WO2008140499A2 - Method for treating sepsis - Google Patents

Abstract

The present invention provides a method of treating and/or preventing sepsis or a sepsis-induced condition in a subject in need thereof or who is at risk of developing sepsis or a sepsis-induced condition, e.g. an inflammatory condition, by the systemic administration of high-molecular weight hyaluronan.

Description

METHODS FOR TREATING SEPSIS

RELATED APPLICATION

This application claims the benefit of U.S. provisional application Ser. No. 60/861,461, filed November 29, 2006, the entire disclosure of which is incorporated herein by reference.

INCORPORATION BY REFERENCE

Each of the applications and patents cited in this text, as well as each document or reference cited in each of the applications and patents (including during the prosecution of each issued patent; "application cited documents"), and each of the PCT and foreign applications or patents corresponding to and/or paragraphing priority from any of these applications and patents, and each of the documents cited or referenced in each of the application cited documents, are hereby expressly incorporated herein by reference. More generally, documents or references are cited in this text, either in a Reference List before the paragraphs, or in the text itself; and, each of these documents or references ("herein-cited references"), as well as each document or reference cited in each of the herein-cited references (including any manufacturer's specifications, instructions, etc.), is hereby expressly incorporated herein by reference.

BACKGROUND OF THE INVENTION

Sepsis is a form of severe, overwhelming infection having an associated systemic inflammatory response that affects the entire body, resulting in fever, rapid heart rate, low blood pressure, organ ischemia and other systemic symptoms. In many cases, sepsis is accompanied by organ failure, such as failure of the kidneys and/or the lungs.

Ultimately, if the body fails to remove the primary infection, the continued presence of the invading organism can cause the body to go into septic shock. In the United States, there are over half a million patients hospitalized with septic shock each year and the incidence of sepsis has been increasing. A prevalent condition associated with sepsis is acute lung injury. More in particular, sepsis is the most frequent cause of admission to the intensive care unit and is the cause of 40% of all cases of acute lung injury. Acute lung injury is characterized by hypoxemia, bilateral infiltrates on chest x-ray, non- cardiogenic lung edema, and inflammation in the lung. Current treatments for sepsis and/or sepsis-induced acute lung injury include antibiotics, fluid resuscitation, blood pressure support with vasoactive agents, replacement of steroids for patients with adrenal insufficiency, and inhibition of the clotting cascade, as well as, respiratory assistive procedures, such as intubation and mechanical ventilation. Steroids, in particular, are widely used to treat sepsis and sepsis-induced conditions. They are typically administered in an attempt to inhibit the harmful inflammation associated with sepsis. However, steroids may have undesirable adverse effects on the body. Moreover, while steroids have been shown to improve oxygenation in acute lung injury with sepsis, the outcome has not been improved. Further still, steroids given late in the course of acute lung injury due to sepsis have been shown to be detrimental.

Despite the progress in understanding sepsis, its pathophysiology, and the variety of therapies for sepsis and sepsis-induced conditions, such as, septic shock and acute lung injury anti-sepsis treatments, the mortality rate of sepsis remains high (e.g. 30- 40%).

Accordingly, new and improved treatments for sepsis and sepsis-related conditions are desired, in particular, treatments that are safe, effective and have a minimum of adverse effects. Such new and improved treatments would represent an advancement in the art.

SUMMARY OF THE INVENTION

The present invention relates to methods of treating or preventing sepsis or a sepsis-induced condition in a subject in need thereof having, or who is at risk of developing, sepsis or a sepsis-induced condition. In one aspect, the invention provides a method of treating sepsis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of hyaluronan, thereby treating sepsis in the subject.

In another aspect, the present invention provides a method of treating a sepsis- induced condition in a subject having sepsis comprising systemically administering to the subject a therapeutically effective amount of hyaluronan, thereby treating the sepsis- induced condition in the subject.

In yet another aspect, the present invention provides a method of treating sepsis in a subject comprising administering high-molecular weight hyaluronan in an amount effective to inhibit cell surface binding of endogenous low-molecular weight hyaluronan, thereby treating sepsis in the subject.

In still a further aspect, the present invention provides a method of treating acute lung injury in a subject having sepsis, comprising administering to the subject a fluid comprising a therapeutically effective amount of hyaluronan, thereby treating acute lung injury in the subject.

In a still further aspect, the present invention provides a pharmaceutical kit for treating sepsis in a subject, comprising a therapeutically effective amount of hyaluronan, a pharmaceutically acceptable carrier, and instructions for use. In various embodiments, the present invention provides a method of coadministering hyaluronan of the invention and at least one anti-sepsis therapy. The antisepsis therapy can be an anti-inflammatory therapy, e.g. NSAIDs, steroids, antibodies against inflammatory components, or a mineralocorticoid, or other suitable compound. In other embodiments, the anti-sepsis therapy can be intubation or mechanical ventilation or a therapy associated with treating acute lung injury, such as, fluid replacement therapy.

In one embodiment, the therapeutically effective amount of hyaluronan is less than about 10% of the circulating volume of the subject. In another aspect, the therapeutically effective amount of hyaluronan is between about 5% and about 20% of the circulating volume of the subject.

In another embodiment, the therapeutically effective amount of hyaluronan is systemically administered.

In a further embodiment, the hyaluronan has a high molecular weight, which, in specific embodiments, can be about 500 kDa, or preferably about 500 kDa to about 3000 kDa or more.

In certain embodiments, the sepsis-induced condition is septic shock, pulmonary inflammation, systemic inflammation, acute lung injury, or dysfunction of at least one bodily organ, e.g. a lung.

Methods of the invention can further comprise obtaining the hyaluronan. These and other embodiments are disclosed or are obvious from and encompassed by, the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings, in which: Figure 1 shows, in bar graph form, neutrophil infiltration in a model of acute lung injury related to over-distention of the lung by mechanical ventilation with varying tidal volumes and administration of HMW HA. * indicates p<0.05 versus all other groups.

Figure 2 shows, in a model using infusions of lipopolysaccharide (LPS) to mimic sepsis, that HMW HA blocked the influx of neutrophils into the bronchoalveolar lavage (BAL) both in animals ventilated with a small tidal volume (7 ml/kg) and a relatively large tidal volume (14 ml/kg). * p<0.05 vs. 7 ml/kg -LPS; # p<0.05 vs. 7 ml/kg +LPS; + p<0.05 vs. 14 ml/kg +LPS.

Figure 3 shows, in a model using infusions of lipopolysaccharide (LPS) to mimic sepsis, a trend toward HMW HA decreasing lactate production in the animals injected with LPS and low tidal ventilation (7 ml/kg) but not with high tidal volume ventilation (14 ml/kg). Systemic blood pressure was maintained with infusion of normal saline. There was no difference in systemic blood pressure (data not show). * p< 0.05 vs. 7 ml/kg -LPS

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a new use of hyaluronan ("HA") in treating and/or preventing sepsis and/or diseases, conditions or symptoms associated with sepsis, including acute lung injury or septic shock. It has now been discovered by the present inventors that hyaluronan (e.g. high-molecular weight) can be systemically administered to a subject as a treatment regime and/or preventative therapy to treat and/or prevent sepsis or a sepsis-induced condition and especially to reduce, alleviate or eradicate sepsis-induced inflammation. Without being bound by theory, it is believed that that hyaluronan, in particular, high-molecular weight hyaluronan ("HMW HA"), may block hyaluronan cell surface binding, in particular, low-molecular weight hyaluronan ("LMW HA") cell surface binding, thereby inhibiting the inflammatory response associated with or induced by bacteria, lipopolysaccharide or low-molecular weight hyaluronan. Accordingly, the present invention is useful to treat sepsis and septic shock, as well as other associated shock responses, including, but not limited to, cardiogenic shock, hypovaolemic shock, obstructive shock or distributive shock, and in particular, any type of shock that involves or includes in its pathophysiology inflammation or an inflammatory cascade that involves and/or is triggered by low-molecular weight hyaluronan. The present invention overcomes disadvantages associated with current treatments, such as undesirable side-effects, low-effectiveness, and discomfort.

In particular, systemic treatment with HMW HA blocks sepsis-induced lung inflammation, including inflammation triggered by bacteria, lipopolysaccharide or LMW HA. The use of HMW HA in fluids for volume replacement during the initial phase of sepsis will decrease sepsis-induced systemic and pulmonary inflammation and thereby decrease the mortality caused by sepsis.

1. Definitions

For the purposes of the present invention, definitions of the following terms are provided. Additional terms are defined in context elsewhere in this disclosure.

As used herein, the term "sepsis" refers to the condition of an individual which results from a severe infection or from the continued presence of a viable microorganism in the blood stream (i.e. bacteremia) and which is associated with widespread inflammation. Sepsis is a condition which is secondary to an infection, i.e. results from the infection.

As used herein, the term "sepsis-related inflammation" or "sepsis-induced inflammation" refers to the inflammatory response that is associated with sepsis.

As used herein, the term "septic shock" refers to a severe systemic inflammatory reaction to infection that results in a variety of physiologic conditions, including vasodilation, hypotension, maldistribution of blood flow, or tissue and/or organ hypoxia. As used herein, the term "sepsis-related condition" or "sepsis-induced condition" or "sepsis-related complication" are used interchangeably herein to refer to the downstream effects of prolonged septic shock on the body, including acute respiratory distress syndrome (ARDS), disseminated intravascular coagulation (DIC), acute renal failure, multiple organ dysfunction syndrome (MODS), and death. The above terms can also refer to systemic inflammation associated with sepsis and septic shock, including any inflammatory cascade associated with sepsis. As used herein, the terms "hyaluronan," or, "hyaluronic acid" or "hyaluronate," (or HA) are used interchangeably refer to a negatively charged, linear glycosaminoglycan (GAG) composed of alternating N-acetyl glucosamine and glucuronic acid in repeating disaccharide units, with no covalently linked protein core. The term "hyaluronan derivative" is meant to encompass any homolog, analog, mimetic, modification, complex, or fragment of hyaluronan which retains at least a substantial amount of the activity and/or function of hyaluronan and can be obtained from any natural or unnatural (e.g. chemical synthetic process) source.

As used herein, the term "obtaining," as in obtaining HA, can refer to synthesizing, purchasing, or otherwise acquiring the HA.

As used herein, the term "circulating volume" refers to the total volume of blood contained in an individual's cardiovascular system.

