WO2009151714A2 - Therapeutic for treatment of circulatory shock, ischemia, inflammatory disease and related conditions - Google Patents

Abstract

Methods of treatment of circulatory shock, ischemia, reperfusion injury, inflammatory diseases and related conditions by administration of a therapeutically effective amount of a pharmaceutical preparation including the peptide Ac-Nle-cyclo(-Asp-His-D-Phe-Arg-Trp-Lys)-OH (bremelanotide), and formulations and preparations for therapeutic treatment of such conditions.

Description

Therapeutic for Treatment of Circulatory Shock, Ischemia, Inflammatory Disease and Related Conditions

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of the filing of U.S. Provisional Patent Application Serial No. 61/038,800 entitled "Treatment of Circulatory Shock, Ischemia, Inflammatory Diseases and Related Conditions", filed on March 24, 2008, and the specification and claims thereof are incorporated herein by reference.

A related application entitled "Pharmaceutical for Ocular Indications" is being filed concurrently herewith, U.S. Patent Application , Attorney Docket No. 0903-093, and the specification and claims thereof are incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention (Technical Field):

The present invention relates to compounds, compositions, formulations and methods for treatment of circulatory shock, ischemia, reperfusion injury, hemorrhagic shock, inflammatory diseases and related diseases, indications, conditions and syndromes by administration of a pharmaceutical composition including a melanocortin receptor agonist such as Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp- Lys)-OH. Description of Related Art: Note that the following discussion refers to a number of publications by authors and year of publication, and that due to recent publication dates certain publications are not to be considered as prior art vis-a-vis the present invention. Discussion of such publications herein is given for more complete background and is not to be construed as an admission that such publications are prior art for patentability determination purposes. It is reported in the literature that administration of certain melanocortin-related peptides increase the survival rate of humans and animals in a state of circulatory shock, such as hemorrhagic shock, ischemia or related conditions. See, for example, D. Giuliani et al., Selective melanocortin MC₄ receptor agonists reverse haemorrhagic shock and prevent multiple organ damage. Brit. J. Pharmacology 150:595-603, 2007. The 2007 Giuliani paper reported on use of NDP-α-MSH, which has the sequence Ac-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂, a peptide called RO27- 3225 with the sequence n-Butanoyl-His-D-Phe-Arg-Trp-Sar-NH₂, and a cyclic peptide called PG-931 with the sequence Ac-Nle-cyc/o(-Asp-Pro-D-Phe-Arg-Trp-Lys)-Pro-Val-NH₂. The foregoing are the only melanocortin peptides to be tested for circulatory shock, and each are conventional melanocortin peptides with a C-terminus primary amide. Additionally, RO27-3225 and PG-931 are described to be highly selective to the melanocortin-4 receptor (MC4-R), as opposed to selective for the melanocortin-1 receptor (MC1-R) or other known melanocortin receptors. In J. E. S. Wikberg et al., New aspects on the melanocortins and their receptors. Pharmacol. Res. 42:393-420, 2000, it is reported that the Ki values of RO27-3325 at human MC1-R is 33,000 nM, and at human MC4-R is 250,000 nM. While the Ki values of PG-931 are not reported, the IC₅₀ value at human MC4-R is reported at 0.6 nM. P. Grieco et al., Extensive structure-activity studies of lactam derivatives of MT-II and SHU-9119: their activity and selectivity at human melanocortin receptors 3, 4 and 5. J. Peptide Res. 62:199-206, 2003.

A reported study in humans involved administration of a bolus injection of the melanocortin adrenocorticotrophic hormone (ACTH) (1-24) to patients with aortic-dissection-induced hemorrhagic shock, resulting in significantly improved cardiovascular function and increased survival rate. G. Noera et al.: Survival rate after early treatment for acute type-A aortic dissection with ACTH-(I -24). The Lancet 358:469-470, 2001. ACTH (1-24) is a linear peptide.

It is also reported that α-MSH and, for some indications, ACTH modulate or control inflammation and inflammatory responses. A. Catania, The melanocortin system in leukocyte biology. J. Leukoc. Biol. 81 :383-392, 2007; A. Catania et al., Targeting melanocortin receptors as a novel strategy to control inflammation. Pharm. Rev. 56:1-29, 2004. While mechanisms of action are not fully elucidated, it is believed that the anti-inflammatory mechanism is at least partially central, involving activation of receptors within the brain, with resulting adrenergic and cholinergic signaling. Induced α-MSH inhibition of activation of the nuclear factor-κB (NF-κB) has been described, as has decreased production of Tumor Necrosis Factor-α (TNF-α), interleukin-1 β (IL-1 ) and interleukin-6 (IL-6). Increased production and expression of interleukin-10 (IL-10) has also been described in response to α-MSH, and IL-10 is known to reduce proinflammatory cytokine production, such as in macrophages.

The peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH is disclosed and claimed in U.S. Patent Nos. 6,579,968 and 6,794,489. However, in the patents the peptide is described as useful for sexual dysfunction, such as male erectile dysfunction and female sexual dysfunction. There is no disclosure of utility for treatment of circulatory shock, ischemia, reperfusion injury, hemorrhagic shock, inflammation, inflammatory diseases, autoimmune diseases, or related diseases, indications, conditions or syndromes. The peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH is also known as bremelanotide, a nonproprietary name adopted for the peptide drug by the United States Adopted Names Council.

BRIEF SUMMARY OF THE INVENTION

This invention relates to use of a melanocortin receptor-specific pharmaceutical composition for use in treatment of circulatory shock, ischemia, reperfusion injury, hemorrhagic shock and related diseases, indications, conditions and syndromes. The melanocortin receptor-specific agent in the pharmaceutical composition may be the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof. This invention also relates to use of a melanocortin receptor-specific pharmaceutical composition for use in treatment of inflammation, inflammatory diseases, autoimmune diseases and related diseases, indications, conditions and syndromes. The melanocortin receptor-specific agent in the pharmaceutical composition may be the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof.

This invention also relates to a pharmaceutical composition including the peptide Ac-Nle-cyc/o(- Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof. Because this peptide has a C-terminal carboxylic acid group rather than an art conventional C-terminal amide group, this peptide exhibits desirable pharmacological characteristics compared to a peptide with an art conventional C-terminal amide group, including one or more of reduced susceptibility to in vivo degradation and an improved time to therapeutic effect following administration.

This invention also relates to a pharmaceutical composition including the peptide Ac-Nle-cyc/o(- Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof for use in treatment of circulatory shock, ischemia, reperfusion injury, hemorrhagic shock and related diseases, indications, conditions and syndromes, which peptide is both a potent human MC1-R agonist and a human MC4-R agonist, thereby providing desirable anti-inflammatory, blood volume and pressor therapeutic effects.

This invention also relates to a pharmaceutical composition including the peptide Ac-Nle-cyc/o(- Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof for use in treatment of circulatory shock, ischemia, reperfusion injury, hemorrhagic shock and related diseases, indications, conditions and syndromes, which peptide is an agonist at each of MC1-R, MC3-R, MC4-R and MC5-R, thereby providing desirable anti-inflammatory, blood volume and pressor therapeutic effects.

This invention also relates to a pharmaceutical composition including a peptide melanocortin receptor agonist wherein the binding at human MC1-R, as determined by Ki values, is at least ten times as great as the binding at human MC4-R, or alternatively wherein the binding at human MC1-R, as determined by Ki values, is more than ten times as great as the binding at human MC4-R.

This invention also relates to a pharmaceutical composition including a peptide melanocortin receptor agonist wherein the Ki at human MC1-R is less than about 1.0 nM and the Ki at human MC4-R is greater than about 1.0 nM but less than about 10 nM.

This invention also relates to a pharmaceutical composition including the peptide Ac-Nle-cyc/o(- Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof for use in treatment of circulatory shock, ischemia, reperfusion injury, hemorrhagic shock, inflammatory diseases, autoimmune diseases and related diseases, indications, conditions and syndromes which results in a decrease in certain circulating pro-inflammatory cytokine levels, including without limitation a decrease in circulating TNF-α or IL-1 or both. This invention also relates to a pharmaceutical composition including the peptide Ac-Nle-cyc/o(-

Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof for use in treatment of circulatory shock, ischemia, reperfusion injury, hemorrhagic shock, inflammatory diseases, autoimmune diseases and related diseases, indications, conditions and syndromes which results in an increase in -A-

certain circulating anti-inflammatory cytokine levels, including without limitation an increase in circulating IL-10.

Other aspects and novel features, and the further scope of applicability of the present invention will be set forth in part in the detailed description to follow, taken in conjunction with the accompanying drawings, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention. The aspects of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and form a part of the specification, illustrate one or more embodiments of the present invention and, together with the description, serve to explain the principles of the invention. The drawings are only for the purpose of illustrating one or more preferred embodiments of the invention and are not to be construed as limiting the invention. FIG. 1 is a graph depicting mean arterial blood pressure in mm Hg in rats in an induced hypovolemia model, showing arterial blood pressure at normal baseline, hypovolemic baseline, and for one hour after treatment with saline (shock saline control) and bremelanotide administered intravenously at doses of 0.01 , 0.1 and 1.0 μmol/kg, and additionally showing alternial blood pressure following administration of saline and bremelanotide at a dose of 1.0 μmol/kg in animals in which hypovolemia was not induced (sham saline and sham bremelanotide at 1.0, respectively).

FIG. 2 is a graph depicting mean arterial blood pressure (MAP) in mm Hg in rats in an induced hypovolemia model against time in minutes. Selected groups of animals had a bilateral cervical vagotomy (V) approximately ten minutes prior to bleeding to induce hypovolemia. MAP of hypovolemic rats was stabilized for approximately fifteen minutes at 32 mm Hg before assignment to one of three groups. The graph depicts MAP of: animals with a vagotomy but no bleeding to induce hypovolemia or other treatment (line with diamond ♦ markers); animals with no vagotomy, bleeding to induce hypovolemia and treatment with bremelanotide at 1 μmol/kg (line with square ■ markers); animals with vagotomy, bleeding to induce hypovolemia and treatment with bremelanotide at 1 μmol/kg (line with triangle A markers); and animals with vagotomy, bleeding to induce hypovolemia and treatment with saline (line with crossed-line x markers), where between 2 and 7 animals are in each group.

FIG. 3 is a bar graph depicting urine production over four hours in rats with hypovolemia induced by removal of 48-50% of total blood volume followed by stabilization of mean arterial pressure at 40 mm/Hg for one hour, followed by intravenous bolus injection of 1.5 ml_ of a solution of 7.5% sodium chloride, 6% dextran-70 and bremelanotide at 1 μmol/kg (left bar, with downward diagonal lines) or 1.5 ml_ of a solution of 7.5% sodium chloride and 6% dextran-70 without bremelanotide (right bar, with upward diagonal lines). There are three animals per group, with ^** indicating p < 0.01.

FIG. 4 is a graph depicting mean arterial pressure in mm Hg in dog studies (2 dogs/group) in which dogs were ventilated with 98.5% O₂, bled to 40 mm Hg and maintained for 30 minutes, and then administered either saline (line with circle • markers) or bremelanotide at 1 μmol/kg (line with square ■ markers), and observed for two hours, with periodic readings of mean arterial pressure.

FIG. 5 is a bar graph comparing the effects of arginine vasopressin (AVP) (1 IU/kg), bremelanotide (1 μmol/kg) and saline on neutrophil gelatinase-associated lipocalin (NGAL) production, an early biomarker for acute renal failure, in an induced shock model. Baseline and steady shock NGAL levels were determined prior to administration of AVP, bremelanotide or saline; shock was induced by removing blood from rats and stabilizing blood pressure at 35 mm Hg for fifteen minutes, at which point blood was withdrawn for determination of steady shock NGAL levels. After fifteen minutes at steady shock, AVP, bremelanotide or saline was administered, and the 2 hour blood draw for NGAL levels was taken at 2 hours or the time of animal death if earlier. ^* indicates p < 0.05; ^** indicates p < 0.01.

DETAILED DESCRIPTION OF THE INVENTION Definitions. Before proceeding with the description of the invention, certain terms are defined as set forth herein.

In the sequence given for bremelanotide according to the present invention, the amino acid residues have their conventional meaning as given in Chapter 2400 of the Manual of Patent Examining Procedure, 8^th Ed. Thus, "NIe" is norleucine, "Asp" is aspartic acid, "His" is histidine, "D-Phe" is D- phenylalanine, "Arg" is arginine, "Trp" is tryptophan, and "Lys" is lysine. "Ac" refers to a peptide or amino acid sequence that is acetylated.

