass (CPB) and cardioplegia. The surgical procedure will include a separate Coronary Artery Bypass Graft (CABG) procedure with or without mitral valve repair for mild to moderate valve dysfunction. The patient must be identified as at moderate to high risk for subsequent end organ complications associated with CABG surgery in the patient. The risk analysis of the patient must include at least two of the following: the age is more than or equal to 65 years old; moderate renal insufficiency defined as an estimated glomerular filtration rate (eGFR) of 31ml/min to 60 ml/min; a history of diabetes requiring treatment rather than a regular diet; and signs of significant left ventricular dysfunction (LV ejection fraction ≦ 0.40) or congestive heart failure in the absence of any type of cardiac pacemaker.

Treating a patient

The drug was studied. D-Arg-2 '6' -Dmt-Lys-Phe-NH₂Is a small peptide (CAS number 736992-21-5). Its molecular weight is 639.8 (free base). Peptides are stable to light, both in powder form and in fluid form. It is stable and resistant to oxidation up to 40 ℃. The drug substance will be provided in the form of a lyophilized powder in a sterile glass vial. At each site, reconstitution will be performed by a non-conscious pharmacist with 10mL of sterile D5W per vial.

The study will be conducted in an blinded fashion at the study site (both patient and site personnel are blinded). The random group code is created by an independent statistician. Patients will be randomized on average into any of the following treatment groups (0 mg/kg/h, 0.001mg/kg/h, 0.005mg/kg/h, 0.01mg/kg/h, 0.025mg/kg/h, 0.05mg/kg/h, 0.10mg/kg/h, 0.25mg/kg/h, 0.50mg/kg/h or 1 mg/kg/h). The reconstituted, diluted drug was infused at 60ml/h using an infusion pump starting at least 10 minutes prior to the expected reperfusion time and lasting 3 hours after CABG surgery. The start time and finish time of the intravenously administered study drug was recorded. The volume of study solution remaining in the infusion bag (sufficient to be evaluated by eye) was recorded and it was checked whether the appropriate amount of diluted study substance was injected. The plasma concentration of the peptide is determined and will provide the most accurate measure of treatment compliance.

Evaluation of therapeutic efficacy

The primary analysis of efficacy would be to compare the area under the curve (AUC) of the creatine kinase-MB curve over 72 hours between placebo and each peptide dose group for the estimated left ventricular infarct size. Secondary efficacy analysis will focus on the effect of the peptide on myocardial injury as determined by: (1) area under the troponin I enzyme curve at 72 hours; (2) cardiac Magnetic Resonance (CMR) at 4+ -1 day, 30 + -3 days, and 6+1.5 months; (3) incidence of arrhythmias after reperfusion; and (4) microvascular occlusion. These analyses were performed for patients with a baseline TIMI blood flow rating of 0, a TIMI blood flow rating of 1, and all patients (TIMI blood flow rating of 0 or 1).

The immediate effect will be examined after CABG surgery, including: (1) the degree of coronary blood flow and the incidence of arrhythmias; (2) determining the effect of the peptide on myocardial function and myocardial remodeling as measured by CMR for 30 days and 6 months; (3) the effect of the peptide on the occurrence of microvascular obstruction; (4) pharmacokinetics of peptides in patients undergoing successful reperfusion; and (5) the effect of the peptide on renal function immediately, 30 days, 90 days and 6 months as determined by serum creatinine, estimated creatinine clearance, cystatin C and BUN measurements.

Cardiac biomarkers. Blood samples were taken to determine CK-MB and troponin I. The area under the curve (AUC) (expressed in arbitrary units) for CK-MB and troponin I release was measured by computerized planimetry at the following time points (at the time of hospitalization; at the time of sheath crossing before and after CABG; every 4 hours during the first 24 hours after CABG; every 6 hours during the second and third days after CABG; after the third day, as clinically specified). Blood samples for determination of NT-proBNP and CRP concentrations were taken at the following time points: before CABG; 24 hours after CABG; 30 plus or minus 3 days after CABG; 90 plus or minus 14 days after CABG; and 6 ± 1.5 months after CABG.