"Low-molecular weight" or "LMW" includes HA that has a weight average molecular weight of less than 500 kilodaltons, preferably less than about 400 kilodaltons, more preferably less than about 300 kilodaltons, still more preferably less than about 200 kilodaltons, and even more preferably about 200-300 kilodaltons, and still even more preferably about 1-300 kilodaltons. The LMW preferably includes endogenous HA.

As used herein, the term "high-molecular weight" or "HMW," as in high- molecular weight (HMW) hyaluronan, is meant to refer to HA that has a weight average molecular weight that is greater than about 500 kilodaltons, preferably between about 500 kilodaltons and 10,000 kilodaltons, more preferably between about 800 kilodaltons and 8,500 kilodaltons, still more preferably between about 1100 kilodaltons and 5,000 kilodaltons, and even still more preferably between about 1400 kilodaltons and 2,500 kilodaltons.

The term "weight average molecular weight" is well-known to one of ordinary skill in the art. Weight average molecular weight (or "average molecular weight" as is interchangeably used herein in reference to the inventive polymers) refers to the heterogeneity of molecular weights of individual polymer molecules within a polymeric composition. The weight average molecular weight can be calculated by the following formula: T- MM-²

wherein Nj is the number of molecules of molecular weight Mj.

The weight average molecular weight of the inventive polymers can be determined by known techniques that are typically used to measure weight average molecular weight, including, but not limited to, light scattering, small angle neutron scattering (SANS), X-ray scattering, and sedimentation velocity.

As used herein, the term "therapeutically effective amount," as in a therapeutically effective amount of HA, can refer to that amount of an agent of the invention effective to ameliorate, reduce, decrease or alleviate at least one symptom or condition associated with sepsis. The therapeutically effective amount used to practice the present invention for therapeutic treatment and/or prevention of sepsis and/or sepsis- related conditions or complications varies depending upon the manner of administration and a subject's age, body weight and general health. Ultimately, the skilled artisan (e.g. the attending physician) will decide the appropriate amount and dosage regimen based on the particular circumstances at hand and knowledge drawn from experience and the art.

By "decrease," as in a decrease of at least one symptom, is meant a reduction in a disease aspect. For example, a reduction by at least about 5% relative to a reference level. An exemplary decrease may be by about 5%, 10%, 15%, 20%, 25% or 50% or even by as much as 75%, 85%, 95% or more.

As used herein, the term "administration" or "administering" or the like is meant to include an act of providing a pharmaceutical composition or compound, e.g. HA, or pharmaceutical composition of the invention, to a subject in need of treatment.

As used herein, the term "systemically administering" or "systemic administration" or "systemic delivery route" or the like refers to a route of administration that results in the delivery of a pharmaceutical composition or compound, e.g. HA, or pharmaceutical composition of the invention, to the circulatory system of a subject.

As used herein, the term "co-administering" or "co-administration" is meant to refer to the local administration or systemic administration of an agent of the present invention together with a second sepsis-related treatment (a second agent), wherein the administration of each is carried out at about the same time or substantially at about the same time. The co-administered agents can be combined together or kept and delivered separately. The term "substantially at about the same time" is not meant to be limited to delivery of the agents at the same moment, but encompasses separate delivery of the agents at different moments, whereby the administration of one agent occurs seconds, minutes, hours or days before the administration of a second agent.

As used herein, the term "subject" is intended to include vertebrates, preferably a mammal. Mammals include, but are not limited to, humans.

As used herein, the term "anti-sepsis therapy," as in co-administering an anti- sepsis therapy, is meant any treatment aimed at treating and/or preventing sepsis or alleviating at least one symptom or condition associated with sepsis other than the administration of HA. For example, an anti-sepsis therapy can include, but is not limited to, the administration of an antibiotic, an anti-inflammatory drug or compound, a nonsteroidal anti-inflammatory drug, a steroid, a corticosteroid, a mineralocorticoid, isotonic fluid administration, inotropic treatment (e.g. dopamine, dobutamine, epinephrine) to increase cardiac output and oxygen delivery to tissues, vasopressin, and physical interventions, such as, intubation and mechanical ventilation.

As used herein, the term "inflammatory cell infiltration," is meant the stage of an inflammatory response occurring in connection with or in response to tissue damage (e.g. resulting from oxygen or glucose deprivation), whereby an influx or migration of inflammatory cells (e.g. neutrophils, monocytes, and macrophages) into the damage tissue occurs as a normal feature of the inflammatory response.

As used herein, the term "organ dysfunction" refers to an adverse or an abnormal effect on an organ. It is also noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises," "comprised," "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of and "consists essentially of have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention. 2. Sepsis, Sepsis-Induced Conditions, and Acute Lung Injury

Sepsis refers generally to the systemic inflammatory condition that is formed in response to an infection by a microorganism in the blood stream (bacteremia) and which ultimately can lead to septic shock and a variety of other shock-related complications, such as, acute renal failure, acute lung injury, and Multiple Organ Dysfunction Syndrome (MODS). Septic shock has a high rate of mortality, in particular, in immunocompromised individuals and the very young and the elderly. Presently, the mortality rate is at an average of about 45% (Dipiro JT et al., editors: Pharmacotherapy: a pathophysiologic approach, Ed. 5, New York, 2002, McGraw-Hill, p. 2030), due primarily from cardiovascular collapse and multiple organ dysfunction syndrome and acute lung injury (Bone R.C., "The pathogenesis of sepsis," Ann Intern Med, 1991; 115:457-69). Moreover, sepsis is extremely widespread and common, particularly in the hospital environment. Sepsis reportedly occurs in almost 2% of all hospitalizations and accounts for as much as 25% of intensive care unit (ICU) bed utilizations. According to the Centers for Disease Control and Prevention, sepsis was the leading cause of death in non-coronary ICU patients, and the tenth most common cause of death overall in the year 2000.

The etiology or cause of sepsis ultimately stems from a primary infection by a microorganism in the blood stream, e.g. bacteremia. In healthy individuals, most cases of bacteremia do not lead to sepsis or septic shock as a result of the effective destruction of the invading organism by the body's natural defense systems. However, those individuals who are immunocompromised (e.g. debilitated or malnourished persons or those persons subject to chemotherapy or who have a viral infection) and especially those who are very young or elderly are at greater risk of being unable to naturally clear the infection, and consequently, are more prone to developing sepsis and ultimately septic shock and related conditions.

The primary infection is commonly a bacterial infection and can be either a gram-negative or a gram-positive bacterium whose common portal of entry is typically, but not limited to, the genitourinary tract, the gastrointestinal tract, the respiratory tract or the skin. The present invention is useful against any infectious cause of sepsis and/or septic shock, for example, from infection by any of the following gram-negative or gram-positive bacterial causative agents: Enterococcus spp., including E.faecium, E. faecalis, E. raffinosus, E. avium, E. hirae, E. gallinarum, E. casseliflavus, E. durans, E. malodoratus, E. mundtii, and E. solitarius; Staphylococcus spp., including S. aureus, S. epidermidis, S. hominis, S. saprophyticus, S. hemolyticus, S. capitis, S. auricularis, S. lugdenis, S. warneri, S. saccharolyticus, S. caprae, S. pasteurii, S. schleiferi, S. xylosus, S. cohnii, and S. simulans; and Streptococcus spp. including S. pyogenes, S. agalactiae, S. pneumoniae, and S. bovis. Additional gram-negative organisms considered to be commonly associated with gram-negative septicemia and septic shock include Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, Serratia marcescens, Pseudomonas aeruginoas, and Proteus species. Additional gram-positive organisms that are commonly found associated with gram-positive septicemia include Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pneumoniae. In addition, fungi, such as Candida species, are also important causes of sepsis and septic shock.

The pathogenesis of sepsis is complex and involves a variety of cascades of inflammatory response and coagulation pathways. In respect of sepsis caused by gram- negative bacteria, the pathogenesis relates especially to the gram-negative bacterial cell wall, which contains a lipopolysaccharide (LPS) component. LPS is a known endotoxin composed of an O antigen side chain, an R core and an inner lipid portion. The lipid portion, known as lipid A, is the toxic component of LPS and which triggers a chain of pathophysiologic events. In the initial phase, macrophages become stimulated by LPS release from lysed cells and are triggered to release inflammatory cytokines. Two such cytokines, TNF-α and IL-I, are believed to be major factors in the pathogenesis of septic shock in part because of their role in triggering the release of additional cytokines and nitric oxide. The cytokines produced by macrophages in turn stimulate neutrophils and platelets, which are activated to release a plethora of toxic mediators, including, for example, platelet-activating factor, oxygen free radicals, and proteolytic enzymes. In addition, neutrophil and platelet activation leads to the release of prostaglandin, leukotriene, thromboxane, and prostacylin (a vasodilator), all of which affects the vascular smooth muscle such that a low systemic vacular resistance is formed with high cardiac output and hypotension (low blood pressure). In addition, the complement system is activated which can produce microemboli and lead to the destruction of endothelial cells. Further, mast cells release histamine, which as a potent vasodilator, increases capillary permeability, enhancing edema (excessive tissue fluid accumulation) formation. A variety of other inflammatory cascades also come into play which further the pathogenesis of sepsis, the clinical manifestations of which include hypotension, low systemic vascular resistance, high cardiac output, low cardiac preload, high venous oxygen saturation, low urine output and warm skin temperature. While the inflammatory cascades involved in sepsis are normal reactions to microbial invasion and are involved in eradicating an infection, they are harmful in overabundance and can lead to shock.

One important manifestation early in sepsis is a drop in blood pressure which occurs as a consequence of extreme peripheral vasodilation triggered by the array of inflammatory cascades. Blood becomes maldistributed throughout the body — some tissues being underperfused and others being overperfused. Blood maldistribution ultimately leads to tissue and organ oxygenation problems characteristic of septic shock.

Clinically, there are two main stages of septic shock. The hyperdynamic stage occurs first in which the patient experiences high cardiac output, warm extremities, and low blood pressure as a result of the decreased systemic vascular resistance. The abnormal vasodilation results in blood maldistribution and thus, inadequate delivery of oxygenated blood to metabolically active tissues. Lactic acidosis can occur as a result of tissue hypoxemia. In the second stage of septic shock, the patient experiences the hypodynamic phase. This phase is characterized by decreased cardiac output and the development of organ ischemia (deficiency of blood supply). Continued inflammatory response pressure leads to further depression in myocardial activity, worsening organ ischemia, and deteriorating tissue perfusion. The patient's skin becomes cool and clammy.