The term "composition", as in pharmaceutical composition, is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions utilized in the present invention encompass any composition made by admixing an active ingredient and one or more pharmaceutically acceptable carriers.

By a melanocortin receptor "agonist" is meant an endogenous substance, drug substance or compound, including a compound such as bremelanotide, which can interact with a melanocortin receptor and initiate a pharmacological response, including but not limited to adenyl cyclase expression, characteristic of the melanocortin receptor. For this invention, a melanocortin receptor agonist which is an agonist at melanocortin-4 receptor (MC4-R) and melanocortin-1 receptor (MC1-R) is preferred, and most preferred is a melanocortin receptor agonist which is an agonist at MC 1 -R, melanocortin-3 receptor (MC3-R), MC4-R and melanocortin-5 receptor (MC5-R). By "circulatory shock" is meant the general medical condition in which organs and/or tissues of the body of the subject, which subject may be human or animal, are not receiving an adequate flow of blood. Circulatory shock includes conditions such as hypovolemic shock, cardiogenic shock, vasodilatory shock and the like. These conditions or dysfunctions in circulation can in turn have different causes, such as bacterial blood infection (septic shock or infectious), severe allergic reaction (anaphylactic shock), trauma (traumatic shock), severe bleeding or loss of blood (hemorrhagic shock), neurologic dysfunction causing abnormal opening of blood vessels (neurogenic shock) or endocrine related (endocrine shock). Circulatory shock can further result in ischemia and ischemic damage to bodily organs, tissues, cells or parts. Upon reperfusion, or restoration of blood flow, ischemia- reperfusion injury can occur, also resulting in damage to bodily organs, tissues, or cells.

By "inflammatory disease," also sometimes called an "inflammatory condition," is meant a disease or condition characterized in part by inflammatory mechanisms, such as specific T lymphocyte reactions or antibody-antigen interactions causing the recruitment of inflammatory cells and endogenous mediator chemicals, including but not limited to cytokines, which mediator chemicals include but are not limited to one or more of increased NF-κB activity, increased TNF-α production, increased IL-1 production and increased IL-6 production.

By "Stage I shock," also sometimes called "compensated shock" or "non-progressive shock," is meant a condition which occurs when the body detects decreased blood flow or perfusion and begins to activate one or more of several reactive mechanisms to restore perfusion or direct blood flow to the most vital body organs. Stage I shock can be asymptomatic, but may also include, but is not limited to, symptoms such as low blood flow or perfusion, rapid or increased heart rate, shallow or irregular breathing, hypotension, hypertension, pallor and cyanosis.

By "Stage Il shock," also sometimes called "decompensated shock" or "progressive shock," is mean a condition which occurs when the compensatory mechanisms of the body begin to fail and organ perfusion cannot be restored to normal or maintained. Symptoms of Stage Il shock include, but are not limited to, confusion, anxiety, disorientation and other mental disturbances indicating a lack of oxygen to the brain, chest pains, increased heart rate, oliguria, multiple organ dysfunction, falling blood pressure (hypotension), rapid breathing, weakness and pupil dilation. By "Stage III shock," also sometimes called "irreversible shock," is meant a condition which occurs after the state of decreased perfusion or blood flow has existed to such an extent that the organs and tissues of the body are permanently affected. Such symptoms include, but are not limited to, multiple organ failure, kidney failure, coma, blood pooling in the extremities and death.

Clinical Applications. The compositions and methods disclosed herein can be used for both medical applications and animal husbandry or veterinary applications. Typically, the methods are used in humans, but may also be used in other mammals. The term "patient" is intended to denote a mammalian individual, and is so used throughout the specification and in the claims. The primary applications of this invention involve human patients, but this invention may be applied to laboratory, farm, zoo, wildlife, pet, sport or other animals. Circulatory Shock. Compositions and methods of the current invention are directed towards the treatment of circulatory shock in a subject. The compositions and methods provided herein may be employed to treat Stage I shock, Stage Il shock or Stage III shock. In one particular embodiment, the methods of the present invention are used to treat the initial stage of shock, which initial stage of shock is characterized by cardiac output insufficient to meet the body's metabolic needs, but not otherwise low enough to produce significant symptoms. The patient may be anxious and alert, with increased respiration.

The invention provides compositions for use and methods of treating or preventing hemorrhagic shock in a patient, which include administering a composition including bremelanotide to a patient diagnosed as suffering from blood loss. The blood loss may, but need not, be measured as a percentage of the subject's blood volume, such as, for example, a blood loss of greater than about 15% total blood volume, or greater than 20%, 25%, 30%, 35%, 40%, or 50% of the subject's total volume. Alternatively, the blood loss may, but need not, be measured in terms of a drop in blood volume in any amount sufficient to cause hemorrhagic shock in a particular subject, such as, for example, a loss of about 750 ml_, 1000 ml_, of about 1500 ml_, or of about 2000 ml_ or more in a human subject. The blood loss may also be measured in terms of a drop in systolic blood pressure, such as, for example, a drop in systolic blood pressure that is about 20 mm Hg, 30 mm Hg, 40 mm Hg, 50 mm Hg, 60 mm Hg, 70 mm Hg, 80 mm Hg, 90 mm Hg or 100 mm Hg or more than 100 mm Hg lower than the subject's normal systolic blood pressure. In particular embodiments, the subject is undergoing or has undergone a medical procedure, such as, but not limited to, surgery, a transfusion or child birth. In other particular embodiments, the subject has suffered a traumatic injury, such as, but not limited to, resulting from a motor vehicle accident, from an industrial injury, or from a gunshot wound.

In additional embodiments of the current invention, the compositions and methods are used to treat cardiogenic shock, hypovolemic shock and vasodilatory shock, each of which can be in any of the aforementioned stages of shock. In one particular embodiment of the present invention, the methods are used to treat cardiogenic shock. Cardiogenic shock is, generally speaking, low blood flow or perfusion that is caused by heart malfunction where the heart does not pump adequate blood. Causes can include any condition that interferes with ventricular filling or emptying, such as, but not limited to, embolism, ischemia, regurgitation and valve malfunction. In another particular embodiment of the present invention, the methods are used to treat vasodilatory shock. Vasodilatory shock is caused by severe venous or arteriolar dilation, which results in inadequate blood flow. Several known causes contribute to vasodilatory shock including, but not limited to, cerebral trauma, drug or poison toxicity, anaphylaxis, liver failure, bacteremia and sepsis. In another more particular embodiment of the present invention, the methods are used to treat shock resulting from sepsis or bacteremia. In an even more particular embodiment, the compositions and methods are used to treat septic shock or bacteremic shock in Stage I, Il or III. In yet another embodiment, the compositions and methods of the present invention are used to treat hypovolemic shock. Hypovolemic shock is, generally speaking, decreased intravascular volume, which decrease in intravascular volume can be relative or absolute. Hemorrhage from conditions such as, but not limited to, ulcers, gastrointestinal injury, trauma, accidents, surgery, and aneurysm may cause hypovolemic shock; but loss of other body fluids may also cause hypovolemic shock. For instance, renal fluid loss, intravascular fluid loss, water or other peritoneal fluid loss may contribute to hypovolemic shock. In one particular embodiment of the present invention, the compositions and methods, including administration of bremelanotide, are used to treat hypovolemic shock. In an even more particular embodiment, the compositions and methods are used to treat hypovolemic shock in Stage I, Stage Il or Stage III.

Circulatory shock, including hemorrhagic shock, may also result from partially controlled or uncontrolled bleeding within one or more internal organs or vessels of a patient. Bleeding may result from any cause, including by way of example from a ruptured aneurysm, dissected aorta, an ulcer or other gastrointestinal bleeding. In some instances the patient exhibits signs of circulatory shock or hypovolemia, which may include hypotension, but the source of internal bleeding is unknown.

In one embodiment, the invention is directed to methods of using bremelanotide to protect the heart, brain or other organs of a patient against injury caused by circulatory shock. The protective effect against circulatory shock occurs instantaneously or within a short time period following administration of a composition comprising bremelanotide, preferably within at least about 40 minutes following administration, more preferably within 1-20 minutes, more preferably within 1-15 minutes, and most preferably within about 1-10 minutes. Ischemia. Ischemia refers to any decrease or stoppage in the blood supply to any bodily organ, tissue, cell, or part, particularly where that decrease or stoppage leads to or would likely lead to ischemic damage to the bodily organ, tissue, cell, or part. An "ischemic episode" refers to any transient or permanent period of ischemia. Ischemia may result from any constriction or obstruction of the vasculature, or may result from circulatory shock, such as hemorrhagic shock, hypovolemic shock, or the like. The decrease or lack of blood flow results in a decrease or lack of oxygen to the affected part of the body, and may also result in an increase of inflammatory disease mediator chemicals such as various cytokines and other substances. During certain surgical procedures such as cardiac surgery and organ transplantation, the flow of blood is stopped temporarily and then resumed (reperfusion), resulting in ischemia-reperfusion injury. During a heart attack, the blood that supplies the heart is stopped, also resulting in ischemia that can evolve into infarction. Current treatment to relieve heart attacks requires reperfusion of the ischemic area of the heart, such as by using thrombolytic drugs or coronary angioplasty.

The invention has particular application in prevention of injury due to renal ischemia, including lung injury secondary to renal ischemia, preventing or limiting ischemic heart injuries subsequent to a myocardial infarction, preventing or limiting ischemic brain injuries subsequent to a cardiovascular injury, including without limitation myocardial infarction, stroke or the like. Neuroprotection is provided by administration of a composition of the invention to a patient with cerebral ischemia or stroke, particularly patients who are concurrently hypotensive. The invention has further particular application in preventing or limiting ischemic organ damage in organ transplant, including transplant of the heart, kidney, liver, lungs, pancreas or small intestine. In one aspect, the pharmaceutical composition of the present invention may be utilized for perfusion of a transplant organ, which perfusion may be prior to, during or subsequent to transplant of the organ.

In one embodiment, the invention is directed to methods of using bremelanotide to protect the heart, brain, kidneys or other organs of a patient against injury caused by ischemia. The protective effect against ischemia occurs instantaneously or within a short time period following administration of a composition comprising bremelanotide, preferably within at least about 40 minutes following administration, more preferably within 1-20 minutes, more preferably within 1-15 minutes, and most preferably within about 1-10 minutes.

Ischemia may also results from any of a variety of diseases or conditions, and in one embodiment the invention is directed to methods of using bremelanotide to protect the organs of a patient against injury resulting from ischemia, which ischemia is caused by a disease or condition. Such disease or condition may include, by way of example and not limitation, atherosclerotic diseases such as atheromata with thrombosis, embolism from the heart or from blood vessel from any organ, vasospasm, hypotension due to heart disease, hypotension due to systemic disease including infection or allergic reactions, or hypotension resulting from administration, ingestion or other exposure to one or more toxic compounds or drugs. Ischemia may also be secondary ischemia, and in another embodiment the invention is directed to methods of using bremelanotide to protect the organs of a patient against injury resulting from secondary ischemia. Such secondary ischemia may be secondary to a disease or condition such diabetes mellitus, hyperlipidemia, hyperlipoproteinemia, dyslipidemia Buerger's disease, also called thromboangiitis obliterans, Takayasu's arteritis, arteritis temporalis, Kawasaki disease, also called lymph node syndrome, mucocutaneous node disease, infantile polyarteritis, cardiovascular syphilis, and various connective tissue diseases and disorders. Ischemia-Reperfusion Injury. Ischemia-reperfusion is the interruption of blood flow to bodily tissue and the subsequent and often abrupt restoration of blood flow to the tissue. While restoration of blood flow following ischemia is essential to preserve functional tissue, the reperfusion itself is known to be harmful to the tissue. Both ischemia and reperfusion are known to be important contributors to tissue necrosis. Several mechanisms appear to play a causative role in the generation of tissue damage associated with ischemia-reperfusion injury.