Cardiac magnetic resonance imaging (CMR). A 1.5T body magnetic resonance scanner was used to perform CMR to assess ventricular function, myocardial edema (area at risk), microvascular obstruction and infarct size. CMR was performed 4 ± 1 day, 30 ± 3 days, and 6+1.5 months after successful CABG. A particular CMR protocol involves acquiring a film image of the left ventricular volume, mass fraction, and ejection fraction. Cine imaging techniques with steady free precession sequences were performed at 4 ± 1 day, 30 ± 3 days, and 6+1.5 months after successful CABG. T2-weighted images were acquired to evaluate myocardial edema for determining ischemic areas at risk of infarction. The triple inversion recovery fast spin echo sequence was performed only at 4 ± 1 day CMR study. Post-contrast delayed enhancement was used at 4 ± 1 day, 30 ± 3 days and 6+1.5 months post-surgery to quantify the injured myocardium. This is quantitatively defined by the intensity of the post-myocardial contrast signal, which is 2SD higher than the signal intensity in the reference region of the distal non-infarcted myocardium within the same slice. A standard extracellular gadolinium-based contrast agent was used at a dose of 0.2 mmol/kg. The fast gradient echo sequence made by two-dimensional inversion recovery will be used at the following time points: (1) for assessing early stage of microvascular occlusion (approximately 2 minutes after contrast injection), single point techniques are contemplated if available; and (2) for evaluation of late stage of infarct size (approximately 10 minutes after contrast injection).

Blood pressure, heart rate and respiratory rate were measured continuously throughout the experiment. Blood chemistry and urine chemistry and hematology analyses will be measured continuously during the test and include: electrolytes (sodium, potassium, bicarbonate, chloride); liver function (total bilirubin, aspartate aminotransferase [ AST or SGOT ] and alanine aminotransferase [ ALT or SGPT ]); renal function (serum creatinine, cystatin C, and blood urea nitrogen [ BUN ]); estimated glomerular filtration rate (eGFR); the incidence of acute renal failure (AKI) following CABG surgery; and a complete blood count.

By continuous measurement of serum creatinine, cystatin C and BUN; continuous calculation of estimated glomerular filtration rate (eGFR); and the incidence of at least grade 1 contrast nephropathy after CABG (defined as an increase of > 25% of the baseline value of serum creatinine increase and/or an increase of 0.5mg/dl serum creatinine increase occurring within 48 hours of radiographic contrast).

A biomarker for the determined risk factor. The concentrations of N-terminal pro-brain natriuretic peptide (NT-proBNP) and glucose were measured, as well as the estimated glomerular filtration rate (eGFR). These biomarkers all significantly predict all-cause mortality by mean follow-up of approximately two and a half years. Calculating a risk score based on these three biomarkers can identify patients at high risk of death during follow-up. It is predicted that the peptide will reduce the risk score of these biomarkers in patients undergoing CABG compared to patients undergoing CABG but not receiving the peptide.

Predicted results

It is predicted that the peptide will reduce infarct size and reduce the incidence of renal AKI and brain complications relative to subjects undergoing CABG but not receiving the peptide. The primary analysis of efficacy would be to compare LV infarct size estimated by the area under the curve (AUC) of the 72 hour creatine kinase-MB curve between placebo and each of the two peptide dose groups using an ANOVA model (or non-parametric equivalent model, Kruskal-Wallis scale analysis if the distribution is judged to be non-gaussian). It is also predicted that in patients undergoing CABG surgery with planned cardiopulmonary bypass and cardioplegia, when the peptide is administered preoperatively, intraoperatively, and immediately post-operatively, the peptide will act as a multi-organ protectant.

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Equivalents of

The present invention is not limited to the specific embodiments described in this application, but rather serves as a single illustration of separate aspects of the invention. Those skilled in the art will appreciate that various modifications and changes may be made without departing from the spirit and scope of the present application. Functionally equivalent methods and apparatuses within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing description. Such changes and modifications are intended to fall within the scope of the appended claims. The present disclosure is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compositions, and biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Further, where features or aspects of the disclosure are described in terms of markush groups, those skilled in the art will recognize that the disclosure is also described in terms of any single member or subgroup of members of the markush group.