Regardless of the etiologic agent, many patients with septicemia or suspected septicemia exhibit a rapid decline, e.g. a decline in health over a 24-48 hour period. Thus, rapid methods of diagnosis and treatment delivery are essential and needed for effective patient care. A variety of treatments are presently used in the art, any of which can be combined together with the methods of the invention, e.g. the method of administering a therapeutically effective amount of HA to a subject having sepsis or at risk of developing sepsis and/or septic shock. For example, presently used pharmaceutical methods, such as, steroid treatments, antibiotic therapies, anti-toxin therapies (e.g. anti-LPS inactivating antibodies), anti-inflammatory therapies (e.g. targeted blockage of the host's inflammatory response in sepsis), anti-coagulation medicines (e.g. Xigris), immune system inhibitors, immunization against sepsis-causing infections, IL-I inhibitors, TNF inhibitors, utilization of hyperimmune plasma against core glycolipid in patients at high risk of infection (e.g. J.D. Baumgartner et al., Lancet 2:59-63 (1985), incorporated by reference), and monoclonal antibodies against sepsis- causing organisms, can be used in combination with the methods of the invention, e.g. HA administration to treat sepsis.

One serious complication that commonly arises in sepsis is acute lung injury. In fact, sepsis is the cause of 40% of all cases of acute lung injury, a condition that typically involves immediate intervention, such as, by intubation and/or mechanical ventilation.

Acute lung injury, which is also known as acute respiratory distress syndrome (ARDS), is an illness characterized by acute lung injury leading to permeability pulmonary edema and respiratory failure. The clinical scenarios that place patients at risk for ARDS are as diverse as trauma, hemorrhage or sepsis, however, sepsis is the most common cause of ARDS. Despite significant advances in critical care management, mortality from ARDS remains over 40%. Each year over 100,000 people die in the United States from complications of ARDS.

Circulating inflammatory cells, particularly neutrophils, play a major role in the onset and progression of acute lung injury in both humans and experimental animal models. Several investigators have demonstrated extensive intrapulmonary accumulation of neutrophils (i.e. neutrophil infiltration) in cases of ARDS. Once activated, these neutrophils release proteases, including matrix metalloproteinases, and other mediators of lung injury.

In one aspect of the present invention, the HA is administered to a subject having acute lung injury, e.g. sepsis-induced acute lung injury, to treat and/or ameliorate the disorder. Without being bound by theory, the HA is believed to, at least in part, block the infiltration of neutrophils to the lungs, thereby treating and/or ameliorating acute lung injury.

Accordingly, in another aspect, the present invention provides a method of administering HA to a subject having acute lung injury from sepsis and the administration of a second therapy for the treatment of acute lung injury. Such other therapies can include any therapy in the art used to treat acute lung injury and/or a symptom thereof. For example, such second therapy can include the administration of a compound to neutralize bacterial or microbial endotoxins, such as that method found in PCT International Publication No. WO95/03057 (Chugai Pharmaceuticals), which relates to an endotoxin neutralizer which contains, as an active ingredient, a tetracycline or its derivative. The tetracycline, or its derivative, need not have antibacterial activity. This endotoxin neutralizer is said to be used therapeutically or preventively for conditions caused by endotoxins. In another example, the second therapy can include a neutorphil-elastase inhibitor, such as that disclosed in Sakamaki et al., Am. J. Respir. Crit. Care Med. 153, 391-397 (1996) or Searles et al., AmSECT, 35th International Conference, (Abstract), (1997), the contents of each of which are incorporated herein by reference.

3. Hyaluronan

Hyaluronan is a glycosaminoglucan consisting of repeating disaccharides of alternating D-glucuronic acid and N-acetylglucosamine. These structures are joined by a β-(l,3)-D linkage while the glucosamine to glucuronic acid linkage is β-(l,4)-D and have a molecular weight ranging from about 1 and 50 X 10 Da (Brimacombe, J S., et al., in Mucopolysaccarides. (Elsevier, Amsterdam, 1964)) depending upon its source. For example, its has been determined that HA averages between 3-5 X lO⁶ Da, or 6-7 X 10⁶ Da, when isolated from rheumatoid fluid, or normal synovial fluid, respectively (Laurent, T C, et al., Immunol Cell Biol., 74:1-7, (1996)). In addition, dilute solutions of HA (<1 mg/mL) are known to result in highly entangled networks which instill unique rheological characteristics to the solution in hand (Laurent, T C, Immuno Cell Biol., 74:1-7, (1996)). For example, solutions of hyaluronan are viscoelastic with the viscosity maintaining a pronounced dependency on shear forces (Ogston, A G., et al., J. Physiol., 199:244-52, (1953)). Therefore, considering the increased localization of HA in the body between surfaces that move against each other, combined with the mechanicauphysical characteristics ascribed above, HA has been attributed the primary role of lubrication and protection of joints and tissues, cartilage surfaces and muscle bundles. Further, HA has also been associated with the scavenging of free radicals and debris (Myint, P., et al., Biochim. Biophys. Acta, 925:194-202, (1987), and Laurent, T C, Ann. Rheum. Dis., 54:429-32, (1995), respectively), keeping the joint cavities open (Edwards, J C W., et al., J. Anat., 185:355-67, 1994), forming flow barriers in the synovium (McDonald, J N., et al., J. Physiol., 485.1:179-93, (1995)), and the prevention of capillary growth (Sattar, A., Sernin. Arthritis Rheum., 22:37-43, (1992)).

HA is synthesized ubiquitously in the plasma membrane of all vertebrate tissues and in some bacteria (Fraser, J R E, J. Intern Med., 242:27-33, (1997)). It is catabolized locally through receptor-mediated endocytosis and lysosomal degradation, in addition to, the lymph nodes and endothelial cells of the liver sinusoids. HA is commonly isolated from the vitreous body of the eye, synovial fluid, rheumatoid fluid, umbilical cord, and skin. And, as noted above, several physiological functions have been associated with HA, in particular, water homeostasis; mitosis, cell migration, differentiation, angiogenesis (Rooney P and Kumar S (1994) EXS (Switzerland) 70: 179-90); and tissue remodeling, both in normal or tumor-associated events.

Synthesis of HA is carried out in animals by HA synthase (HAS), which is comprised of three proteins on the cell surface that link together N-acetylglucosamine and D-glucuronic acid in repeating disaccharide units and excretes long chains into the extracellular space (Itano et al., J Bio Chem, 1999, 274(35):25085-25092). HAS exists as 3 isoforms (HASl, HAS2 and HAS3). HAS3 forms LMW HA, while the products of HASl and HAS2 are HMW HA. Several HA receptors have been identified, but the two best studied that have been shown to be involved in cell signaling are CD44 and RHAMM, also identified as CD168 (Turley et al., J Biol Chem, 2002, 277:4789-4592). RHAMM has been found on the cell surface, attached to the cytoskeleton, and within the mitochondria and cell nucleus. CD44 is a transmembrane receptor that plays an important role in cell signaling (Jing et al., Nature Med, 2005, 11(11):1173-1179). Recently, HA has also been found to bind to TLRs (Toll-like receptors), a group of innate immune receptors belonging to the IL-I receptor family (Taylor et al., J Biol Chem, 2004, 279(17):17079-17084). TLRs are also binding sites for lipopolysaccharide (LPS), an inflammatory protein released by bacteria, which causes systemic inflammation and lung injury in sepsis. LPS stimulation of cytokine production has been found to be dependent on TLR binding with subsequent activation of the JNK and NFKB pathway (Paik et al., Hepatology, 2003, 37(5): 1043-55). HMW HA and LMW HA have been shown to compete for the same binding sites (McKee et al., J Clin Invest, 1996, 98(10):2403-13; Scheibner et al., J Immunol, 2006, 177(2):1272-81).

LMW HA (e.g. 200-300 kD) can function as an intracellular signaling molecule in inflammation (Jiang et al., Nature Med., 2005, 11(11):1173-1179; Noble PW, Proteoglycans in Lung Disease, New York, Marcel Dekker, 2002, pages 23-26). HMW HA (e.g. greater than about 500 kDa) is a major component of the extracellular matrix and functions to maintain the structural integrity. HMW HA treatment has been shown to decrease inflammation in osteoarthritis (Asari et al., Arch. Histol. Cytol., 1998, 61(2): 125-135; Gotoh et al., Ann. Rheum. Dis., 1993, 52(l l):817-822; Brandt et al., Arthritis. Rheum., 2000, 43(6): 1192-1203) and liver injury (Nakamura et al., J.Gastroenterol., 2004, 39(4): 346-354), to decrease protein permeability, increase ultrafiltration, decrease cytokine levels with peritoneal dialysis (Polubinska et al., Kidney Int., 2000, 57(3): 1182-1189) to block cigarette-induced emphysema in mice (Cantor et al., Exper. Lung Res., 1997, 23:229-244) and to block LMW HA-induced cytokine production (Scheibner et al., J. Immunol., 2006, 15:177(2):1272-1281).

The present invention contemplates any suitable source, method, process or technology known and/or used and/or described in the art to isolate, purify, manufacture, process or otherwise obtain hyaluronan for use in the methods of the present invention. In particular, it will be readily appreciated that such methods, technologies and processes can be especially designed to provide or obtain high-molecular weight hyaluronan in accordance with the invention.

In one aspect, the hyaluronan of the present invention can be obtained from any natural source. For example, the HA of the invention can be obtained from an animal tissue comprising HA (e.g. umbilical cord or joint tissue), hi another example, the HA can be prepared by bacteria using bacterial fermentation processes. The HA of the invention can also by obtained by using any suitably known or available chemical synthesis approach and/or enzymatic process. The enzymatic process can be driven using the HA biosynthetic enzymes (e.g. isolated from an HA producing cell or tissue or prepared by well-known recombinant means) and the appropriate substrates.

Accordingly, the HA of the invention can be obtained from any known natural source, e.g. isolated and purified from a biological source, or produced in vitro or ex vivo using chemical synthetic processes and/or enzymatic methods. In addition, the present invention further contemplates any suitable derivative, analog, mimetic etc. of the high- molecular weight HA of the invention which can be prepared using any known means or process and so long as the derivative, analog, mimetic etc. of HA has the same or substantially the same function of HA as it pertains to the present invention, i.e. the use of HA as a treatment and/or therapy for sepsis and/or a sepsis-related condition. Exemplary references describing different ways for obtaining HA are as follows, each of which is incorporated herein by reference.