Various methods of limiting reperfusion injury have been described, such as induced hypothermia, controlled reperfusion, and ischemic preconditioning. Induced hypothermia is the induction of moderate hypothermia, thought to suppress many of the chemical reactions associated with reperfusion injury. Controlled reperfusion refers to controlling the initial period of reperfusion by reperfusing the tissue at a low pressure using blood that has been modified to be hyperosmolar, alkalotic, and substrate-enriched. Ischemic preconditioning is the purposeful causing of short ischemic events to have protective effect by slowing cell metabolism during a longer ischemic event. Although these treatments may be useful in surgical settings (e.g., before or after planned heart surgery), they are not possible in emergency settings. The invention has particular application in preventing or limiting the severity of renal reperfusion injury, including lung injury secondary to renal reperfusion, preventing or limiting reperfusion heart injuries subsequent to a myocardial infarction, preventing or limiting reperfusion brain injuries subsequent to a cardiovascular injury, including without limitation myocardial infarction, stroke or the like. Renal injury may occur as a result of trauma or disease or as a result of surgery or other medical procedures. Acute renal injury, also know as acute kidney injury (AKI), is a recognized major complication secondary to surgery, particularly cardiovascular surgery, more particularly coronary artery bypass graft (CABG) surgery, and is strongly associated with in-hospital mortality. M. Swaminathan et al.: Trends in acute renal failure associated with coronary artery bypass graft surgery in the United States. Crit. Care Med. 35:2286-2291 , 2007.

The invention has further particular application in preventing or limiting reperfusion organ damage in organ transplant, including transplant of the heart, kidney, liver, lungs, pancreas or small intestine. In one aspect, the pharmaceutical composition of the present invention may be utilized for perfusion of a transplant organ, which perfusion may be prior to, during or subsequent to transplant of the organ.

In one embodiment, the invention is directed to methods of using bremelanotide to protect the heart, brain, kidneys or other organs of a patient against injury caused by ischemia-reperfusion injury, including injury caused by or during reperfusion. The protective effect against ischemia-reperfusion injury occurs instantaneously or within a short time period following administration of a composition comprising bremelanotide, preferably within at least about 40 minutes following administration, more preferably within 1-20 minutes, more preferably within 1-15 minutes, and most preferably within about 1-10 minutes.

Increased Cytokine Expression. Expression of various cytokines is increased during an inflammatory process, including an inflammatory process secondary to circulatory shock, ischemia, reperfusion injury and the like. TNF-α is a pleiotropic cytokine produced mainly by macrophages, and also by other types of cells. Other cytokines which increase during an inflammatory process, including an inflammatory process secondary to circulatory shock, ischemia, reperfusion injury and the like, include IL-1 and IL-6. While cytokines such as TNF-α have beneficial effects in many instances, significantly increased levels, such as secondary to circulatory shock, ischemia, reperfusion injury and the like, can have pathological effects. In one aspect, reperfusion of hypoxic or ischemic tissues, such hypoxic as secondary to circulatory shock, results in inflammatory responses, including increased cytokine expression.

In one embodiment, the invention is directed to methods of using bremelanotide to decrease pro-inflammatory cytokine production and expression, including decreasing pro-inflammatory cytokine production and expression secondary to circulatory shock, ischemia, reperfusion injury and the like. The decrease in pro-inflammatory cytokine production and expression, including without limitation one or more of TNF-α, IL-1 and IL-6, occurs instantaneously or within a short time period following administration of a composition comprising bremelanotide, preferably within at least about 40 minutes following administration, more preferably within 1-20 minutes, more preferably within 1-15 minutes, and most preferably within about 1-10 minutes.

In a related embodiment, the invention is directed to methods of using bremelanotide to increase anti-inflammatory cytokine production and expression. The increase in anti-inflammatory cytokine production and expression, including without limitation IL-10 production and expression, occurs instantaneously or within a short time period following administration of a composition comprising bremelanotide, preferably within at least about 40 minutes following administration, more preferably within 1-20 minutes, more preferably within 1-15 minutes, and most preferably within about 1-10 minutes.

Hemodialysis. The compositions and methods of this invention may be employed to prevent hypotension while subjects are undergoing hemodialysis, as an adjunct in the removal of excess extracellular fluid during hemodialysis by preventing or minimizing hypotension secondary to removal of excess fluid, to stabilize high blood pressures between hemodialysis treatments by removal of excess extracellular fluid, and for similar and related indications. In hemodialysis, blood is pumped through the blood compartment of a dialyzer, exposing it to a semipermeable membrane. The cleansed blood is then returned via the circuit back to the body. Ultrafiltration occurs by increasing the hydrostatic pressure across the dialyzer membrane, generally by applying a negative pressure to the dialysate compartment of the dialyzer. This pressure gradient causes water and dissolved solutes to move from blood to the dialysate, and allows removal of up to several liters of excess extracellular fluid during a typical 3 to 5 hour treatment.

Removal of excess extracellular fluid in subjects on long-term hemodialysis is critical because the presence of chronic volume expansion of excess fluid results in hypertension. However, it is frequently not possible to remove all accumulated excess fluid during hemodialysis because of intradialytic (during dialysis) hypotension, lntradialytic hypotension is defined as a decrease in systolic blood pressure by > 20 mm Hg or a decrease in mean arterial pressure by 10 mm Hg more. Intradialytic hypotension is associated with symptoms such as abdominal discomfort, nausea, vomiting, muscle cramps, dizziness or fainting, and anxiety. Intradialytic hypotension can induce cardiac arrhythmias, and predispose the subject to coronary ischemic events or cerebral ischemic events. Estimates are that intradialytic hypotension occurs in about 25% to 50% of all hemodialysis sessions. The primary cause of intradialytic hypotension is believed to be the rapid removal of circulatory volume during hemodialysis, resulting in hemodynamic instability. The most common current treatment for intradialytic hypotension is either to decrease the rate of fluid removal or infuse fluid, but both methods result in insufficient dialysis and resulting volume overload. Pharmacological interventions have been considered, such as use of midodrine, an α-adrenergic agent. However, this is associated with a number of side effects, including induction of supine systolic hypertension. In addition, various vasopressin receptor agonists have been considered for this indication, as disclosed in U.S. Patent No. 7,183,255, issued February 27, 2007.

Thus in one aspect the present invention provides a method for reducing excess extracellular fluid in a subject undergoing hemodialysis, by limiting, entirely or partially, intradialytic hypotension by administration of a pharmaceutical composition including a melanocortin receptor-specific compound, and preferably a melanocortin receptor-specific compound that is an agonist at melanocortin-4 receptor, more preferable bremelanotide. Acute Blood Loss Secondary to Surgery. The pharmaceutical compositions and methods of this invention can be utilized for subjects with acute blood loss that occurs during surgery. In one embodiment, the subject can be undergoing a surgery that can cause acute blood loss. In another embodiment, the subject can be scheduled to undergo a surgery that can cause acute blood loss. In another embodiment, the subject can be predisposed to or at high risk of needing a surgery that can cause acute blood loss as a result of genetic factors (e.g., family history) and/or environmental factors (e.g., diet).

As used herein, the term "surgery" is used interchangeable with the term "operation." A surgery that causes acute blood loss can involve any type of cell (e.g., somatic cell, germ cell, embryonic cell, stem cell), tissue (e.g., bone, muscle, connective, blood), and/or organ (e.g., brain, kidney, lung, heart, pancreas, prostate, ovary, uterus, gastrointestinal tract). Examples of surgeries that can cause acute blood loss include, but are not limited to, an elective surgery.

There are many different types of surgery including, but is not limited to, optional or elective surgery, required surgery, and urgent or emergency surgery. Many surgical procedures are associated with a high risk of hemorrhage or blood loss. These include cardiac surgeries, coronary artery bypass graft surgery, abdominal hysterectomies, cerebral amyloid angiopathy, repair of a brain aneurysm, radiosurgery for arteriovenous malformations, endovascular treatment of posteri or circulation aneurysms, proliferative vitreoretinopathy, lipoma excision, and sinus surgery. As such, the subject can be one who is undergoing, scheduled to undergo, or has undergone one of the foregoing surgical procedures, or any other surgical procedure with a high risk of hemorrhage or blood loss, or any other surgical procedure in which there is intra-operative or post-operation hemorrhage, blood loss, hypovolemia or hypotension.

In one aspect, the compositions and methods of the invention have particular application in cardiac surgery, such as CABG surgery. Over 500,000 bypass surgeries are performed per year in the United States, but there are no approved cardioprotective drugs for reduction of cardiovascular events post surgery. Further, ischemic or reperfusion injury to other other organs or tissues may result, including injury to the brain, kidneys or lungs. Additionally, a large proportion of bypass patients are hypotensive prior to or during surgery, and are currently administered conventional vasopressor drugs. The subject can be administered compositions including bremelanotide by the methods of the present invention before, during and/or after the surgery. The timing and quantity of bremelanotide compositions administered can be selected by the skilled practitioner using ordinary skill taking into account, for example, the degree of blood loss in the subject.

Acute Blood Loss Secondary to Trauma. The pharmaceutical compositions and methods of this invention can be utilized for subjects with acute blood loss from trauma. In one embodiment, the subject is suffering from or diagnosed with a trauma that can cause acute blood loss. In another embodiment, the subject can be predisposed to or at risk of suffering a trauma that causes acute blood loss as a result of genetic factors (e.g., triple-X syndrome) and/or environmental factors (e.g., living in a high crime neighborhood). As used herein, the term "trauma" is used interchangeable with the term "injury." A trauma that causes acute blood loss can involve any type of cell (e.g., somatic cell, germ cell, embryonic cell, stem cell), tissue (e.g., bone, muscle, connective, blood), and/or organ (e.g., brain, kidney, lung, heart, pancreas, prostate, ovary, uterus, gastrointestinal tract). Examples of trauma that can cause acute blood loss include, but are not limited to, a burn, a gunshot wound, and a stab wound.

There are many different types of trauma including, but not limited to, accidental injury and criminal injury. An accidental injury is injury sustain in any type of accident (e.g., automobile accident injury, whiplash, drowning, fall, sports injury, burn, machinery accident, suffocation, natural accident, occupational injury, toy-related injury). Criminal injury is injury caused by criminal activity (e.g., child abuse, assault), and in particular, gunshot wound and stab wound.

Trauma includes battlefield trauma. Battlefield trauma includes trauma secondary to gunshot or an explosive device, including but not limited to rockets, mortars, mines, improvised explosive devices and the like. Uncontrolled hemorrhage is the leading cause of preventable combat-related deaths. The vast majority of these deaths occur in the field before the injured can be transported to a treatment facility. It has been estimated that the most common cause single cause of preventable death on the battlefield results from bleeding from extremity wounds. Bleeding from torso wounds is another cause of preventable death. Battlefield trauma also includes penetrating head wounds and injuries.

The subject can be administered compositions including bremelanotide by the methods of the present invention, which administration may be before the onset of symptoms of conditions such as hypovolemic shock, traumatic shock or hemorrhagic shock, or after the onset of symptoms. The timing and quantity of compositions administered can be selected by the skilled practitioner using ordinary skill taking into account, for example, the degree of blood loss in the subject.

Inflammatory Diseases and Conditions. Compositions and methods of the current invention are further directed towards the treatment of inflammatory diseases and inflammatory conditions in a subject. There are a number of inflammatory diseases and inflammatory conditions which may be so treated. In one aspect, the inflammatory condition results from a disease including a form of arthritis, including but not limited to osteoarthritis, rheumatoid arthritis, septic arthritis, gout and pseudogout, juvenile idiopathic arthritis, Still's disease and ankylosing spondylitis, as well as arthritis secondary to other diseases, such as arthritis secondary to lupus erythematosus, Henoch-Schonlein purpura, psoriatic arthritis, reactive arthritis, haemochromatosis, hepatitis, Wegener's granulomatosis, vasculitis syndromes, Lyme disease, familial Mediterranean fever, hyperimmunoglobulinemia D with recurrent fever, TNF receptor-associated periodic syndrome and inflammatory bowel disease, including Crohn's disease and ulcerative colitis. In another aspect, the inflammatory condition results from a disease including a form of inflammatory bowel disease, such as Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behget's syndrome, infective colitis and indeterminate colitis. In another aspect, the inflammatory condition results from an autoimmune disease, including but not limited to systemic syndromes such as systemic lupus erythematosus, Sjogren's syndrome, scleroderma, rheumatoid arthritis and polymyositis, or a syndrome affecting only a local body system, such as the endocrine system (diabetes mellitus type 1 , Hashimoto's thyroiditis, Addison's disease, etc.), dermatologic system (pemphigus vulgaris), hematologic system (autoimmune hemolytic anemia), or neural system (multiple sclerosis). Thus autoimmune diseases include, in addition to the general syndromes discussed above, such diseases and conditions as acute disseminated encephalomyelitis, Addison's disease, ankylosing spondylitis, antiphospholipid antibody syndrome, aplastic anemia, autoimmune hepatitis, autoimmune oophoritis, celiac disease, Crohn's disease, gestational pemphigoid, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, idiopathic thrombocytopenic purpura, Kawasaki disease, lupus erythematosus, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, opsoclonus myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus, pernicious anaemia, primary biliary cirrhosis, Reiter's syndrome, Sjogren's syndrome, Takayasu's arteritis, temporal arteritis warm autoimmune hemolytic anemia and Wegener's granulomatosis.