Those skilled in the art will appreciate that all ranges described herein can also encompass any and all possible subranges and combinations of subranges thereof for any or all purposes, particularly to provide support for the description. Any listed range can be easily viewed as fully disclosed and enabling it to be divided into sub-ranges of at least equal halves, thirds, quarters, fifths, tenths, etc. of the same range. As a non-limiting example, each range described herein may be readily divided into a lower third, a middle third, an upper third, and the like. Those skilled in the art will also appreciate that all language such as "up to," "at least," "greater than," "less than," and the like encompass the same and applies to ranges that may be subsequently separated into the aforementioned sub-ranges. Finally, those skilled in the art will appreciate that a range includes each individual member. Thus, for example, a group having 1 to 3 cells refers to a group having 1 cell, 2 cells, or 3 cells. Similarly, a group having 1 to 5 cells refers to a group having 1 cell, 2 cells, 3 cells, 4 cells, or 5 cells, and so forth.

All patents, patent applications, prior applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, so that they are not inconsistent with the explicit teachings of this specification. Other embodiments are within the scope of the following claims.

Claims (20)

1. A method for treating obstructive coronary artery disease, the method comprising:

(a) a therapeutically effective amount of the peptide D-Arg-2 '6' -Dmt-Lys-Phe-NH₂Or a pharmaceutically acceptable salt thereof, to a mammalian subject; and

(b) performing a coronary artery bypass graft CABG procedure on the subject.

2. The method of claim 1, wherein the peptide is administered to the subject prior to the CABG procedure.

3. The method of claim 1, wherein the peptide is administered to the subject after the CABG surgery.

4. The method of claim 1, wherein the peptide is administered to the subject during and after the CABG surgery.

5. The method of claim 1, wherein the peptide is administered to the subject sequentially before, during, and after the CABG surgery.

6. The method of claim 5, wherein the peptide is administered to the subject at least 3 hours after the CABG surgery.

7. The method of claim 5, wherein the peptide is administered to the subject at least 5 hours after the CABG surgery.

8. The method of claim 5, wherein the peptide is administered to the subject at least 8 hours after the CABG surgery.

9. The method of claim 5, wherein the peptide is administered to the subject at least 12 hours after the CABG surgery.

10. The method of claim 5, wherein the peptide is administered to the subject at least 24 hours after the CABG surgery.

11. The method of claim 5, wherein administration of the peptide to the subject is initiated at least 8 hours prior to the CABG procedure.

12. The method of claim 5, wherein administration of the peptide to the subject is initiated at least 5 hours prior to the CABG procedure.

13. The method of claim 5, wherein administration of the peptide to the subject is initiated at least 2 hours prior to the CABG procedure.

14. The method of claim 5, wherein administration of the peptide to the subject is initiated at least 1 hour prior to the CABG procedure.

15. The method of claim 5, wherein administration of the peptide to the subject is initiated at least 30 minutes prior to the CABG procedure.

16. The method of claim 1, wherein the administration of the peptide by systemic intravenous infusion is initiated about 30 minutes prior to induction of anesthesia.

17. The method of claim 1, wherein said peptide is administered in combination with cardioplegic fluid.

18. The method of claim 1, wherein the peptide is administered as part of a priming solution of a heart-lung machine during cardiopulmonary bypass.

19. The method of claim 1, wherein the level of one or more of N-terminal pro-brain natriuretic peptide, glucose, and estimated glomerular filtration rate is decreased in a subject administered the peptide compared to a comparable subject undergoing CABG surgery without administration of the peptide.

20. A method for preventing renal or brain complications during a coronary artery bypass graft CABG surgery, the method comprising:

(a) a therapeutically effective amount of the peptide D-Arg-2 '6' -Dmt-Lys-Phe-NH₂Or a pharmaceutically acceptable salt thereof, to a mammalian subject; and

(b) performing a coronary artery bypass graft CABG procedure on the subject.