U.S Patent No. 4,517,295, entitled, "Hyaluronic acid from bacterial culture," relates to the preparation of hyaluronic acid in high yield from streptococcus bacteria by fermenting the bacteria under anaerobic conditions in a CO₂ -enriched growth medium, separating the bacteria from the resulting broth and isolating the hyaluronic acid from the remaining constituents of the broth. The bacteria may be grown free of endotoxins by filtering all ingredients through a 1OK filter prior to inoculation of the medium and subsequently maintaining pyrogen-free conditions. The reference further describes facilitating the separation of the microorganisms from the polysaccharide by killing the bacteria with trichloroacetic acid. After removal of the bacterial cells and concentration of the higher molecular weight fermentation products, the hyaluronic acid is isolated and purified by precipitation, resuspension and reprecipitation.

U.S. Patent No. 4,782,046, entitled, "Ultrapure hyaluronic acid and method of making it," relates to the preparation of hyaluronic acid having controlled molecular weight and which is substantially free of protein and nucleic acids by using a hyaluronidase-negative or hyaluronidase inhibited microbiological source.

U.S. Patent No. 4,801,539, entitled, "Fermentation method for producing hyaluronic acid," relates to the preparation of streptolysin-free hyaluronic acid by cultivating a microorganism belonging to a genus Streptococcus which is anhemolytic and is capable of producing hyaluronic acid (e.g., Streptococcus zooepidemicus FERM BP-784).

U.S. Patent Nos. 5,411,874 and 5,563,051, entitled, "Production of hyaluronic acid," relates to a process for the production of hyaluronic acid by continuous fermentation of Streptococcus equi in a chemostat culture which provides high yields of high molecular weight hyaluronic acid uncontaminated by toxic impurities. The process reportedly solves the problem of traditional batch culture in which degradation enzymes can begin to break down the cell walls of Streptococcus releasing cell contents into the fermenter broth, leading to purification difficulties. U.S. Patent No. 5,559,104, entitled, "Procedure for the purification of hyaluronic acid and fraction of pure hyaluronic acid for ophthalmic use," relates to the preparation of a highly pure fraction of hyaluronic acid which is non-inflammatory and avoids postoperative complications in ocular surgery. Also disclosed is a process for the preparation of hyaluronic acid characterized by converting hyaluronic acid into a corresponding quaternary ammonium salt and, following purification procedures, reconverting the quaternary ammonium salt into a sodium salt of hyaluronic acid.

U.S. Patent No. RE 37,336, entitled, "Method for providing hyaluronic acid," relates to a method for the recombinant production of hyaluronic acid. Disclosed are DNA segments encoding hyaluronic acid synthase which are employed to construct recombinant cells useful in the production of hyaluronate synthase or hyaluronic acid. The reference also relates to chromosomal DNA from Streptococcus equisimilis that is partially digested with EcoRI and the resultant fragments are ligated to form recombinant vectors. It is reported that the vectors are useful in the transformation of host cells such as E. coli and or Streptococcal hosts. Resultant transformants are screened by the screening assays to identify colonies which have incorporated HA synthase DNA in a form that is being actively transcribed into the corresponding HA synthase enzyme. These colonies may be selected and employed in the production of the enzyme itself or its product, HA.

U.S. Patent No. 6,537,795, entitled, "Method and means for the production of hyaluronic acid," relates to a method for selecting streptococcus strains capable of producing hyaluronic acid with molecular weight exceeding six million includes the steps of cultivating strains of streptococci individually in culture medium which is free of metal ions which promote degradation of hyaluronic acid and which does not release from the reactor metal ions which promote the degradation of hyaluronic acid.

U.S. Patent No. 6,660,853, entitled, "Method for purifying high molecular weight hyaluronic acid," relates to a method for purifying high molecular weight hyaluronic acid, including the steps of providing one or more raw hyaluronic acid sources; extracting hyaluronic acid from the source; precipitating hyaluronic acid extracts; dissolving extract precipitates in water; extracting enzyme inhibitors from the dissolved precipitates with chloroform, and centrifuging to isolate an aqueous portion; adding one or more protein hydrolyzing enzymes to the aqueous portion in a reactor; adding a solution of CPC and NaCl to the reactor; filtering reactor contents through at least one membrane filter; filtering membrane filtered solution through at least one diafilter having a molecular weight cutoff of about 30 kDa; precipitating the diafiltered solution and isolating purified hyaluronic acid precipitate; and formulating isolated precipitate to about 10 mg/ml hyaluronic acid. U.S. Patent No. 7,105,320, entitled, "Process for producing hyaluronic acid or its derivative," relates to a chemical method for producing hyaluronic acid or a hyaluronic acid derivative comprises acting a hyaluronidase on an oxazoline derivative. The method reportedly is an improvement over traditional methods of preparation method such as cockscomb extraction methods or fermentation methods which have conventionally been employed industrially.

U.S. Patent No. 5,652,347, entitled, "Method for making functionalized derivatives of hyaluronic acid," relates to derivatized hyaluronate that has been functionalized with dihydrazide and which may be cross-linked. The references further relates to a method for producing hyaluronate functionalized with dihydrazide includes mixing hyaluronate and dihydrazide in aqueous solution, then adding carbodiimide so that the hyaluronate and dihydrazide react to form functionalized hyaluronate.

Accordingly, any method known in the art, such as those particular methods referenced above, or any other known or suitable method can be employed to obtain, provide, or manufacture the hyaluronan of the invention. In specific embodiments, high- molecular weight HA is desirable. Thus, those methods which are capable of providing such HA are preferred. In addition, any know method for concentrating, purifying, or isolating HA of a particular molecular weight or a particular molecular weight range, mean, distribution or average can be employed. Such methods can include any suitable separation technology, such as, for example, centrifugation and/or filtration methods. Further, new techniques have been developed, such as molecular ultrafiltration, which can be used in accordance with the present invention. By this means of purification it is possible to discard those HA fractions with a molecular weight coming within the higher or lower margins of a desired range of molecular sizes. For example, in the EPO patent No. 01238572, a procedure is described to obtain fractions of sodium hyaluronate with mean molecular weights of between 250,000 and 350,000 Daltons, by exposing the product directly obtained by extraction of organic material and subsequent enzymatic deproteinization with papain, to two molecular ultrafiltrations through membranes with a molecular cutoff of 30,000, that is, with membranes which trap only those fractions with molecular weights of over 30,000. This fractioning appears to be important to the obtainment of a product free from secondary actions of an inflammatory nature, since the fractions responsible for such effects are those with low molecular weights, for example about 30,000 Daltons. After further molecular filtration, using membranes with an exclusion limit of 200,000 (that is, membranes which trap those fractions with molecular weights of over 200,000 Daltons) the obtained filtered product is a fraction (called in the patent HY AL ASTINE) with a mean molecular weight between 50,000 and 100,000 Daltons, while the portion left on the membrane is a sodium hyaluronate fraction which has a mean molecular weight between 500,000 and 730,000 (the fraction called HYALRCTIN). Such methods can be implemented by the skilled artisan to obtain HA at a desired molecular weight (or desired weight average molecular weight) in accordance with the present invention.

The skilled artisan will readily keep in mind that the molecular weight of naturally-obtained HA will vary depending on the source. For example, HA obtained from synovial fluid can have a molecular weight of about 1 to 8 million (1,000 to 8,000 kilodaltons). In another example, HA obtained from human umbilical cord tissue can have a molecular weight around 3.6-4.5 million (3,600 to 4,500 kilodaltons). In a yet another example, HA obtained from the common source of rooster combs can occur with very high values, e.g. up to 12-14 million (12,000 to 14,000 kilodaltons), or even higher. As the chemical composition of hyaluronic acid from natural sources is the same regardless of its particular organismal source, HA from any source is essentially non- immunogenic (Brimacombe and Webber (1964)).

It will be further readily appreciated by the skilled artisan that HA obtained from a biological system, e.g. rooster combs, likely will be associated with proteins and other glycosaminoglycans, for example, chondroitin sulphate. Accordingly, it may be desirous to purify the HA from any contaminates, such as unwanted proteins, in order to provide a non-immunogenic HA preparation for use in the present invention.

The HA of the present invention can also be obtained from any commercial source and such commercial sources will be readily available to the person of ordinary skill in the art. For example, HEALON® (Pharmacia AB, Uppsala, Sweden) provides a commercial HA product which has a molecular weight of around 3.5 million to 5 million daltons. This product is prepared from rooster combs according to a method based on the disclosure of U.S. Pat. No. 4,141,973, which is incorporated herein by reference. It will be appreciated that a primary source of HA will be from bacterial fermentation processes. The use of bacteria for biotechnological production of HA has been advocated for several reasons, technical, economical as well as ethical. The production by Streptococcus spp. has been known for more than 50 years and most of the systems utilize group A and C streptococci. Examples of particular strains include Streptococcus pyogenes (group A), which is a human pathogen, and Streptococcus equi and Streptococcus equisimilis (group C), which are animal pathogens.

The skilled artisan will readily appreciate and consider the available guidance, knowledge and information available in the art, as exemplified above, in obtaining HA for the purposes of the present invention. 4. Pharmaceutical Compositions, Dosages and Administrative Modes

The invention provides, in one aspect, pharmaceutical compositions and formulations comprising hyaluronan (e.g. high-molecular weight HA) and/or functional derivative, analog or mimetic thereof, and methods of using same for treating an inflammatory condition caused by or in part by the binding of low-molecular weight hyaluronan on a cell, such as an immune system cell or epithelial cell (e.g. a lung epithelial cell as in acute lung injury treatment). The invention further provides, in another aspect, pharmaceutical compositions and formulations and methods of using same to systemically administer hyaluronan (e.g. high-molecular weight HA) and/or a functional derivative, analog or mimetic thereof, to treat sepsis and/or a sepsis-induced condition, such as sepsis-induced systemic inflammation, or acute lung injury, or MODS.

The pharmaceutical compositions of the invention can comprise any pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention are particularly suitable for liquid or fluid replacement therapy, or can be administered intravenously by injection, or the like, such that the administration achieved is systemic in nature.