In another aspect, the inflammatory condition results from or is related to chronic obstructive pulmonary disease (COPD), also known as chronic obstructive airway diseases, including but not limited to diseases characterized by the pathological limitation of airflow in the airway that is not fully reversible, such as for example chronic bronchitis, emphysema, pneumoconiosis, pulmonary neoplasms and other lung disorders. Other inflammatory conditions include upper or lower airway diseases and disorders, such as allergic asthma, non-allergic asthma, allergic rhinitis, vasomotor rhinitis, allergic conjunctivitis, non-allergic conjunctivitis, and the like, as well as airway diseases related to external toxins or substances, such as various forms of pneumoconiosis (coalworker's pneumoconiosis, asbestosis, silicosis, bauxite fibrosis, berylliosis, or siderosis), byssinosis or hypersensitivity pneumonitis (farmer's lung or bird fancier's lung).

In yet another aspect, the inflammatory condition results from or is related to some form of transplant-related condition or syndrome, such as graft-versus-host disease, hyperacute rejection, acute rejection, or chronic rejection. Graft-versus-host disease is a common complication of allogeneic bone marrow transplantation, but can occur with other transplantations, and particularly those with T cells present in the graft, either as contaminants or intentionally introduced. Hyperacute, acute or chronic rejection can occur with bodily organs such as kidneys, liver, pancreas, spleen, uterus, heart or lungs, as well as transplantation of bone, cornea, face, hand, penis or skin. In one embodiment, a pharmaceutical composition including bremelanotide is given prophylactically to limit or prevent a transplant-related condition or syndrome, such as immediately before, during or after transplantation of a bodily fluid, organ or part. In another embodiment, the bodily fluid, organ or part being transplanted is perfused with a solution of a pharmaceutical composition including bremelanotide. In yet another embodiment, bremelanotide is administered in conjunction with, combination with or series with one or more other agents for transplant rejection, such as calcineurin inhibitors including cyclosporin or tacrolimus, mTOR inhibitors including sirolimus or everolimus, anti-proliferatives including azathioprine or mycophenolic acid, corticosteroids including prednisolone or hydrocortisone, antibodies such as monoclonal anti-IL-2Rα receptor antibodies, basiliximab or daclizumab, or polyclonal anti-T-cell antibodies such as anti-thymocyte globulin or anti-lymphocyte globulin.

Clinical Utility and Applications. In one aspect the invention provides a method for treatment of a mammalian patient with circulatory shock, including administration to the patient with circulatory shock of a therapeutically effective amount of a pharmaceutical composition including the peptide Ac- Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof. Administering may include intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, intrathoracic, intrathecal, intraosseous, intracranial, intracerebroventricular, or intracerebral administration. The therapeutically effective amount may be between about 0.5 mg and about 50 mg, preferably between about 1 mg and about 10 mg, and more preferably less than about 6 mg of the peptide or pharmaceutically acceptable salt thereof. Circulatory shock may include hypovolemic shock, cardiogenic shock, vasodilatory shock, septic shock, hemorrhagic shock, traumatic shock, or neurogenic shock. Hemorrhagic shock may include hypovolemic shock secondary to trauma, aortic dissection, ruptured aneurysm, and stroke. In another aspect, the invention provides a method of limiting hemorrhagic shock in a mammal, preferably a person, with bleeding secondary to trauma, the method including administering a therapeutically effective amount of a pharmaceutical composition including the peptide Ac-Nle-cyc/o(- Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof. This may include the step of establishing an intravenous line in a person with bleeding secondary to trauma, wherein administering includes intravenous administration. In one aspect the person is to be transported to a trauma care facility and the intravenous line is established prior to transport of the person to the trauma care facility. In a related aspect, intravenous administration of the pharmaceutical composition is initiated prior to transport of the person to the trauma care facility. In another aspect, intravenous administration of the pharmaceutical composition is done during transport of the person to the trauma care facility. In another aspect, the pharmaceutical composition is administered concurrent with or prior to the onset of metabolic acidosis. The composition may be in an aqueous solution. Intravenous administration may comprise intravenous administration of a bolus of the pharmaceutical composition. The therapeutically effective amount may comprise between about 0.5 mg and about 50 mg of the peptide or pharmaceutically acceptable salt thereof, or between about 1 mg and about 10 mg of the peptide or pharmaceutically acceptable salt thereof, or less than about 6 mg of the peptide or pharmaceutically acceptable salt thereof. In another aspect, administering for this method may include intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, intrathoracic, intrathecal, intraosseous, intracranial, intracerebroventricular, or intracerebral administration.

In another aspect, the invention provides a method of limiting injury in a person with spinal cord injury, the method includes intrathecal administration of a therapeutically effective amount of a pharmaceutical composition including the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof. In this method, intrathecal administration includes administration proximal the site of spinal cord injury. For this aspect, the therapeutically effective amount may include less than about 1 mg of the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a method for reducing excess extracellular fluid in a subject undergoing hemodialysis, including the steps of: administering a pharmaceutical composition including a melanocortin agonist to the subject in a therapeutically effective amount to maintain blood pressure during hemodialysis; and reducing excess extracellular fluid in the subject during hemodialysis.

The melanocortin agonist may be a peptide melanocortin receptor agonist with binding at human MC1- R, as determined by Ki values, at least ten times as great as the binding at human MC4-R. The melanocortin agonist may further be a peptide melanocortin receptor agonist wherein the binding at human MC1-R, as determined by Ki values, is more than ten times as great as the binding at human MC4-R. In one aspect, the melanocortin agonist is a peptide melanocortin receptor agonist wherein the Ki at human MC1-R is less than 1.0 nM and the Ki at human MC4-R is greater than 1.0 nM but less than about 10 nM. In a related aspect, the melanocortin agonist is the peptide Ac-Nle-cyc/o(-Asp-His- D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a method for preventing hypotension in a subject during hemodialysis, including the step of administering a pharmaceutical composition including a melanocortin agonist to the subject in a therapeutically effective amount to maintain blood pressure during hemodialysis, whereby excess extracellular fluid in the subject may be removed during hemodialysis without inducing hypotension. The melanocortin agonist may be a peptide melanocortin receptor agonist with binding at human MC1-R, as determined by Ki values, at least ten times as great as the binding at human MC4-R. The melanocortin agonist may further be a peptide melanocortin receptor agonist wherein the binding at human MC1-R, as determined by Ki values, is more than ten times as great as the binding at human MC4-R. In one aspect, the melanocortin agonist is a peptide melanocortin receptor agonist wherein the Ki at human MC1-R is less than 1.0 nM and the Ki at human MC4-R is greater than 1.0 nM but less than about 10 nM. In a related aspect, the melanocortin agonist is the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof. In one aspect the pharmaceutical composition is administered by intravenous means and the therapeutically effective amount of the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or pharmaceutically acceptable salt thereof comprises between about 1 mg and about 10 mg. In another aspect, the pharmaceutical composition is administered by subcutaneous or intramuscular means and the therapeutically effective amount of the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or pharmaceutically acceptable salt thereof comprises between about 1 mg and about 50 mg. In another aspect, the invention provides a method for limiting hypertension between hemodialysis treatments in a subject undergoing hemodialysis by reducing excess extracellular fluid in the subject, including the steps of: administering a pharmaceutical composition including the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe- Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof to the subject in a therapeutically effective amount to inhibit hypotension secondary to extracellular fluid reduction during hemodialysis; and reducing excess extracellular fluid in the subject during hemodialysis; whereby the removal of excess extracellular fluid in the subject limits hypertension between hemodialysis treatments.

In another aspect, the invention provides a method for treatment of a mammalian patient with inflammatory disease, including administration to the patient with an inflammatory disease of a therapeutically effective amount of a pharmaceutical composition including the peptide Ac-Nle-cyc/o(- Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof. The inflammatory disease may be osteoarthritis, rheumatoid arthritis, septic arthritis, gout and pseudogout, juvenile idiopathic arthritis, Still's disease, ankylosing spondylitis, as psoriatic arthritis, reactive arthritis, inflammatory bowel disease, Crohn's disease, or ulcerative colitis. Alternatively, the inflammatory disease may be secondary to a form of inflammatory bowel disease, including Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischemic colitis, diversion colitis, Behget's syndrome, infective colitis and indeterminate colitis, an autoimmune disease, including systemic lupus erythematosus, Sjogren's syndrome, scleroderma, rheumatoid arthritis and polymyositis, an endocrine system disease including diabetes mellitus type 1 , Hashimoto's thyroiditis, and Addison's disease, a dermatologic disease including pemphigus vulgaris, a hematologic disease including autoimmune hemolytic anemia, a neural disease including multiple sclerosis, a chronic obstructive pulmonary disease, including chronic bronchitis, emphysema, pneumoconiosis, and pulmonary neoplasms, an upper or lower airway disease or disorder, including allergic asthma, non-allergic asthma, allergic rhinitis, vasomotor rhinitis, allergic conjunctivitis, and non-allergic conjunctivitis, an airway disease related to external toxins or substances, including pneumoconiosis, coalworker's pneumoconiosis, asbestosis, silicosis, bauxite fibrosis, berylliosis, or siderosis, byssinosis andhypersensitivity pneumonitis (farmer's lung or bird fancier's lung), or a transplant-related condition or syndrome. Administering may include intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, intrathoracic, intrathecal, intraosseous, intracranial, intracerebroventricular, or intracerebral administration. In one aspect, administration comprises intravenous administration of a bolus of the pharmaceutical composition in an aqueous solution. The therapeutically effective amount may comprise between about 0.5 mg and about 50 mg of the peptide or pharmaceutically acceptable salt thereof, or between about 1 mg and about 10 mg of the peptide or pharmaceutically acceptable salt thereof, or less than about 6 mg of the peptide or pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a method for treatment of a mammalian patient with increased cytokine expression, including administration to the patient with increased cytokine expression of a therapeutically effective amount of a pharmaceutical composition including the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof. The increased cytokine expression may be secondary to circulatory shock, ischemia, or reperfusion injury. Administering may comprise intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, intrathoracic, intrathecal, intraosseous, intracranial, intracerebroventricular, or intracerebral administration. Administering may additionally comprise intravenous administration of a bolus of the pharmaceutical composition in an aqueous solution. The therapeutically effective amount may comprise between about 0.5 mg and about 50 mg of the peptide or pharmaceutically acceptable salt thereof, or between about 1 mg and about 10 mg of the peptide or pharmaceutically acceptable salt thereof, or less than about 6 mg of the peptide or pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a method for prophylactic treatment of a mammalian patient at risk of increased cytokine expression, including administration to the patient at risk of increased cytokine expression of a therapeutically effective amount of a pharmaceutical composition including the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a method of protecting mammalian tissue or organs from the deleterious effects of ischemia, including the step of administering a pharmaceutical composition including the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof to a mammalian patient in a therapeutically amount effective sufficient to reduce the deleterious effects of ischemia, wherein the patient's mean arterial pressure increases less than about 10%.

In another aspect, the invention provides a method of preventing or reducing ischemia- reperfusion injury to a mammalian tissue or organ, including the step of administering a pharmaceutical composition including the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof to a mammalian patient at risk for ischemia-reperfusion injury in a therapeutically effective amount sufficient to prevent or reduce ischemia-reperfusion injury.

In another aspect, the invention provides a pharmaceutical composition adapted for intravenous administration, including an aqueous buffered and isotonic solution including a pharmaceutically acceptable salt of the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH. In yet another aspect, the invention provides a pharmaceutical composition adapted for intravenous administration, including an aqueous hypertonic solution including a pharmaceutically acceptable salt of the peptide Ac-NIe- cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH.

Methods of Administration and Use. Circulatory Shock. In yet another aspect, the invention includes methods which optionally include monitoring the subject for symptoms of circulatory shock both before and after administration of a pharmaceutical composition including bremelanotide. Thus a subject may be administered bremelanotide by one of the methods of the invention after suffering an injury likely to induce circulatory shock but prior to the manifestation of overt symptoms of cardiovascular shock, including prior to manifestation of circulatory shock in Stage I, Stage Il or Stage III.