The phrase "pharmaceutically acceptable carrier" is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals. The carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier is advantageously "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, marmitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Administration of the pharmaceutical compositions of the invention can be by any suitable means, such as, for example oral administration, parenteral administration, intravenous administration, transdermal administration, nasal administration, topical administration or by direct injection into the blood. Preferably, the route of administration achieves a systemic delivery of the HA of the invention. Administration of the pharmaceutical compositions of the invention can also be carried at or substantially at the same time (i.e. co-administration) as the administration of one or more anti-sepsis agent or compound or therapy, such as, for example, an antiinflammatory agent, an antibiotic, a steroid, a mineralocorticoid, an inhibitory antibody against an immune system function, an antibody against a microbial toxin, e.g. antibody against LPS, etc. Such co-administered therapies can be formulated in any suitable configuration, e.g. as a single pharmaceutical composition or prepared and administered as separate compositions. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that inhibits, reduces, alleviates, or eradicates at least one symptom of sepsis or a sepsis-related condition. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient (e.g. HMW HA), preferably from about 5 percent to about 70 percent, more preferably from about 10 percent to about 30 percent.

While HA is preferably administered systemically due to the nature of sepsis or sepsis-induced conditions, the pharmaceutical compositions can also be administered by other routes as well, such as by oral administration. HA for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration and which are in such an amount or dosage which is sufficient to treat at least one symptom of sepsis or a sepsis-related condition. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, caplets, liquids, gels, gel caps, syrups, slurries, suspensions and the like, for ingestion by the subject. Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl- cellulose, or sodium carboxymethyl cellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push- fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers. Pharmaceutical formulations for parenteral or intravenous administration (e.g. by fluid replacement therapy) include aqueous solutions of the active compounds of the invention, e.g. HMW HA. For injection, the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer' solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Injection of the compositions of the invention can be carried out by directly injecting the compositions into the blood stream or circulatory system. Preferably, the HA compounds of the invention are formulated for fluid replacement therapy suitable for administration to a subject suffering from sepsis, septic shock, or a sepsis-induced condition, in particular, acute lung injury or MODS. Fluid replacement therapy is known in the art and any suitable method, apparatus, or fluid used in the fluid replacement therapy is contemplated.

The term fluid replacement therapy, as used herein, refers to the intravenous infusion of fluid. It will be readily appreciated that fluid replacement therapy is typically used to administer to subjects in need (e.g. trauma patients, acute lung injury patients, septic shock patients) to reverse the effects of hypovalaemia by increasing circulatory blood volume and blood pressure back towards a normal level, in order to maintain the perfusion of vital organs and to reduce the risk of death from multiple organ failure. Compounds of the invention can also be delivered directly to selected sites in the body, e.g. a site of severe inflammation, by a variety of means, including injection, infusion, catheterization and topical application, among others. Compounds of the invention also may be bound to carrier bio-compatible particles, e.g., autologous, allogenic or zeno genie cells, to facilitate targeted delivery of the active agents.

The compounds of the invention may also be formulated with liposomes. Liposomes are vesicles in which an aqueous compartment or volume is entirely enclosed by a membrane of lipid molecules which are usually phospholipids. Liposomes may be formed spontaneously when lipids are dispersed in aqueous media, producing a population of liposome vesicles having average maximum diameters ranging from nanometers to microns. Liposomes can be formed such that they will entrap molecules, such as the HMW HA of the invention, within one or both of the aqueous compartment and the membrane. In fact, liposomes can be formed from natural constituents such that their membrane or membranes forms or form a bi-layer which is similar to the lipid arrangement in natural cell membranes. It is possible that this similarity can be exploited in the delivery of the compounds of the invention because the liposome's ability to mimic the behavior of natural membranes make liposomes an extremely safe and efficacious vehicle for medical use. Apart from the chemical constituents of liposomes which determine their fluidity, charge density, and permeability, liposomes can be characterized by size and shape. Liposomes have average maximum diameters ranging from 25 nanometers to greater than 1,000 nanometers, which coincide with the average maximum diameters of living cells. As indicated above, liposomes may include a single bi-layer membrane. However, they may also include multiple concentric membrane lamella successively surrounding one another. It is possible, therefore, to group liposomes into one of the following categories based on the number of layers of membranes and relative average diameters: multilamellar vesicle (MLV) liposomes, small unilamellar vesicle (SUV) liposomes, large unilamellar vesicle (LUV) liposomes, and intermediate-sized unilamellar vesicle (IUV) liposomes. See New, R. C, "Liposomes~A Practical Approach," Oxford University Press, Oxford, pp. 1-33, 1990.

Several factors such as lamellarity (that is, the number of lamella), lipid composition, charge on the liposomal surface, and the total lipid concentration have been proven to influence drug deposition within the deeper skin strata. See, for example, Weiner et al. "Topical Delivery of Liposomally Encapsulated Interferon in a Herpes Guinea Pig Model," Antimicrob. Agents Chemother., 33: 1217-1221, 1989. There has also been much discussion on the mechanism of liposome diffusion in skin. Originally, it was thought that liposomes diffused intact through to the dermis where they became localized as set forth in Mezei, M. and Gulasekharam, V., "Liposomes~A Selective Drug Delivery System for the Topical Route of Administration," Life Sci., 26: 1473-1477, 1980. Later, this theory was criticized by Ganesan et al. "Influence of Liposomal Drug Entrapment on Percutaneous Absorption," Int. J. Pharm., 20: 139-154, 1984; and Ho et al. "Mechanism of Topical Delivery of Liposomally Entrapped Drugs," J. Controlled ReI., 2: 61-65, 1985, as it was thought that the densely packed stratum comeum would not allow the passage of liposomes through to the epidermis and dermis. Egbaria, K. and Weiner, N., "Topical Application of Liposomal Preparations," Cosmet. Toilet., 106: 79-93, 1991, postulated that molecular mixing of the bi-layers of the liposome and the stratum corneum takes place. There have also been indications that the follicular pathway contributes to the liposomal delivery of compounds into the skin as discussed in Du Plessis et al. "Topical Delivery of Liposomally Encapsulated Gamma- Interferon," Antiviral Res., 18: 259-265, 1992; and evidence that the size of the liposome is important as described in Du Plessis et al. "The Influence of Particle Size of Liposomes on the Deposition of Drug into Skin," 103: 277-282, 1994.

The efficacious pharmaceutical compositions of the invention may comprise a mixture of liposomes with a sufficient amount of HA to function as an effective delivery vehicle for the systemic delivery of HMW HA. The amount of HA effective for that purpose is dependent on the molecular weight fraction of HA. For example, where the molecular weight is relatively low, the concentration may be relatively high, and vice versa. HA may have an average molecular weight in the range of 10,000 to 1,000,000 daltons or more, preferably the fraction being greater than 500,000 daltons. Hyaluronic acid is usually provided in the form of aqueous solutions of HA salts, such as sodium or potassium hyaluronate, with an HA concentration in the range of 0.3 to 2.5% by weight. Compositions having HA within these ranges of concentrations and average molecular weights will generally be effective in accordance with the methods of the invention.

Liposomes are generally prepared using two techniques, a Freeze Thaw Cycle technique and a Conventional Film technique. Both techniques produce predominantly multilamellar vesicle (MLV) liposomes (MLVs typically include five or more concentric lamellae and have average maximum diameters in the range of 100 to 1,000 nanometers). Other techniques of liposome preparation may also be used. The present invention contemplates liposomes made by any suitable means. The phrases "parenteral administration" and "administered parenterally," as used herein, refer to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. The administration of the HA compositions of the present invention are preferably by any means that achieves a systemic administration, i.e. delivery to the blood stream of a subject.

The phrases "systemic administration" and "administered systemically," as used herein, refer to the administration of a compound of the invention, such that it enters the patient's circulatory system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

In another aspect, systemic administration may be achievable through nasal administration. For nasal administration, penetrants appropriate to the particular barrier to be permeated can be used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention may be manufactured in a manner known in the art, e.g. by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms. In other cases, a preferred preparation may be a lyophilized powder in lmM-50 mM histidine, 0. l%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to use.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the compounds of the invention from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the compounds then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally- administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. In certain cases of the present invention, in particular those cases of rapid decline of a patient's health due to the rapid onset of sepsis or a sepsis-induced condition, such prolonged effect may not be preferred and instead, rapid delivery of the HA of the invention is preferred. Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

The present invention further contemplates the co-administration of the HMW HA of the invention, or a pharmaceutical composition thereof, together with one or more anti-sepsis or anti-inflammatory compounds or compounds that might be useful in treating any type of shock syndrome, including septic shock. For the purposes of this invention, "co-administering" is administration of two or more compounds, or pharmaceutical compositions comprising the compounds at the same time or at about the same time, e.g. sequential administration. Sequential administration also encompasses an administration regimen occurring in some pattern over the course of days, weeks, or months, such as, for example, administering on a first day an HA or composition thereof followed by on a second day an anti-sepsis therapy. There is no intended limitation as to the exact manner by which co-administration may occur and the skilled artisan will be able to competently design a suitable co-administration regimen comprising the agents of the invention and one or more additional compounds.

In one aspect, the present invention contemplates a method of co-administering the HMW HA of the invention and an antibiotic used to treat the underlying primary bacterial infection or any secondary infection arising from the sepsis conditions, hi addition, such compounds can be expanded to include any known anti-viral, anti-fungal, anti-parasitic, or anti-bacterial compound that might be useful in combating the sepsis and/or sepsis-induced condition at hand, e.g. opportunistic infections or secondary viral infections. For example, the compounds can be anti-bacterial drugs. Anti-bacterial antibiotic drugs are well known and can include: penicillin G, penicillin V, ampicillin, amoxicillin, bacampicillin, cyclacillin, epicillin, hetacillin, pivampicillin, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, carbenicillin, ticarcillin, avlocillin, mezlocillin, piperacillin, amdinocillin, cephalexin, cephradine, cefadoxil, cefaclor, cefazolin, cefuroxime axetil, cefamandole, cefonicid, cefoxitin, cefotaxime, ceftizoxime, cefinenoxine, ceftriaxone, moxalactam, cefotetan, cefoperazone, ceftazidme, imipenem, clavulanate, timentin, sulbactam, neomycin, erythromycin, metronidazole, chloramphenicol, clindamycin, lincomycin, vancomycin, trimethoprim- sulfamethoxazole, aminoglycosides, quinolones, tetracyclines and rifampin and antibacterial antibodies. (See Goodman and Gilman's, Pharmacological Basics of Therapeutics, 8th Ed., 1993, McGraw Hill Inc.). It is further conceivable that the present invention can be administered together with an ongoing or existing cancer treatment as sepsis can be a condition that results from or is linked to cancer. The anticancer compounds contemplated by the present invention are limitless and include any of those known in the art. Exemplary cancer therapeutic agents include, but are not limited to, chemical or biological reagents that inhibit the growth of proliferating cells or tissues wherein the growth of such cells or tissues is undesirable. Chemotherapeutic agents are well known in the art (see e.g. , Gilman A. G., et ai, The Pharmacological Basis of Therapeutics, 8th Ed., Sec 12:1202- 1263 (1990) and Teicher, B. A. Cancer Therapeutics: Experimental and Clinical Agents (1996) Humana Press, Totowa, NJ), and are typically used to treat neoplastic diseases. Other similar examples of chemotherapeutic agents include: bleomycin, docetaxel (Taxotere), doxorubicin, edatrexate, erlotinib (Tarceva), etoposide, finasteride (Proscar), flutamide (Eulexin), gemcitabine (Gemzar), genitinib (Irresa), goserelin acetate (Zoladex), granisetron (Kytril), imatinib (Gleevec), irinotecan (Campto/Camptosar), ondansetron (Zofran), paclitaxel (Taxol), pegaspargase (Oncaspar), pilocarpine hydrochloride (Salagen), porfimer sodium (Photofrin), interleukin-2 (Proleukin), rituximab (Rituxan), topotecan (Hycamtin), trastuzumab (Herceptin), tretinoin (Retin- A), Triapine, vincristine, and vinorelbine tartrate (Navelbine). The present invention also contemplates in yet another aspect a method of coadministering the HMW HA of the invention with other known anti-sepsis and/or antiinflammatory compounds, including steroids, mineralocorticoids, antibodies, toxin inhibitors/neutralizers (e.g. WO 95/03057, incorporated by reference), antibiotics, vasopressin, inotropic treatments (e.g. dopamine, dobutamine, epinephrine), compounds to block clotting cascades (e.g. the anticoagulant drug, drotrecogin alfa (Xigris)), antibodies and/or compounds which inhibit or block the activities of known components of the sepsis-induced inflammatory response, septic shock, or inflammation caused by low-molecular weight HA, generally, and sepsis-related physical therapies and/or interventions, such as fluid replacement therapy (of which HA can be a component), intubation, and mechanical ventilation, in particular where acute lung injury is a problem. More particularly, monoclonal antibody against endotoxin A (or any bacterial toxin which plays a causative role in the sepsis-induced inflammatory response), antagonist of interleukin-1 receptor, inhibitor of platelet-activating factor, antagonist of bradykinin, antibody against tumor necrosis factor, suppressor of nitric oxide synthases, inhibitor of endothelin receptor, activated protein C (see Pathophysiology. Ed. Copstead and Banasik, 3d ed., Elsevier Inc., 2005, pp.528-548).

Methods of and devices for mechanical ventilation are known in the art and is a widely accepted form of therapy and means for treating patients with respiratory failure. Ventilation is the process of delivering oxygen to and washing carbon dioxide from the alveoli in the lungs. When receiving ventilatory support, the patient becomes part of a complex interactive system which is expected to provide adequate ventilation and promote gas exchange to aid in the stabilization and recovery of the patient. Clinical treatment of a ventilated patient often calls for monitoring a patient's breathing to detect an interruption or an irregularity in the breathing pattern, for triggering a ventilator to initiate assisted breathing, and for interrupting the assisted breathing periodically to wean the patient off of the assisted breathing regime, thereby restoring the patient's ability to breath independently. A wide variety of mechanical ventilators are available. Most modern ventilators allow the clinician to select and use several modes of inhalation either individually or in combination via the ventilator setting controls that are common to the ventilators. These modes can be defined in three broad categories: spontaneous, assisted or controlled. During spontaneous ventilation without other modes of ventilation, the patient breathes at his own pace, but other interventions may affect other parameters of ventilation including the tidal volume and the baseline pressure, above ambient, within the system. Li assisted ventilation, the patient initiates the inhalation by lowering the baseline pressure by varying degrees, and then the ventilator "assists" the patient by completing the breath by the application of positive pressure. During controlled ventilation, the patient is unable to breathe spontaneously or initiate a breath, and is therefore dependent on the ventilator for every breath. During spontaneous or assisted ventilation, the patient "works" (to varying degrees) by using the respiratory muscles in order to breath. The present invention contemplates any known medical or mechanical ventilator, such as, in U.S. Patent Nos. 5,307,795, 5,161,525, 5,678,539, 5,931,160, and 7,066,173, each of which are incorporated herein by reference. After pharmaceutical compositions comprising a compound of the invention formulated in an acceptable carrier have been prepared, they can be placed in an appropriate container and labeled for use in accordance with the methods described herein along with information including amount, frequency and method of administration in accordance with the invention. The pharmaceutical composition may be formulated from a range of preferred doses, as necessitated by the condition of the patient being treated. For example, the compounds described herein may preferably be 60%, 61%, 62%, 63%, 64%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, and any percentage between 60% and 90%, and preferably between about 0.05% to about 0.35%, and even more preferably between about 0.05% and about 90%, of the weight per volume (w/v) of the composition. It will be within the capabilities of one of ordinary skill in the art to formulate the herein described compounds in liquid form such that they are not prohibitive against infusion or intravenous administration or the like. In embodiments involving the co-administration of another active ingredient, such as, for example, an anti-cancer agent or an anti-bacterial agent, the agents of the invention can be administered in combination therewith in a ratio in the range of 1:1-1:5, 1:1-1:10, 1:1-1:25, 1:1-1:50. 1:1-1:100, 1:1-1:500, 1:1-1 :1000, 1:1-1:10,000, 5:1-1:1, 10:1-1:1, 25:1-1-1, 50:1-1:1, 100:1-1:1, 500:1-1:1, 1000:1-1:1 or 10,000:1-1:1.

The amount of an agent used in accordance with the methods disclosed herein will depend upon the body mass of the patient, the nature and severity of the condition being treated, the nature of therapeutic treatments which the patient has undergone, and on the idiosyncratic responses of the patient, and the state or status of the disease or condition being treated. Ultimately, the attending physician will decide the amount of HA or HA composition to administer to each individual patient and the duration thereof.

5. Kits and/or Pharmaceutical Packages

The kits contemplated by the invention can comprise HA of a particular molecular weight or a range of molecular weights (e.g. HMW HA). The kits can also comprise, together or separate from the HA of the invention, additional active ingredients useful in treating sepsis or a sepsis-induced condition, such as, for example, systemic inflammation or septic shock or acute lung injury. Such additional active ingredients are described herein elsewhere. The kits can comprise any suitable container comprising any compound of the invention as described herein previously or within the ambit of the invention. The kits may also include instructions for using the compounds of the invention in the methods described herein. The kits can also include the pharmaceutical compositions of the invention described herein and can include instructions and any devices which are advantageous or useful for the administration of the pharmaceutical compositions or inventive compounds, e.g. a syringe or delivery implement. The container is not intended to be limited to any particular form, shape, or size and its construction can be of any suitable material in the art that is not detrimental to the contents contained therein.

All the essential materials and reagents involved in administering the compounds of the invention can be assembled together in the herewith kits. When the components of the kit are provided in one or more liquid solutions, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being particularly preferred. In a preferred aspect, the kit comprises an HA composition that is suitable for use in liquid replacement therapy typical in the treatment of subjects having sepsis or a sepsis- induced condition.

The components of these kits may be provided in dried or lyophilized forms. When reagents or components are provided in dried form, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in another container means. The kits of the invention may also include an instruction sheet defining administration of the compounds of the invention or for explaining the desired procedures contemplated by the present invention.

The kits of the present invention also will typically include a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained. Irrespective of the number or type of containers, the kits of the invention also may comprise, or be packaged with, an instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal, e.g. implements for fluid replacement therapy with the HA and/or other compounds of the invention. Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle. hi another embodiment, the kits of the invention may contain the compounds of the invention which have been covalently or non-covalently combined with a chelating agent; an auxiliary molecule such as mannitol, gluconate, glucoheptonate, tartrate, and the like; and a reducing agent such as SnCl₂, Na dithionite or tin tartrate. The HA compound/chelating agent and the auxiliary molecule may be present as separate components of the kit or they may be combined into one kit component. The HA compound/chelating agent, the auxiliary molecule, and the reducing agent may be provided in solution or in lyophilized form, and these components of the kit of the invention may optionally contain stabilizers such as NaCl, silicate, phosphate buffers, ascorbic acid, gentisic acid, and the like. Additional stabilization of kit components may be provided in this embodiment, for example, by providing the reducing agent in an oxidation-resistant form. Determination and optimization of such stabilizers and stabilization methods are well within the level of skill in the art. When the compounds of the invention are in lyophilized form, the kit may optionally contain a sterile and physiologically acceptable reconstitution medium such as water, saline, buffered saline, and the like. The amounts of an HA compound/chelating agent, auxiliary molecule, and reducing agent in this embodiment are optimized in accordance with the methods described herein.

Agents of the invention may be used in accordance with the methods of the invention by one of skill in the art, e.g., by specialists in infectious disease medicine, to treat patients having sepsis or a sepsis-induced condition.

The present invention also provide packaged pharmaceutical compositions comprising a pharmaceutical acceptable carrier and a compound or salt of any one of the herein disclosed compounds. Other packaged pharmaceutical compositions provided by the present invention further comprise indicia comprising at least one of: instructions for using the composition in accordance with the herein described methods.

***

This invention is further illustrated by the following examples, which should not be construed as limiting. All documents mentioned herein are incorporated herein by reference.

EXAMPLES

The invention is further described by way of the following non-limiting examples.

Example 1. Systemic administration of HMW HA before ventilation in model of acute lung injury.

LMW HA has been found to play a role in the models of acute lung injury described herein HMW HA (3 ml, 0.35%) was administered systemically 18 hours prior to mechanical ventilation of the lung with large tidal volumes (high Vx = 20 ml/kg) and found to block high Vτ-induced lung neutrophil infiltration (Figure 1). This was compared to non-ventilated controls (Non-Vent), Vj 7, and Vy 20 + HMW HA. Lung neutrophil infiltration is measured as the number of neutrophils in the bronchoalveolar lavage (BAL).

Example 2. Systemic HMW HA pretreatment blocks lactic acid production with sepsis.