Methods of treating or preventing shock described herein comprise administering a therapeutically effective amount of bremelanotide to a subject. As used herein, the term "administer" and "administering" are used to mean introducing bremelanotide into a subject. When administration is for the purpose of treatment, bremelanotide is provided at, or after the onset of, a symptom of shock. The therapeutic administration of bremelanotide serves to attenuate any symptom, or prevent additional symptoms from arising. When administration is for the purposes of preventing shock ("prophylactic administration"), bremelanotide is provided in advance of any visible or detectable symptom. The prophylactic administration of bremelanotide serves to attenuate subsequently arising symptoms or prevent symptoms from arising altogether. The route of administration of bremelanotide includes, but is not limited to, topical, transdermal, intranasal, vaginal, rectal, oral, subcutaneous, intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, epidural and intrathecal.

Furthermore, the methods of treating or preventing circulatory shock of the present invention also relate to coadministering one or more substances to the subject in addition to bremelanotide. The term "coadminister" indicates that each of at least two compounds is administered during a time frame wherein the respective periods of biological activity or effects overlap. Thus the term includes sequential as well as concurrent administration of compounds where one compound is bremelanotide. If more than one compound is coadministered, the routes of administration of the two or more compounds need not be the same. The scope of the invention is not limited by the identity of the compound which may be coadministered. For example, bremelanotide may be coadministered with androstenetriol, androstenediol or derivatives thereof, various vasopressin agonists, or other pharmaceutically active substances, such as catecholamines or other α adrenergic agonists, α₂ adrenergic agonists, β adrenergic agonists or β₂ adrenergic agonists, including but not limited to epinephrine, norepinephrine, dopamine, isoproterenol, vasopressin and dobutamine. Alternatively, bremelanotide may be coadministered with fluids or other substances that are capable of alleviating, attenuating, preventing or removing symptoms in a subject suffering from, exhibiting the symptoms of, or at risk of suffering from hypovolemic shock, vasodilatory shock or cardiogenic shock. Types of fluid that can be coadministered with bremelanotide should be specific to the circumstances surrounding the particular subject that is suffering from, exhibiting the symptoms of, or at risk of suffering from shock. For example, fluids that may be coadministered with bremelanotide include, but are not limited to, salt solutions — such as sodium chloride and sodium bicarbonate — as well as whole blood, synthetic blood substitutes, plasma, serum, serum albumin and colloid solutions. Colloid solutions include, but are not limited to, solutions containing hetastarch, albumin or plasma. In one particular embodiment of the present invention, fluids such as one or more of salt solutions, colloidal solutions, whole blood, synthetic blood substitutes, plasma or serum are coadministered with bremelanotide in patients suffering from or exhibiting the symptoms of hypovolemia or a hypovolemic shock, such as hemorrhagic shock.

Particular embodiments of the coadministration methods of the present invention include methods of performing a transfusion in a subject, with the transfusion methods comprising providing blood or synthetic blood substitutes that comprise bremelanotide to a subject. The blood used in the transfusion methods can be whole blood, synthetic blood substitutes, or any fractionated portion of whole blood, such as plasma, serum, or red blood cells. Prophylactic Use. The invention also relates to methods of preventing or preventing the progression of shock in a subject at risk of suffering from shock by administering a therapeutically effective amount of bremelanotide to the subject, prior to or immediately at the onset of the first symptoms of shock. As used herein, the term "prevent," as it relates to shock, indicates that a substance of the present invention is administered to a subject to prohibit one or more symptoms of shock from detectably appearing or to attenuate the effects of one or more symptoms of shock. The term "prevent" also encompasses prohibiting entirely shock or any of its associated symptoms, from detectably appearing. Thus a subject may be "pretreated," such as a subject in a surgical setting, by using the substances of the present invention to prevent shock from arising. The phrase "preventing the progression," as it relates to shock, is used to mean a procedure designed to prohibit the detectable appearance of one or more additional symptoms of shock in a patient already exhibiting one or more symptoms of shock, and is also used to mean prohibiting the already-present symptoms of shock from worsening in the subject. The symptoms of shock that are included in preventative methods of the present invention include, but are not limited to, such symptoms of shock as highlighted herein, such as tachycardia, shallow or erratic breathing and death. A subject that is "at risk of shock" may be recognized based upon the specific circumstances surrounding a subject. For example, a surgery patient or a subject that has been wounded and begun losing blood would be at risk of shock. Similarly, a patient with a bacterial infection and exhibiting a fever or low blood pressure may also be at risk of shock or an inflammatory disease or condition. In additional embodiments of the current invention, the methods are used to prevent cardiogenic shock, hypovolemic shock and vasodilatory shock, each of which can be in any of the three aforementioned stages of shock. In one particular embodiment of the present invention, the methods are used to prevent cardiogenic shock. In another particular embodiment of the present invention, the methods are used to prevent vasodilatory shock. In another more particular embodiment of the present invention, the methods are used to prevent shock resulting from sepsis or bacteremia. In an even more particular embodiment, the methods are used to prevent septic shock or bacteremic shock in Stage I, Il or III shock. In yet another embodiment, the methods of the present invention are used to prevent hypovolemic shock. In one particular embodiment of the present invention, the methods are used prevent hemorrhagic shock. In an even more particular embodiment, the methods are used to prevent hemorrhagic shock in Stage I, Stage Il or Stage III.

Similar to the methods of treating shock described herein, one embodiment of the methods of preventing shock of the present invention comprises coadministering another substance with bremelanotide or a derivative thereof. The scope of the invention is not limited by the identity of the substance which may be coadministered with bremelanotide to prevent shock. For example, bremelanotide may be coadministered with androstenetriol, androstenediol or derivatives thereof, various vasopressin agonists, or other pharmaceutically active substances, such as catecholamines or other α adrenergic agonists, α₂ adrenergic agonists, β adrenergic agonists or β₂ adrenergic agonists, including but not limited to epinephrine, norepinephrine, dopamine, isoproterenol, vasopressin and dobutamine, to prevent shock.

Alternatively, bremelanotide may be coadministered with fluids or other substances that are capable of preventing or removing symptoms in a subject at risk of suffering from hypovolemic shock, vasodilatory shock or cardiogenic shock. The types of fluid that can be coadministered with bremelanotide to prevent shock should be specific to the circumstances surrounding the particular subject that is at risk of suffering from shock. For example, fluids that may be coadministered with bremelanotide include, but are not limited to, salt solutions - such as sodium chloride and sodium bicarbonate - as well as whole blood, synthetic blood substitutes, plasma, serum, serum albumin and colloid solutions. Colloid solutions include, but are not limited to, solutions containing hetastarch, albumin or plasma. In one particular embodiment of the present invention, fluids including one or more of salt solutions, colloidal solutions, whole blood, synthetic blood substitutes, plasma or serum are coadministered with bremelanotide or a derivative thereof in subjects at risk of suffering a hypovolemic shock, such as hemorrhagic shock. Inflammation-Related Applications. In yet another aspect, the invention includes methods which optionally include monitoring the subject for signs or symptoms of inflammation, inflammatory diseases or inflammatory conditions both before and after administration of bremelanotide. Thus a subject may be administered bremelanotide by one of the methods of the invention after being diagnosed with a condition, disease or syndrome likely to induce an inflammatory response, but prior to the manifestation of overt symptoms of inflammation, inflammatory disease or inflammatory condition. Methods of treating or preventing inflammation, inflammatory diseases or inflammatory conditions described herein comprise administering a therapeutically effective amount of bremelanotide to a subject. As used herein, the term "administer" and "administering" are used to mean introducing at least one compound into a subject. When administration is for the purpose of treatment, the substance is provided at, or after the onset of, a sign or symptom of inflammation, inflammatory disease or inflammatory condition. The therapeutic administration of this substance serves to attenuate any symptom, or prevent additional symptoms from arising. When administration is prophylactic administration for the purposes of preventing or limiting inflammation, inflammatory disease or an inflammatory condition, a pharmaceutical composition including bremelanotide is provided in advance of any visible or detectable symptom. The prophylactic administration of bremelanotide serves to attenuate subsequently arising symptoms or prevent symptoms from arising altogether. The route of administration of bremelanotide includes, but is not limited to, topical, transdermal, intranasal, vaginal, rectal, oral, subcutaneous intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, epidural and intrathecal. Devices for Administration. In certain aspects, special devices may be provided for delivery and administration of a pharmaceutical composition including bremelanotide. Thus in one aspect a prefilled syringe may be adapted for use in military applications for emergency treatment under battlefield conditions, such as for treatment of battlefield trauma, or use by paramedic personnel responding to a trauma victim. The prefilled syringe may include a lyophilized component including bremelanotide and an aqueous solubilizing component, such that the pharmaceutical composition may be reconstituted immediately prior to use. The resulting reconstituted pharmaceutical composition may be an isotonic or hypertonic solution. In a related aspect, the prefilled syringe may include bremelanotide in solution with the pharmaceutical composition including one or more preservatives or stabilizers, such that the prefilled syringe may be stored for a specified period at ambient temperature, such as room temperature, without substantial degradation.

Compounds of the Invention. In one aspect, the invention comprises a pharmaceutical composition including bremelanotide with the following general structure:

The peptide has a formula Of C₅₀H₆₈N₁₄O₁₀, and a net molecular weight of 1025.18. Bremelanotide may be synthesized by conventional means, including either solid-phase or liquid-phase techniques, and purified to greater than 99% purity, yielding a white powder that is a clear, colorless solution in water.

Bremelanotide is an agonist at MC1-R, MC3-R, MC4-R and MC5-R. Utilizing NDP-α-MSH as a competitor, bremelanotide exhibits binding selectivity with a Ki value of approximately 0.2 nM at human MC1-R, of approximately 9.0 nM at human MC4-R, of approximately 72.0 nM at human MC3-R and of approximately 159 nM at human MC5-R. Bremelanotide has an EC₅₀ of approximately 3.0 nM at MC4- R under cell receptor densities approximating normal physiological systems with a relative efficacy, compared to NDP-α-MSH, in excess of 90%. In determining the foregoing, it is advantageous to use human melanocortin receptors, such as employing cells, such as HEK-293 cells, that express recombinant hMC1-R, hMC3-R, hMC4-R, or hMC5-R. NDP-α-MSH may be iodinated, such as [I¹²⁵]- NDP-α-MSH (Perkin Elmer), or NDP-α-MSH may be labeled with a lanthanide chelate, such as Eu- NDP-α-MSH (PerkinElmer Life Sciences catalog No. AD0225).

Where [l¹²⁵]-NDP-α-MSH is employed, a competitive inhibition binding assay may be performed using membrane homogenates prepared from HEK-293 cells that express hMC1-R, hMC3-R, hMC4-R, or hMC5-R. Assays are performed in 96 well GF/B Millipore multiscreen filtration plates (MAFB NOB10) pre-coated with 0.5% bovine serum albumin (Fraction V). Membrane homogenates are incubated with 0.2nM (for hMC4-R) 0.4 nM (for MC3-R and MC5-R) or 0.1 nM (for hMC1-R) [I¹²⁵J-NDP- α-MSH (Perkin Elmer) and increasing concentrations of the test melanocortin agonist, such as bremelanotide, in buffer containing 25 mM HEPES buffer (pH 7.5) with 100 mM NaCI, 2 mM CaCI₂, 2 mM MgCI₂, 0.3 mM 1 ,10-phenanthroline, and 0.2% bovine serum albumin. After incubation for 60 minutes at 37° C, the assay mixture is filtered and the membranes washed three times with ice-cold buffer. Filters are dried and counted in a gamma counter for bound radioactivity. Non-specific binding is measured by inhibition of binding of [l¹²⁵]-NDP-α-MSH in the presence of 1 μM NDP-α-MSH. Maximal specific binding (100%) is defined as the difference in radioactivity (cpm) bound to cell membranes in the absence and presence of 1 μM NDP-α-MSH. Radioactivity (cpm) obtained in the presence of test compounds is normalized with respect to 100% specific binding to determine the percent inhibition of [l¹²⁵]-NDP-α-MSH binding. Ki values are determined using Graph-Pad Prism® curve-fitting software. Alternatively, a competitive inhibition binding assay is performed employing Eu-NDP-α-MSH

(with determination by time-resolved fluorometry (TRF) of the lanthanide chelate. In comparison studies with [l¹²⁵]-NDP-α-MSH, the same values, within experimental error ranges, are obtained for percent inhibition and Ki. Typically competition experiments to determine Ki values are conducted by incubating membrane homogenates prepared from HEK-293 cells that express recombinant hMC4-R with 9 different concentrations of test compounds of interest and 2 nM of Eu-NDP-α-MSH in a solution containing 25 mM HEPES buffer with 100 mM NaCI, 2 mM CaCI₂, 2 mM MgCI₂ and 0.3 mM 1 ,10- phenanthroline. After incubation for 90 minutes at 37°C, the reaction is stopped by filtration over AcroWell 96-well filter plates (Pall Life Sciences). The filter plates are washed 4 times with 200 μl_ of ice-cold phosphate-buffered saline. DELFIA Enhancement solution (PerkinElmer Life Sciences) is added to each well. The plates are incubated on a shaker for 15 minutes and read at 340 nm excitation and 615 nm emission wavelengths. Ki values are determined by curve-fitting with Graph-Pad Prism® software using a one-site fixed-slope competition binding model.

Peptide Synthesis. Bremelanotide may be readily synthesized by any known conventional procedure for the formation of a peptide linkage between amino acids. Such conventional procedures include, for example, any solution phase procedure permitting a condensation between the free alpha amino group of an amino acid having its carboxyl group and optionally other reactive groups protected and the free primary carboxyl group of another amino acid having its amino group and optionally other reactive groups protected. In a preferred conventional procedure, bremelanotide may be synthesized by solid-phase synthesis and purified according to methods known in the art. Any of a number of well- known procedures utilizing a variety of resins and reagents may be used to prepare bremelanotide.

The process for synthesizing bremelanotide may be carried out by a procedure whereby each amino acid in the desired sequence is added one at a time in succession to another amino acid or by a procedure whereby peptide fragments with the desired amino acid sequence are first synthesized conventionally and then condensed to provide bremelanotide. The resulting peptide is then cyclized to yield a cyclic peptide of the invention.

Solid phase peptide synthesis methods are well known and practiced in the art. In such a method the synthesis of bremelanotide can be carried out by sequentially incorporating the desired amino acid residues one at a time into the growing peptide chain according to the general principles of solid phase methods. These methods are disclosed in numerous references, including, Merrifield, R. B., Solid phase synthesis (Nobel lecture). Angew Chem 24:799-810, 1985 and Barany et al., The Peptides, Analysis, Synthesis and Biology, Vol. 2, Gross, E. and Meienhofer, J., Eds. Academic Press 1-284 (1980). Reactive side chain groups of the various amino acid residues are protected with suitable protecting groups, which prevent a chemical reaction from occurring at that site until the protecting group is removed. Usually also common is the protection of the alpha amino group of an amino acid residue or fragment while that entity reacts at the carboxyl group, followed by the selective removal of the alpha amino protecting group to allow a subsequent reaction to take place at that site. Specific protecting groups have been disclosed and are known in solid phase synthesis methods and solution phase synthesis methods.

Alpha amino groups may be protected by a suitable protecting group, including a urethane-type protecting group, such as benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl, such as p- chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-biphenyl- isopropoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl (Moz); aliphatic urethane-type protecting groups, such as t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropoxycarbonyl, and allyloxycarbonyl. Fmoc is preferred for alpha amino protection.

Guanidino groups may be protected by a suitable protecting group, such as nitro, p- toluenesulfonyl (Tos), Z, pentamethylchromanesulfonyl (Pmc), adamantyloxycarbonyl, and Boc. Pmc is a preferred protecting group for Arg.

Bremelanotide described herein was primarily prepared using solid phase synthesis. Solid phase synthesis was commenced from the C-terminal end of the peptide by coupling a protected alpha amino acid to a suitable resin. Such a starting material can be prepared by attaching an alpha amino- protected Lys amino acid by an ester linkage to a p-benzyloxybenzyl alcohol (Wang) resin or a 2- chlorotrityl chloride resin, by an amide bond between an Fmoc-Linker, such as p-[(R, S)-α-[1-(9H-fluor- en-9-yl)-methoxyformamido]-2,4-dimethyloxybenzyl]-phenoxyacetic acid (Rink linker) to a benzhydrylamine (BHA) resin, or by other means well known in the art. Fmoc-Linker-BHA resin supports are commercially available and generally used when feasible. The resins are carried through repetitive cycles as necessary to add amino acids sequentially. The alpha amino Fmoc protecting groups are removed under basic conditions. Piperidine, piperazine, diethylamine, or morpholine (20- 40% v/v) in DMF may be used for this purpose. Following removal of the alpha amino protecting group, the subsequent protected amino acids are coupled stepwise in the desired order to obtain an intermediate, protected peptide-resin. The activating reagents used for coupling of the amino acids in the solid phase synthesis of peptides are well known in the art. After bremelanotide is synthesized, if desired, the orthogonally protected side chain protecting groups may be removed using methods well known in the art for further derivatization of the peptide.

Reactive groups in a peptide can be selectively modified, either during solid phase synthesis or after removal from the resin. For example, the N-terminus acetylation may be accomplished while the peptide is on resin, or the peptide may be removed from the resin by use of a cleaving reagent and then modified. Methods for N-terminus modification, such as acetylation are well known in the art.

Bremelanotide can, in one embodiment, be cyclized prior to cleavage from the peptide resin. For cyclization through reactive side chain moieties, the desired side chains are deprotected, and the peptide suspended in a suitable solvent and a cyclic coupling agent added. Suitable solvents include, for example DMF, dichloromethane (DCM) or 1-methyl-2-pyrrolidone (NMP). Suitable cyclic coupling reagents include, for example, 2-(1 H-benzotriazol-1-yl)-1 ,1 ,3,3-tetramethyluronium tetrafluoroborate (TBTU), 2-(7-aza-1 H-benzotriazol-1-yl)-1 ,1 ,3,3-tetramethyluronium tetrafluoroborate (TATU), 2-(2-oxo- 1(2H)-pyridyl)-1 ,1 ,3,3-tetramethyluronium tetrafluoroborate (TBTU) or N.N'-dicyclohexylcarbodiimide/i- hydroxybenzotriazole (DCCI/HOBt). Coupling is conventionally initiated by use of a suitable base, such as N,N-diispropylethylamine (DIPEA), sym-collidine or N-methylmorpholine (NMM). Following cleavage bremelanotide from the solid phase following synthesis, bremelanotide can be purified by any number of methods, such as reverse phase high performance liquid chromatography (RP-HPLC), using a suitable column, such as a Ci₈ column. Other methods of separation or purification, such as methods based on the size or charge of the peptide, can also be employed. Once purified, bremelanotide can be characterized by any number of methods, such as high performance liquid chromatograph (HPLC), amino acid analysis, mass spectrometry, and the like.

Salt Forms. Bremelanotide may be in the form of any pharmaceutically acceptable salt. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, lithium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl- morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like. An acid addition salt, prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids, is particularly preferred. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, carboxylic, citric, ethanesulfonic, formic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, malonic, mucic, nitric, pamoic, pantothenic, phosphoric, propionic, succinic, sulfuric, tartaric, p-toluenesulfonic acid, trifluoroacetic acid, and the like. Acid addition salts of bremelanotide are prepared in a suitable solvent and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, trifluoroacetic, citric, tartaric, maleic, succinic or methanesulfonic acid. In one preferred embodiment, bremelanotide is in an acetate salt form, and is formulated in an aqueous solution, optionally including one or more salts, such as sodium chloride.

Pharmaceutical Compositions. The invention provides a pharmaceutical composition that includes bremelanotide and a pharmaceutically acceptable carrier. The carrier may be a liquid formulation, and is preferably a buffered, isotonic, aqueous solution. Pharmaceutically acceptable carriers also include excipients, such as diluents, carriers and the like, and additives, such as stabilizing agents, preservatives, solubilizing agents, buffers and the like, as hereafter described. For injection administration formulations, water containing at least one or more buffering constituents is preferred, and stabilizing agents, preservatives and solubilizing agents may also be employed. For most pharmaceutical formulations, non-active ingredients will constitute the greater part, by weight or volume, of the preparation. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that it may be administered by syringe. The form must be stable under the conditions of manufacture and storage and may optionally contain one or more agents to preserve the pharmaceutical form against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol, for example glycerol, propylene glycol or liquid polyethylene glycol, suitable mixtures thereof, and vegetable oils. If in an aqueous solution, bremelanotide may be appropriately buffered by means of saline, acetate, phosphate, citrate, or other buffering agents, which may be at any physiologically acceptable pH, generally from about pH 4 to about pH 7. A combination of buffering agents may also be employed, such as phosphate buffered saline, a saline and acetate buffer, and the like. In the case of saline, a 0.9% saline solution may be employed. In the case of acetate, phosphate, citrate, acetate and the like, a 50 mM solution may be employed. In addition to buffering agents, a suitable preservative may be employed, to prevent or limit bacteria and other microbial growth. One such preservative that may be employed is 0.05% benzalkonium chloride. Another such preservative that may be employed is 1.0 - 1.5% benzyl alcohol.

Among pharmaceutical buffers that may be employed in formulations of bremelanotide are calcium carbonate, ammonia solution, sodium hydroxide, monoethanolamine, malic acid, diethanolamine, monobasic sodium phosphate, citric acid monohydrate, boric acid, dibasic sodium phosphate, succinic acid, hydrochloric acid, trishydroxymethylaminomethane (TRIS), acetic acid, adipic acid, arginine, benzoic acid, boric acid, carbonic acid, citric acid, ethanolamine, ethylenediamine, glutamic acid, glycine, lactic acid, lysine, maleic acid, phosphoric acid, tartaric acid, triethanolamine and tromethamine. Any of a number of compounds may be used for tonicity adjustment, such as saline, dextrose, mannitol, glycerine, potassium chloride and the like. Water soluble organic solvents and surfactants may be employed, including propylene glycol, ethanol, polyethylene glycol 300, polyethylene glycol 400, glycerine, dimethylacetamide, Λ/-methyl-2-pyrrolidone, dimethylsulfoxide, Solutol HS 15, Cremophor EL, Cremophor RH 60, polysorbate 80 and the like. For intravenous bolus injection, the amount of organic solvent administered can be, for example, up to about 70% propylene glycol, up to about 50% polyethylene glycol 300, about to about 20% ethanol, up to about 15% glycerine, or up to about 10% polyethylene glycol 400. For intravenous infusion, the amount of organic solvent administered can be, for example, up to about 25% polysorbate 80, up to about 15% glycerin, up to about 10% Cremophor EL, up to about 15% ethanol or up to about 10% propylene glycol. Other formulation components can include, for example, various cyclodextrins, such as α-cyclodextrin or sulfobutylether-β-cyclodextrin, benzyl alcohol, α-tocopherol, disodium succinate, sucrose, dextrose, sodium ascorbate, EDTA, Tween 80, and the like.

In one aspect, formulations adapted for rapid bolus intravenous injection or alternatively slow infusion intravenous injection are provided, and the following pharmaceutical compositions are exemplary: Formulation 1 : A sterile solution of 1 - 200 mg of bremelanotide active ingredient in 1 mL of

Water for Injection. The pH of the solution is adjusted between 4 and 7.5. The formulation may be packaged in Type 1 glass vials, or Type 1 glass syringes, or plastic ampoules or syringes molded from specially formulated polypropylene.

Formulation 2: A sterile solution of 1 - 200 mg of bremelanotide active ingredient and 9 mg of sodium chloride in 1 mL of Water for Injection. The pH of the solution is adjusted between 4 and 7.5. The formulation may be packaged in Type 1 glass vials, or Type 1 glass syringes, or plastic ampoules or syringes molded from specially formulated polypropylene.

Formulation 3: A sterile solution of 1 - 200 mg of bremelanotide active ingredient with 0 - 25 mg of glycerin in 1 mL of Water for Injection. The pH of the solution is adjusted between 4 and 7.5. The formulation may be packaged in Type 1 glass vials, or Type 1 glass syringes, or plastic ampoules or syringes molded from specially formulated polypropylene.

Formulation 4: A sterile solution of 1 - 200 mg of bremelanotide active ingredient with 1 - 10 mg of benzyl alcohol and 0 - 25 mg of glycerin in 1 mL of Water for Injection. The pH of the solution is adjusted between 4 and 7.5. The formulation may be packaged in Type 1 glass vials, or Type 1 glass syringes, or plastic ampoules or syringes molded from specially formulated polypropylene.