In an effort to determine whether HMW HA would also have protective effects as an anti-inflammatory agent in sepsis, animals were pretreated with HMW HA by intraperitoneal injection of HMW HA (3 ml of 0.35% 1600 kDa HA). Eighteen hours after injection of HA, to simulate sepsis, bacterial lipopolysaccharide (LPS, 1 mg/kg) was injected through the carotid arterial line. After 1 hour to allow development of LPS-induced inflammation, mechanical ventilation was initiated at 7 ml/kg (the standard of care for human sepsis) (Figure 2), since the mortality of animals with arterial LPS and no mechanical ventilation was 50%. Arterial blood pressure and heart rate were monitored continuously. Systemic blood pressure was maintained by periodic infusion of normal saline. Systemic blood pressure and heart rate were constant in all groups. Following 4 hours of mechanical ventilation, the animals were euthanized. At the time of initiation of mechanical ventilation, an increase was observed in lactic acid production that was blocked by pretreatment with HMW HA (Figure 2).

After 4 hours of mechanical ventilation, there was a significant increase in lung neutrophil infiltration with LPS infusion, which was blocked by pretreatment with HMW HA (Figure 2). In effect, systemic administration of HMW HA (1600 kD) blocked bacterial lipopolysaccharide (LPS)-induced lung neutrophil infiltration, as measured by the number of neutrophils in the bronchoalveolar lavage (BAL) fluid. HMW HA had no effect on lung neutrophil infiltration in rats ventilated with 7 ml/kg but no LPS. HMW HA did not affect lung neutrophil infiltration in rats without ventilation and no LPS (data not shown). These data indicate that systemic administration of HMW HA is an effective new treatment for sepsis.

Example 3. Determination of efficacy of HMW HA in preventing pulmonary and systemic inflammation and decreasing mortality in sepsis. hi order to determine whether intravenous administration of HMW HA after LPS infusion blocks both pulmonary and systemic inflammation, end-points are selected as follows: systemic and pulmonary edema, as measured by Evans blue dye, release of cytokines in serum, BAL and lung tissue, and mortality.

Screening for optimal dosing and size range of HMWHA. To determine the optimal dosing and size range, a non-lethal, pseudo-sepsis mouse model of intraperitoneal injection of LPS is used. One hour prior to LPS (1 mg/kg) injection, the HMW HA is infused by tail vein injection. Two hours after LPS injection, serum cytokines MIP-2, TNFα and IL-6 and total serum HA are measured with increasing doses and sizes of HMW HA (500-3000 kDa). This is compared to control mice with infusion of carrier only control mice treated with compound only and no LPS and mice treated with dexamethasone (3 mg/kg) via tail vein one hour prior to LPS as a positive control of cytokine and HA inhibition. This model develops systemic release of cytokines in a short period of time, allowing large-scale rapid determination of optimal dosing and size range of HMW HA.

Efficacy of HMWHA in inhibiting inflammation post LPS infusion. The optimal dosing range and size range determined above is subsequently tested for efficacy in a rat model of sepsis that develops systemic hypotension and acute lung injury. One hour after arterial injection of LPS (1 mg/kg) in anesthetized rats, HMW HA is given by continuous intravenous infusion. Markers of pulmonary and systemic inflammation are measured 4 hours after LPS injection. The use of rats allows the provision of larger amounts of lung tissue for measurement of the HA amounts and size, allows easier placement of arterial and central lines for constant monitoring of arterial and airway pressure, and allows for constant central venous infusion of HMW HA with less mortality than mice.

LPS-induced lung injury model.

Sprague-Dawley rats are anesthetized with intraperitoneal ketamine (50 mg/kg) and diazepam (5 mg/kg) and then receive either 1 mg/kg of Salmonella typhosa LPS (Lot 81H4018; Sigma Chemical Co., St. Louis, MO) or an equivalent volume of normal saline as a control via the jugular artery. Arterial injection has previously been determined to cause acute lung injury and hypotension within 4 hours of injection. After one hour of spontaneous respiration to allow for development of a septic response, the rat has a tracheostomy performed and is placed on a ventilator set to deliver a V_T 7 ml/kg in room air without PEEP (positive end-expiratory pressure). Mechanical ventilation is used. The respiratory rate is set at 85 to 100 breaths per minute to maintain pCO2 between 35 and 45 torr. Arterial pressure and airway pressure are monitored continuously. Normal saline is infused to maintain mean arterial pressure greater than 60 mm Hg, and that volume is recorded. Rats are sacrificed after 4 hours of ventilation. The left lung is lavaged with normal saline for measurement of HA, cytokines, and cell counts. The right lung is flash frozen for extraction of HA, Western Blot analysis, and extraction of RNA for measurement of HASl, 2 and 3, and cytokines. Serum is collected for measurement of systemic inflammation, including cytokines and HA. Separate groups of animals are used to determine lung pathology by perfusion fixation through the airway at 30 cm H₂O water pressure, as well as to determine of capillary leak by Evans blue dye.

Determination of the effect of the HMW HA on LPS-induced mortality. To measure effects on mortality, non-ventilated, anesthetized rats are treated with three hours of continuous infusion of HMW HA (at the optimal dose and size determined above) or carrier alone begun 1 hour after treatment with high-dose LPS (5 mg/kg). After the completion of the infusion, the animals are allowed to recover from anesthesia and observed. The length of survival is measured from the time of the completion of infusion.

Example 4. Determination of effect of HMW HA administration on composition of circulating HA and lung HA.

To determine whether systemic administration of HMW HA decreases the ratio of LMW HA to HMW HA, the amount and size of HA are measured in the serum and lung tissue of LPS-treated rats with and without systemic administration of HMW HA. Extraction and Sizing of HA.

HA is extracted and isolated as previously described (Lago et al., Carbohydrate Polymers, 2005, 62:321-326). Lung tissue is treated with 0.2% sodium chloride solution and then filtered. The supernatant is treated with 1% cetrltriumethlamonium bromide (CTAB) solution and centrifuged. The precipitate is re-suspended by adding 0.9 M calcium chloride to solution to dissociate the HA-CTAB complex; the suspension is then treated with 25% v/v aqueous ethanol, and the nucleic acids are precipitated and separated by centrifugation. The resulting suspension is deproteinized and defatted four times by chloroform solvent (10% of the total volume). HA is precipitated by first adding 75% v/v aqueous ethanol and then acetone. HA size is determined by size exclusion chromatography/multiangle laser light scattering (SEC/MALLS) (Shiedlin et al., Biomacromolecules, 2004, 5(6):2122-7).

Example 5. Exploration of mechanism of HMW HA inhibition of LPS and

LMW HA induced endothelial cell dysfunction.

LPS and LMW HA induce production of cytokines and upregulate expression of neutrophil binding receptors, VCAM and ICAM, by binding to Toll-like receptors (TLR) with subsequent activation JNK and NFKB pathways. In an effort to investigate whether HMW HA inhibits systemic inflammation by blocking LMW HA and LPS binding to TLR on endothelial cells, EC from the lung and aorta are treated with HMW HA or a control compound for the effect of charge followed by stimulation with LMW HA or LPS. Study endpoints include cytokine production, VCAM and ICAM expression, and activation of the JNK and NFkB pathways. To explore the role of TLR, specific blocking antibodies are used, and EC are isolated from the main pulmonary artery and aorta from TLR2 and TLR4 +/+, +/-, and -/- mice. The role of the JNK and NFkB pathways is explored using specific blockers. Endothelial cells.

Human main pulmonary endothelial cells (PAEC), microvascular pulmonary endothelial cells (MVEC), and aortic endothelial cells (AEC) are purchased from Clonetics (Walkerville, MD). Mouse PAEC and EC are isolated from TLR2 and TLR4 +/+, +/-, and -/- mice (Jackson Laboratories, Bar Harbor, Maine, USA). EC are characterized by positive staining for factor VIII and negative staining for alpha-actin smooth muscle (Moore et al., Am J Physiol, 1998, 275:L574-L582). Cell Culture Media for Maintenance of Cultured Cells. EC are grown in Clonetics defined culture media (Walkerville, Maryland, USA) supplemented with growth factors and antimicrobial agents. Human PAEC were grown in Endothelial Cell Basal Medium-2 with (EBM-2, CC-3156) supplemented with (CC4176) 2% FBS, human epidermal growth factor, hydrocortisone, vascular endothelial growth factor, human fibroblast growth factor, human recombinant insulin growth factor, ascorbic acid, GA- 1000 (gentamicin and amphotericin B), and heparin. Stimulation of EC cells with HA and LPS. Endotoxin- free, protein-free and DNA- free HA is provided by the Genzyme

Corporation. EC are treated with HMW HA (1600 or 3000 kDa at 50, 100, 500, 1000 or 3000 ug/ml) or with carboxy methyl cellulose at the same doses (to control for effects of charge) for 24 hours. EC are then stimulated with LMW HA (35 or 180 kDa, 50 and 100 ug/ml) or Salmonella typhosa LPS (Lot 81H4018; Sigma Chemical Co., St. Louis, MO, 100 ng/ml) for 6 or 24 hours at 37⁰C. Cell supernatants are harvested for measurement of IL-8 in human cells and MIP-2 in mouse cells. Cells are harvested for measurement of mRNA expression of IL-8, JNK and NF&B pathway activation by Western Blot, and VCAM and ICAM expression by flow cytometry. Results are compared in human PAEC, MVEC and AEC, and mouse PAEC and AEC isolated from TLR2 and TLR4 +/+, +/-, and -/- mice.

Measurement of cytokines.

ELISA is used to measure cytokine production in cell supernatants (IL-8: R&D, Minneapolis, MN; MIP-2: (Biosource International, Camarillo, CA).

Measurements of JNK and NFKB activation.

Western Blot is used to measure activation of the JNK and NFKB pathways with p-JNK, NFKB/IKB, and JNK, (Santa Cruz Biotechnology, Santa Cruz, CA). For measurement of the NFKB pathway activation, nuclear and cytosolic proteins are analyzed separately. DNA binding is measured with EMSA to confirm activation.

Inhibitors of JNK and NFKB/IKB pathways.

To explore the role of the JNK and NFκB/IκB pathways, the JNK II inhibitor SP600125 is used; at 10 uM, the NFKB peptide inhibitor SN 50, and the NFKB control peptide SN 50 as an inactive control (EMD Biosciences, La Jolla, CA). The cells are preincubated for one hour followed by stimulation with LMW HA and LPS. The LDH- based in vitro toxicology assay (Sigma Chemical Co, St. Louis, MO) is performed on the cell culture supernatants to rule out toxic effects of the inhibitors.