Formulation 5: A sterile lyophilized powder containing 1 - 200 mg of bremelanotide active ingredient and 10 - 35 mg of mannitol. When reconstituted with Water for Injection, the pH is between 4 and 7.5. The formulation may be packaged in Type 1 glass vials or syringes. Formulation 6: A sterile solution of 1 - 200 mg of bremelanotide active ingredient with 1 - 10 mg of polysorbate 80 and 0 - 25 mg of glycerin in 1 ml_ of Water for Injection. The formulation may be packaged in Type 1 glass vials, or Type 1 glass syringes, or plastic ampoules or syringes molded from specially formulated polypropylene. Formulation 7: A sterile solution of 1 - 200 mg of bremelanotide active ingredient with 1 - 5 mg of glycocholic acid and 0 - 25 mg of glycerin in 1 ml_ of Water for Injection. The formulation may be packaged in Type 1 glass vials, or Type 1 glass syringes, or plastic ampoules or syringes molded from specially formulated polypropylene.

Formulation 8: A sterile solution of 1 - 200 mg of bremelanotide active ingredient with 1 - 25 mg of propylene glycol and 1 - 10 mg of polysorbate 80 in 1 ml_ of Water for Injection. The formulation may be packaged in Type 1 glass vials, or Type 1 glass syringes, or plastic ampoules or syringes molded from specially formulated polypropylene.

Routes of Administration. If it is administered by injection, the injection may be intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, intrathoracic, intrathecal, intraosseous, intracranial, intracerebroventricular, intracerebral or other means known in the art. Bremelanotide may be formulated for administration by any means known in the art, including but not limited to formulation as tablets, capsules, caplets, suspensions, powders, ointments, creams, lyophilized preparations, suppositories, ocular drops, skin patches, oral soluble formulations, sprays, aerosols and the like. In general, any route of administration by which the bremelanotide is introduced across an epidermal layer of cells may be employed. Administration means may thus include administration through mucous membranes, buccal administration, oral administration, dermal administration, inhalation administration, intranasal administration, urethral administration, vaginal administration, and the like. For treatment of circulatory shock, intravenous administration is particularly preferred.

Therapeutically Effective Amount. In general, the actual quantity of bremelanotide administered to a patient will vary between fairly wide ranges depending upon the mode of administration, the formulation used, and the response desired. The dosage for treatment is administration, by any of the foregoing means or any other means known in the art, of an amount sufficient to bring about the desired therapeutic effect. Thus a therapeutically effective amount includes an amount of bremelanotide that is sufficient to therapeutically alleviate circulatory shock, increased cytokine levels, hypovolemia, hypotension, inflammatory response or related conditions in a patient, or to prevent or delay onset or recurrence of circulatory shock, increased cytokine levels, hypovolemia, hypotension, inflammatory response or related conditions. The response to the therapeutically effective amount may be a cellular, organ or tissue-specific response, or a system response. Thus in one aspect, a therapeutically effective amount of bremelanotide administered to a patient with circulatory shock is such amount as results in an increase in blood pressure, a decrease in signs and symptoms of shock, or other responses relevant to circulatory shock. In another aspect, a therapeutically effective amount of bremelanotide administered to a patient with ischemia or ischemia-reperfusion injury is such amount as results in a decrease in cytokine levels or expression, such as a decrease in TNF-α, IL-2 or another inflammatory cytokine, a decrease in signs and symptoms of ischemia or ischemia-reperfusion injury, or other responses relevant to ischemia or ischemia-reperfusion injury. In yet another aspect, a therapeutically effective amount of bremelanotide administered to a patient undergoing hemodialysis for the purpose of reducing excess extracellular fluid is such amount as permits removal of excess extracellular fluid during hemodialysis without inducing hypotension.

In general, bremelanotide is highly active when used for treatment of circulatory shock, ischemia, reperfusion injury, inflammatory disease or autoimmune disease, with dose responses at doses below 0.01 μmol/kg, and optimal or peak dose responses between about 0.05 μmol/kg and about 1 μmol/kg. In one aspect bremelanotide may be administered at 0.01 , 0.05, 0.1 , 0.5, 1 , or 5 μmol/kg body weight, depending on the desired therapeutic response, the route of administration, the formulation and other factors known to those of skill in the art.

Thus in one aspect, for treatment of circulatory shock, increased cytokine levels, hypovolemia, hypotension, inflammatory disease or related conditions bremelanotide may be administered by intravenous administration as a bolus. The dose of bremelanotide administered to an adult patient in need thereof may between about 0.5 mg and 50 mg of bremelanotide, preferably between about 1 mg and 10 mg of bremelanotide, and more preferably between about 3 mg and 6 mg of bremelanotide. Doses administered to children and infants may be appropriately scaled.

In a second aspect, for treatment of circulatory shock, ischemia, reperfusion injury, increased cytokine levels, hypovolemia, hypotension, inflammatory disease or related conditions bremelanotide may be administered by intravenous administration as part of a solution that is rapidly infused. The quantity of bremelanotide administered may be in any suitable quantity of an aqueous formulation, such that the liquid volume intravenously administered may be between about 1 ml_ and about 1 L, more preferably between about 50 ml_ and about 250 ml_. In another aspect, for treatment of circulatory shock, ischemia, reperfusion injury, increased cytokine levels, hypovolemia, hypotension, inflammatory disease or related conditions bremelanotide may be administered by intravenous administration over a period of time. A suitable rate for intravenous administration may be employed, such as between about 10 μg/minute and about 1 mg/minute, more preferably between about 100 μg/minute and about 500 μg/minute. Administration over a period of time at a suitable rate provides for concurrent fluid volume replacement and permits discontinuation of administration of bremelanotide when the desired therapeutic effect has been obtained.

For administration of bremelanotide by intravenous means, either as a bolus or as a component of a rapidly infused solution, the bremelanotide may in one aspect be in a solution that is hypertonic. The solution may be a hypertonic crystalloid or colloid solution. In one aspect the hypertonic solution includes sodium chloride and a second sodium source, such as sodium acetate. In another aspect, the hypertonic solution includes a source of sodium and a source of chloride, with a ratio of sodium to chloride of between about 1 :0.7 and 1 :1 , the sources of sodium and chloride including NaCI, KCI, NaHCO₃, Na₂SO₄, Na₂HPO₄, MgCI₂, Na acetate, Na lactate, and the like. The hypertonic formulation may include other ingredients, including KHCO₃, K acetate, K lactate, MgSO₄, K₂HPO₄ or, in general, any ingredient or compound listed above in the discussion on pharmaceutical compositions.

Alternative Melanocortin Receptor Agonists. While certain embodiments of the present invention are described primarily in the context of bremelanotide, it is to be understood that other melanocortin receptor agonists may be employed in some aspects. For example, the metallopeptide melanocortin receptor agonists disclosed in WO 02/064091 , filed on February 13, 2001 , and U.S. Patent No. 7,307,063, issued on December 1 1 , 2007, both entitled Melanocortin Metallopeptides for Treatment of Sexual Dysfunction; and WO 01/13112, filed on June 14, 2000, entitled Melanocortin Metallopeptide Constructs, Combinatorial Libraries and Applications, may be employed. In addition, the peptidomimetic melanocortin receptor agonists disclosed in U.S. Serial No. 10/776,419, filed on February 10, 2004, entitled Peptidomimetics of Biologically Active Metallopeptides; the pyrrolidine melanocortin receptor agonists disclosed in U.S. Patent No. 7,189,755, issued on March 13, 2007, entitled Pyrrolidine Melanocortin-Specific Compounds; and the bicyclic melanocortin receptor agonists disclosed in WO 05/017574, filed on January 20, 2004, entitled Bicyclic Melanocortin-Specific Compounds, may also be employed. Also particular preferred are the piperazine melanocortin agonists disclosed in WO 05/102340, filed on January 20, 2004 and U.S. Patent No. 7,354,923, issued on April 8, 2008, both entitled Piperazine Melanocortin-Specific Compounds; the melanocortin agonists disclosed in WO 03/006620, filed on July 11 , 2002, entitled Linear and Cyclic Melanocortin Receptor- Specific Peptides; WO 04/005324, filed on July 9, 2003, entitled Peptide Compositions for Treatment of Sexual Dysfunction; WO 01/00224, filed on June 29, 2000 and U.S. Patent No. 6,794,489, issued on September 21 , 2004, entitled Compositions and Methods for Treatment of Sexual Dysfunction; and U.S. Patent No. 7,176,279, issued on February 13, 2007, entitled Cyclic Peptide Compositions and Methods for Treatment of Sexual Dysfunction. The entire disclosure of each of the foregoing are incorporated here by reference. It is to be understood that the foregoing listing of patents and patent applications disclosing melanocortin receptor agonists is intended to only be exemplary, and that other melanocortin receptor agonists, whether heretofore known or hereafter developed, may similarly be used in the practice of aspects of this invention. It is to be understood that the contents of the above published patents and patent applications, and in particular the general formulas and exemplified compounds therein, are incorporated in this specification in their entirety by reference thereto. It is further to be understood that the foregoing listing is intended to only be exemplary, and that other melanocortin receptor agonists, whether heretofore known or hereafter developed, may similarly be used in the practice of this invention.

The invention is further exemplified by the following non-limiting examples: Example 1

A rat hemorrhagic shock model, utilizing male Sprague Dawley rats weighing approximately 350 grams, was established. Each rat was anesthetized with 1.5% isoflurane carried by medical oxygen. The jugular vein was cannulated for blood withdrawal and subsequent intravenous administration, and a blood pressure transducer was implanted in the left femoral artery. Heparin was administered (600 IU/kg intravenous). Animals were maintained under anesthesia during the procedure, with body temperature maintained by a heating pad and monitored by a rectal probe. Baseline blood pressure, pulse pressure, heart rate, hematocrit, temperature and related parameters were obtained. Approximately 50% to 60% of blood (approximately 2.5 to 3 ml_ blood/100 g body weight) was then withdrawn over a ten to twenty minute period, and the animals were stabilized when mean arterial pressure (MAP) was at approximately 35 to 40 mm Hg for a period of fifteen minutes. Animals were then administered test substances, and observed for a period of up to two hours, including repeat measurements of blood pressure, pulse pressure, heart rate, hematocrit, temperature and related parameters. Animals were sacrificed and tissue samples obtained for examination. Example 2

Rats as in Example 1 , which were cannulated but which did not have blood withdrawn, were intravenously administered either saline as or 1.0 μmol/kg bremelanotide in a saline solution, to determine the effect of bremelanotide in normal rats as shown in FIG. 1 (sham saline and sham bremelanotide at 1.0), normal baseline pressures were maintained for one hour post injection, with a small increase in mean arterial pressure following administration of bremelanotide. Example 3

Utilizing rats as in Example 1 , the effect of bremelanotide in saline administered intravenously at doses of 0.01 , 0.1 and 1.0 μmol/kg on shock-induced animals was determined. A total volume of 0.150 ml_ was injected, with the same amount of saline not containing bremelanotide utilized in the sham saline and shock saline injections. MAP increased at all three bremelanotide dose levels, with a p value for results at 60 minutes post treatment compared to the saline control of less than 0.01 for the 0.1 and 1.0 μmol/kg bremelanotide doses, and less than 0.05 for the 0.01 μmol/kg bremelanotide dose. As shown in FIG. 1 similar results were obtained by plotting mean arterial pressure, with the mean arterial pressure at 60 minutes post dose with the 1.0 μmol/kg bremelanotide dose exceeding the sham saline mean arterial pressure (animals as in Example 2), and the mean arterial pressure at 60 minutes post dose with the 0.1 μmol/kg bremelanotide dose approximating the sham saline mean arterial pressure. Ischemic organ injury as determined by histologic examination showed substantially less injury in hemorrhagic shock induced animals administered 1.0 μmol/kg bremelanotide doses as compared to animals administered saline control. Example 4

Utilizing rats as in Example 1 , levels of cytokine expression and concentrations, including TNF-α and IL-2, are determined following intravenous administration of bremelanotide at doses of 0.01 , 0.1 and 1.0 μmol/kg in shock-induced animals. Results obtained are compared to results in shock-induced animals administered only an equal quantity of saline, and in animals in which shock was not induced but which were administered either saline or bremelanotide. Example 5