Example 6. High-molecular weight hyaluronan (HMW HA) attenuates high tidal volume ventilation induced lung inflammatory cell infiltration in rat model of sepsis.

Mechanical ventilation (MV) with high tidal volumes (V_T) leads to ventilator- induced lung injury. Furthermore, MV with even moderately high tidal volumes and systemic endotoxin synergistically increases lung inflammatory cells and cytokine production. High molecular weight hyaluronan (HMW HA), a component of the extracellular matrix, has been found to have anti-inflammatory properties, as described herein elsewhere. Mice that overexpress HMW HA are protected against bleomycin- induced lung injury. Here, the efficacy of HA infusions in preventing lung inflammation in rats with MV and indirect lung injury from endotoxin (a model of sepsis) is examined. Methods.

Sprague-Dawley rats weighing 180-280 g were randomly divided into the following groups: control group (MV only), sepsis group (MV+LPS) and pretreatment group (MV+LPS+ HA, 1600, 200 and 35 kDa). Each group was divided into low V_x (7 ml/kg) and high VT (14 ml/kg) subgroups (n=5-6/group) and exposed to MV for 4 h. LPS (1 mg/kg), to simulate endo toxemia, or equivalent volume of normal saline, as placebo, was infused one hour prior to ventilation. Animals received 1600 kDa, 200 kDa or 35 kDa (3 ml of 0.35% 1600 kDa, Genzyme Corp. Cambridge, MA) via intraperitoneal route 18 h prior to the beginning of study. During MV, hemodynamic and respiratory parameters (BP, HR, AP) were recorded every 30 min. Arterial blood gases and lactate were measured 5 min and 4 h after start of MV. Following 4 h of MV, rats were sacrificed and bronchoalveolar lavage (BAL) was performed. Total cell counts in BAL were performed by use of a hemacytometer. To measure cell differentials, cells in BAL were fixed on glass slides using cytospin and then stained with a hematologic staining kit.

Results.

In rats with and without LPS, rats with high VT had increased BAL neutrophils compared to rats with low V_x (3.6+1.3 vs. 1.2±0.6 x 10⁴/ml,p<0.0001 for rats without LPS and 8.2±1.3 vs. 4.7±0.3 x 10⁴,/?<0.0001 for rats with LPS). In comparison between rats with and without LPS of the same V_T, rats receiving LPS had increased BAL neutrophils as compared to rats without LPS (8.2±1.3 vs. 3.6±1.3 x 10⁴,/?<0.0001 for high V_T and 4.7±0.3 vs. 1.2±0.6 x 10⁴,/?<0.0001 for low V_x). HMW HA 1600 kDa pretreatment decreased BAL neutrophils in both high V_τ and low V_τ with LPS (8.2±1.3 vs. 2.3+0.2 x 10⁴,jo<0.0001 for high V_x and 4.7±0.3 vs. 1.5±0.2 2 x 10⁴,/?<0.0001 for low V_x). The results were the same with the use of 200 kDa HA or 35 kDa HA. As with BAL neutrophils, high V_x also increased BAL monocytes as compared to low V_x (20.7xl0⁴ vs. 9.3±2.2 x 10⁴/ml, pO.OOOl), and LPS further augmented these changes (36.4 x 10⁴ vs. 20.6 x 10⁴, p, p<0.001). However only 1600 kDa, and not 200 kDa or 35 kDa HA completely blocked the increase in BAL macrophages with high V_T (17.2+1.6 x 10⁴/ml), high V_x+ LPS (16.6+0.5 x 10⁴/ml) and low V_x + LPS (11.8±0.5 x 10⁴/ml). Both 200 and 35 kDa HA only partially blocked BAL monocyte infiltration with low and high V_x ventilation. Conclusions.

Endotoxin enhanced ventilator-induced lung injury by recruiting increased inflammatory cells (neutrophils and monocytes) into the lungs. Only pretreatment with HMW HA blocked both neutrophil and monocyte BAL infiltration in rat model of sepsis with both low and high tidal volume ventilation. These results indicate HMW HA as an effective treatment strategy for sepsis induced lung injury.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

WHAT IS CLAIMED IS;

1. A method of treating sepsis in a subject in need thereof comprising administering to the subject a therapeutically effective amount of hyaluronan, thereby treating sepsis in the subject.

2. The method according to claim 1, wherein the therapeutically effective amount of hyaluronan is less than about 10% of the circulating blood volume of the subject.

3. The method according to claim 1, wherein the therapeutically effective amount of hyaluronan is between about 5% and 20% of the circulating blood volume of the subject.

4. The method according to claim 1 , wherein the hyaluronan is systemically administered.

5. The method according to claim 1, wherein the hyaluronan is high molecular weight hyaluronan.

6. The method according to claim 5, wherein the high molecular weight hyaluronan is greater than about 500 kDa.

7. The method according to claim 5, wherein the high molecular weight hyaluronan is in the range of about 500 kDa to about 10,000 kDa.

8. The method according to claim 1, wherein the subject is a human.

9. A method of treating a sepsis-induced condition in a subject having sepsis comprising systemically administering to the subject a therapeutically effective amount of hyaluronan, thereby treating the sepsis-induced condition in the subject.

10. The method according to claim 9, wherein the sepsis-induced condition is septic shock.

11. The method according to claim 9, wherein the sepsis-induced condition is pulmonary inflammation.

12. The method according to claim 9, wherein the sepsis-induced condition is acute lung injury.

13. The method according to claim 9, wherein the sepsis-induced condition is organ dysfunction of at least one bodily organ.

14. The method according to claim 13, wherein the organ is a lung.

15. The method according to claim 9, wherein the sepsis-induced condition is systemic inflammation.

16. The method according to claim 9, wherein the therapeutically effective amount of hyaluronan is less than about 10% of the circulating blood volume of the subject.

17. The method according to claim 9, wherein the therapeutically effective amount of hyaluronan is between about 5% and 20% of the circulating blood volume of the subject.

18. The method according to claim 9, wherein the hyaluronan is high-molecular weight hyaluronan.

19. The method according to claim 18, wherein the high-molecular weight hyaluronan is greater than about 500 kDa.

20. The method according to claim 18, wherein the high-molecular weight hyaluronan is in the range of about 500 kDa to about 10,000 kDa.

21. The method according to claim 9, wherein the subject is a human.

22. The method according to claim 9, further comprising the step of co- administering an anti-inflammatory therapy.

23. The method according to claim 22, further comprising the step of obtaining the anti-inflammatory therapy.

24. The method according to claim 22, wherein the anti-inflammatory therapy is selected from the group consisting of a nonsteroidal anti-inflammatory drug, steroid, antibody, and a mineralocorticoid.

25. The method according to claim 9, further comprising the step of co- administering an anti-sepsis therapy.

26. The method according to claim 25, further comprising the step of obtaining the anti-sepsis therapy.

27. The method according to claim 25, wherein the anti-sepsis therapy is intubation or mechanical ventilation.

28. A method of treating sepsis in a subject comprising administering an amount of high-molecular weight hyaluronan in an amount effective to inhibit the effects of low-molecular weight hyaluronan in sepsis.

29. The method according to claim 28, wherein the high-molecular weight hyaluronan is sytemically administered.

30. The method according to claim 28, wherein the high-molecular weight hyaluronan is greater than about 500 kDa.

31. The method according to claim 28, wherein the high-molecular weight hyaluronan is in the range of about 500 kDa to about 10,000 kDa.

32. The method according to claim 28, wherein the low-molecular weight hyaluronan is less than 500 kDa.

33. The method according to claim 28, wherein the low-molecular weight hyaluronan is in the range of about 10 kDa to about 500 kDa.

34. The method according to claim 28, further comprising the step of coadministering an anti-inflammatory therapy.

35. The method according to claim 34, further comprising obtaining the antiinflammatory therapy.

36. The method according to claim 35, wherein the anti-inflammatory therapy is selected from the group consisting of a nonsteroidal anti-inflammatory drug, steroid, antibody, and a mineralocorticoid.

37. The method according to claim 28, further comprising the step of coadministering an anti-sepsis therapy.

38. The method according to claim 37, further comprising obtaining the antisepsis therapy.

39. The method according to claim 37, wherein the anti-sepsis therapy is intubation or mechanical ventilation.

40. A method of treating acute lung injury in a subject having sepsis, comprising administering to the subject a fluid comprising a therapeutically effective amount of hyaluronan, thereby treating acute lung injury in the subject.

41. The method according to claim 40, wherein the method further comprises coadministering a therapy for acute lung injury.

42. The method according to claim 37, wherein the therapy for acute lung injury is mechanical ventilation or intubation.

43. The method according to claim 41, further comprising obtaining the therapy for acute lung injury.

44. The method according to claim 40, wherein the hyaluronan is high molecular weight hyaluronan.

45. The method according to claim 44, wherein the high-molecular weight hyaluronan is greater than about 500 kDa.

46. The method according to claim 44, wherein the high-molecular weight hyaluronan is in the range of about 500 kDa to about 10,000 kDa.

47. The method according to claim 40, further comprising the step of co- administering an anti-inflammatory therapy.

48. The method according to claim 47, further comprising obtaining the antiinflammatory therapy.

49. The method according to claim 47, wherein the anti-inflammatory therapy is selected from the group consisting of a nonsteroidal anti-inflammatory drug, steroid, antibody, and a mineralocorticoid.

50. The method according to claim 40, further comprising the step of co- administering an anti-sepsis therapy.

51. The method according to claim 50, further comprising obtaining the antisepsis therapy.

52. A kit for treating sepsis in a subject, comprising a therapeutically effective amount of hyaluronan and instructions for use.

53. The kit of claim 52, wherein the hyaluronan is present in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

54. The kit of claim 52, further comprising a therapy for acute lung injury, a therapy for anti-sepsis therapy, or an anti-inflammatory therapy.

55. The kit according to claim 52, wherein the hyaluronan is high molecular weight hyaluronan.

56. The kit according to claim 55, wherein the high-molecular weight hyaluronan is greater than about 500 kDa.

57. The method according to claim 55, wherein the high-molecular weight hyaluronan is in the range of about 500 kDa to about 10,000 kDa.

58. The method according to any one of claims 1, 9, 28 or 40, further comprising obtaining the hyaluronan.