Utilizing rats as in Example 1 , a bilateral cervical vagotomy was performed approximately ten minutes prior to bleeding to induce hypovolemia. A mean arterial blood pressure of approximately 32 mm Hg was stabilized for approximately fifteen minutes before assignment to one of three groups. FIG. 2 depicts mean arterial pressure (MAP) of: animals with a vagotomy but no bleeding to induce hypovolemia or other treatment (line with diamond ♦markers); animals with no vagotomy, bleeding to induce hypovolemia and treatment with bremelanotide at 1 μmol/kg (line with square ■ markers); animals with vagotomy, bleeding to induce hypovolemia and treatment with bremelanotide at 1 μmol/kg (line with triangle A markers); and animals with vagotomy, bleeding to induce hypovolemia and treatment with saline (line with crossed-line x markers), where between 2 and 7 animals are in each group. Example 6

Utilizing rats as in Example 1 , urine production over four hours in rats with induced hypovolemia was determined. A total of six animals had 48-50% of total blood volume removed followed by stabilization of mean arterial pressure at 40 mm/Hg for one hour, followed by assignment to one of two groups of three animals each. In one group, animals received an intravenous bolus injection of 1.5 ml_ of a solution of 7.5% sodium chloride, 6% dextran-70 and bremelanotide at 1 μmol/kg, with urine production measured for four hours after treatment (left bar, with downward diagonal lines). The second group of animals received 1.5 ml_ of a solution of 7.5% sodium chloride and 6% dextran-70 without bremelanotide, also with urine production measured for four hours after treatment (right bar, with upward diagonal lines). Results are as shown in FIG. 3, illustrating rats treated with bremelanotide plus fluid therapy had statistically significantly greater urine production (p < 0.01 ) than rats receiving only fluid therapy. Example 7 Utilizing rats as in Example 1 , core body temperature was measured by a metal probe inserted into the descending colon. Core body temperature decreased uniformly in all animals during induced shock by removal of blood volume, and was dose-responsively increased after treatment with 1 μmol/kg, 0.1 μmol/kg and 0.01 μmol/kg of bremelanotide, but core body temperature continued to decrease in animals administered only saline, suggesting improved blood perfusion in the intestinal tract after treatment with bremelanotide following induced shock by blood removal, but not after treatment with saline. Example 8

In studies as in Example 6, rats had 48-50% of total blood volume removed followed by stabilization of mean arterial pressure at 40 mm/Hg for one hour, followed by assignment to one of two groups of three animals each. In one group, animals received an intravenous bolus injection over fifteen to twenty minutes of 1.5 ml_ of a solution of 7.5% sodium chloride, 6% dextran-70 and bremelanotide at 1 μmol/kg, and in the second group animals received 1.5 ml_ of a solution of 7.5% sodium chloride and 6% dextran-70 without bremelanotide. At all timepoints measured to four hours post intravenous injection, animals receiving bremelanotide plus fluid therapy had higher mean arterial blood pressure. Example 9

Dog studies of induced hypovolemia were conducted by bleeding dogs to 40 mm Hg (from starting mean arterial pressures of between 110 and 120 mm Hg) and maintaining mean arterial pressures at approximately 40 mm Hg for 30 minutes. At all times, dogs were anesthetized with isoflurane and ventilated with 98.5% O₂. Animals were then administered either saline or bremelanotide at 1 μmol/kg, and observed for two hours, with periodic readings of mean arterial pressure. FIG. 4 shows changes in mean arterial pressure in mm Hg over the study course of dogs administered either saline (line with solid • circle) or bremelanotide at 1 μmol/kg (line with solid ■ square). Example 10

In dog studies as in Example 9, serum lactate levels were measured following administration of either saline or bremelanotide at 0.87 μmol/kg after induction of hypovolemia by bleeding. Serum lactate levels in plasma did not significantly increase over a three hour period following administration of bremelanotide, but did significantly increase over a three hour period following administration of saline. Example 11

In rat studies, baseline levels of neutrophil gelatinase-associated lipocalin (NGAL), an early biomarker for acute renal failure, were determined using a commercially available Elisa assay. Shock was then induced by removing blood from rats and stabilizing blood pressure at 35 mm Hg for fifteen minutes, following which blood was again withdrawn for determination of NGAL levels. Animals were then divided into three groups, with one group administered arginine vasopressin (AVP) (1 IU/kg), a second group administered bremelanotide (1 μmol/kg) and a third group administered saline. At two hours post dosing, or earlier in the event of animal death, blood was again withdrawn for determination of NGAL levels, with results as shown in FIG. 5. Example 12

In other studies as in Example 11 , blood pH, base excess, saturated O₂ and blood lactate levels were determined at baseline, at fifteen minutes following stabilization of blood pressure at 35 mm Hg following blood removal, and at two hours post dosing with AVP (1 IU/kg), bremelanotide (1 μmol/kg) or saline. Blood pH, base excess and saturated O₂ all decreased between baseline and stabilization following bleeding at 35 mm Hg, and continued to decrease at two hours post dosing for animals receiving AVP or saline, but increased for animals receiving bremelanotide (p < 0.01 at two hours with between four and seven animals per group). Lactate levels increased between baseline and stabilization following bleeding at 35 mm Hg, and continued to increase at two hours post dosing for animals receiving AVP or saline, but decreased relative to the stabilization levels for animals receiving bremelanotide. Example 13 A patient suffering from severe trauma, such as resulting from a gunshot wound or motor vehicle accident, and at risk of hemorrhagic shock, is administered an intravenous bolus injection of a solution including approximately 4 mg of bremelanotide. The bremelanotide may be administered prior to emergency transport of the patient to a hospital or other acute care medical facility. Example 14

A patient undergoing cardiovascular surgery is administered an intravenous bolus injection of a solution including approximately 4 mg of bremelanotide. The bremelanotide may be administered prior to the onset of symptoms of hypovolemia or other circulatory shock. Example 15 A patient undergoing cardiovascular surgery is administered an intravenous solution including bremelanotide, with the bremelanotide administered at rate from about 10 μg/minute to about 500 μg/minute. The bremelanotide may be administered prior to the onset of symptoms of hypovolemia or other circulatory shock. Example 16 A patient undergoing hemodialysis who has hypertension between hemodialysis procedures, but who has or is at risk of intradialytic hypotension, is administered an intravenous solution including bremelanotide, with the bremelanotide administered at a rate of from about 10 μg/minute to about 500 μg/minute. Concurrent with or subsequent to administration of bremelanotide a total volume of from about 0.5 L to about 2.0 L of excess extracellular fluid is removed from the patient during hemodialysis.

Although the invention has been described in detail with particular reference to these preferred embodiments, other embodiments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art and it is intended to cover all such modifications and equivalents. The entire disclosures of all references, applications, patents, and publications cited above are hereby incorporated by reference.

Claims

CLAIMS What is claimed is:

1. A method of preventing or reducing ischemia or reperfusion injury to a tissue or organ of a mammalian subject, comprising the step of administering a pharmaceutical composition comprising the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof to a mammalian subject at risk for ischemia or reperfusion injury in a therapeutically effective amount sufficient to prevent or reduce ischemia or reperfusion injury.

2. The method of claim 1 , wherein the patient at risk for ischemia or reperfusion injury is undergoing cardiovascular surgery.

3. The method of claim 1 , wherein administering comprises intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, intrathoracic, intrathecal, intraosseous, intracranial, intracerebroventricular, or intracerebral administration.

4. The method of claim 1 wherein administration comprises intravenous administration of a bolus of the pharmaceutical composition in an aqueous solution.

5. The method of claim 1 wherein the therapeutically effective amount comprises between about 0.5 mg and about 50 mg of the peptide or pharmaceutically acceptable salt thereof.

6. A method for treatment of a mammalian subject with circulatory shock, comprising administration to the subject with circulatory shock of a therapeutically effective amount of a pharmaceutical composition comprising the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof.

7. The method of claim 6, wherein administering comprises intravenous, intraarterial, subcutaneous, intramuscular, intraperitoneal, intrathoracic, intrathecal, intraosseous, intracranial, intracerebroventricular, or intracerebral administration.

8. The method of claim 6 wherein the therapeutically effective amount comprises between about 0.5 mg and about 50 mg of the peptide or pharmaceutically acceptable salt thereof.

9. The method of claim 6, wherein circulatory shock is hypovolemic shock, cardiogenic shock, vasodilatory shock, septic shock, hemorrhagic shock, traumatic shock, or neurogenic shock.

10. The method of claim 9, wherein hemorrhagic shock is secondary to trauma, aortic dissection, ruptured aneurysm, or stroke.

11. A method of limiting hemorrhagic shock in a mammalian subject with bleeding secondary to trauma, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof to the subject.

12. A method of limiting injury in a mammalian subject with spinal cord injury, the method comprising intrathecal administration of a therapeutically effective amount of a pharmaceutical composition comprising the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof to the subject

13. A method for reducing excess extracellular fluid in a subject undergoing hemodialysis, comprising the steps of: administering a pharmaceutical composition comprising a melanocortin agonist to the subject in a therapeutically effective amount to maintain blood pressure during hemodialysis; and reducing excess extracellular fluid in the subject during hemodialysis.

14. The method of claim 13 wherein the melanocortin agonist is the peptide Ac-Nle-cyc/o(- Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof.

15. A method for preventing hypotension in a subject during hemodialysis, comprising the step of: administering a pharmaceutical composition comprising a melanocortin agonist to the subject in a therapeutically effective amount to maintain blood pressure during hemodialysis; whereby excess extracellular fluid in the subject may be removed during hemodialysis without inducing hypotension.

16. The method of claim 15 wherein the melanocortin agonist is the peptide Ac-Nle-cyc/o(- Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof.

17. A method for limiting hypertension between hemodialysis treatments in a subject undergoing periodic hemodialysis, comprising the steps of: administering a pharmaceutical composition comprising the peptide Ac-Nle-cyc/o(-Asp-His-D- Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof to the subject in a therapeutically effective amount to inhibit hypotension secondary to extracellular fluid reduction during hemodialysis; and reducing excess extracellular fluid in the subject during hemodialysis; whereby the removal of excess extracellular fluid in the subject limits hypertension between hemodialysis treatments.

18. A method for treatment of a mammalian subject with inflammatory disease, comprising administration to the patient with an inflammatory disease of a therapeutically effective amount of a pharmaceutical composition comprising the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof.

19. A method for prophylactic treatment of a mammalian subject at risk of pro-inflammatory cytokine expression, comprising administration to the patient at risk of pro-inflammatory cytokine expression of a therapeutically effective amount of a pharmaceutical composition comprising the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof.

20. A method of protecting tissue or organs of a mammalian subject from the deleterious effects of ischemia, comprising the step of administering a pharmaceutical composition comprising the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof to a mammalian subject in a therapeutically amount effective sufficient to reduce the deleterious effects of ischemia.

21. A pharmaceutical composition adapted for intravenous administration, comprising an aqueous buffered and isotonic solution including a pharmaceutically acceptable salt of the peptide Ac- Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH.

22. A pharmaceutical composition adapted for intravenous administration, comprising an aqueous hypertonic solution including a pharmaceutically acceptable salt of the peptide Ac-Nle-cyc/o(-

Asp-His-D-Phe-Arg-Trp-Lys)-OH.

23. Use of the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for use in the prevention of ischemia or reperfusion injury to a tissue or organ of a patient at risk of ischemia or reperfusion injury.

24. The use of claim 23, wherein the tissue or organ comprises the kidneys.

25. The use of claim 23, wherein the patient is at risk of ischemia or reperfusion injury as a result of a surgical procedure.

26. The use of claim 25, wherein the surgical procedure comprises artery bypass graft surgery.

27. Use of the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for use in reducing ischemia or reperfusion injury to a tissue or organ of a patient at risk of ischemia or reperfusion injury.

28. The use of claim 27, wherein the tissue or organ comprises the kidneys.

29. Use of the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for use in the treatment of a patient with circulatory shock.

30 Use of the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for use in the treatment of a patient with bleeding secondary to trauma.

31. Use of the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for use in the treatment of a patient with spinal cord injury.

32. Use of the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof for the manufacture of a medicament to prevent hypotension in a patient undergoing hemodialysis.

33. Use of the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof for the manufacture of a medicament to prevent hypertension between hemodialysis treatments in a patient undergoing periodic hemodialysis.

34. Use of the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for use in the treatment of a patient with inflammatory disease.

35. Use of the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for use in the treatment of a patient with pro-inflammatory cytokine expression.

36. Use of the peptide Ac-Nle-cyc/o(-Asp-His-D-Phe-Arg-Trp-Lys)-OH or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for use in the prophylactic treatment of a patient at risk of pro-inflammatory cytokine expression.