Methods For Treatment of Hemorrhagic Shock and Related Disorders

Technical Field

The present invention relates to the use of certain agents for the treatment of hemorrhagic shock and surgical procedures involving a cardiopulmonary by-pass machine.

Federal Support

The present invention arose in part from research funded by the Federal SBL Program, DARPA grant HUOOO 1-05-1 -0001.

Related Applications

This application claims priority to U.S. Provisional Application 60/721,979 (filed on September 30, 2005) which is incorporated by reference in its entirety.

Background of the Invention

The leading, but not well known publicly, cause of death among civilians younger than forty years of age is trauma. In modern warfare, the cause of death in 50% of soldiers killed in action is hemorrhage. In the United States alone, traumatic injuries associated with hemorrhagic shock result in approximately 150,000 civilian deaths per year (Minino et al. (2002) Deaths: Final Data for 2000. National Vital Statistics Reports. Department of Health and Human Services 50, 120). Although our knowledge concerning the molecular and cellular pathophysiology of hemorrhagic shock has advanced greatly, the development of new therapies has not progressed as rapidly. The lack of therapeutic development may be related to complexity of this state, as multiple pathways are involved in its pathogenesis. It is known that prompt control of bleeding and subsequent resuscitation can reduce the mortality rate by as much as 20%. However, the restrictive time window associated with treating of traumatic hemorrhage, the challenges of intravenous access, and the limited availability of resuscitative fluids, especially in the battlefield, limit the clinical effectiveness of fluid resuscitation. Another strategy for treating patients in a state of shock is to develop and assess the efficacy of cytoprotective drugs. Using cytoprotective agents prophylactically or soon after the insult would significantly improve post-hemorrhage outcome.

Studies have been performed using valproic acid in focal ischemia models to assess their capacity in treating stroke (Ren et al. (2004) J. Neurochem. 89, 1358-1367). In addition, experiments with /3-hydroxybutyrate in hemorrhagic shock models resulted in decreased expression in apoptotic markers in rat lungs (Koustova et al. (2003) Surgery 134, 261 -21 A).

Because traumatic hemorrhage disproportionately affects the most productive part of the general population, and because of the shortcomings of available therapies, the research and development of novel and efficacious interventions for this condition is essential.

Summary of the Invention The invention encompasses a method of treating a subject suffering from hemorrhagic shock comprising administering to said subject an effective amount of one or more agents selected from the group consisting of hydroxamic acids and carboxylic acids with greater than five carbon atoms and esters thereof. In some embodiments of the invention, the carboxylic acids with greater than five carbon atoms have about eight to about thirty carbon atoms, hi other embodiments, the hemorrhagic shock is caused by penetrating or blunt trauma. In another embodiment, the hemorrhagic shock is caused by gastrointestinal bleeding. In still a further embodiment, the hemorrhagic shock is caused by obstetrical bleeding due to an obstetrical procedure.

The carboxylic acids with greater than five carbon atoms include, but are not limited to, valproic acid, phenylbutrate and pharmaceutically acceptable salts thereof while the hydroxamic acids include, but are not limited to, suberoylanilide hydroxamic acid, trichostatin A and pharmaceutically acceptable salts thereof.

In some embodiments, the agent is administered in an amount about 150 mg/kg to about 600 mg/kg and/or in an amount sufficient to alleviate cellular injury. The agents used in the methods of the invention may also be administered in a sustained release formulation. The agents may be administered immediately following to about seventy-two hours after onset of hemorrhagic shock and may be administered more than once. The agents may be administered via any acceptable route including, but not limited to, intramuscular, intravenous and subcutaneous injection. The agents may also be administered orally. The invention also encompasses a method of protecting a subject against tissue injury associated with a surgical procedure utilizing a cardiopulmonary bypass machine comprising administering to said subject an effective amount of one or more agents capable of inhibiting deacetylation of one or more proteins, hi some embodiments, the one or more proteins is a histone protein and include, but are not limited to, H2A, H2B, H3 and H4 histone proteins. Thus, in one embodiment of the invention, the agents are histone deacetylase inhibitors. hi some embodiments of the invention, the surgical procedure is selected from the group consisting of cardiac by-pass surgery and organ transplantation. The cardiac by-pass surgery may comprises atrial or ventricle valve replacement.

In other embodiments of the invention related to surgical procedures, the agents include, but are not limited to, valproic acid, sodium butyrate, trichostatin A, suberoylanilide hydroxamic acid, phenylbutyrate, beta-hydroxybutyrate and pharmaceutically acceptable salts thereof and amount of agent administered is about 600 mg/kg to about 75 mg/kg. The agents can administered prior, during and subsequent to the surgical procedure. When the agent is administered prior to the surgical procedure, it administered at least about twenty-four hours to about fifteen minutes prior to said surgical procedure. The agents may also be administered more than once and may be administered via any acceptable route including, but not limited to, intramuscular, intravenous and subcutaneous injection.

Brief Description of Figures

Figure 1 shows that pharmacological treatment of rats using valproic acid (VPA) (300 mg/kg, twice, sc) significantly extends survival. The figure depicts fractional survival (%) in animals pretreated with VPA and pre-injected with vehicle, normal saline (NS) after otherwise lethal hemorrhage (60% loss of the total blood volume). The data presented is as the percent of surviving animals over time and analyzed using the chi square test (p <0.05).

Figure 2 shows that the incidence of pro-survival activity of VPA treatment is observed at concentrations dissimilar from those necessary for anti-convulsive and analgesic actions of this drug. Blood concentration in VPA-pretreated animals ranged from 1.8 to 2.2 mmol/L.

Figure 3 shows that VPA pre-treatment inhibits potencies specific to histone deacetylase (HDAC), manifested in hyperacetylation of histones and non-histone proteins. Figure 3A shows the detection of acteylated lysines based on use of antibodies. Figure 3B shows the results of a densitometric analysis for actetylation of nuclear histones.

Figure 4 shows that VPA pre-treatment induces patterns of histone acetylation attributable to HDAC Class I and II inhibition specifically. Figure 5 shows that VPA and analog influence of survival correlates with HDAC- inhibiting activities. As evident, the structural analog of VPA, 2-methlyl-2-pentenoic acid (2M2P), has the same range of anti-epileptic action, but is significantly less efficacious in inhibiting HDAC.

Figure 6 shows serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) after limited volume (0.5*) resuscitation with normal saline (NS) and VPA-supplemented saline.

Figure 7 shows that effϊacy of VPA-supplemented treatment is dose-dependent.

Figure 8 shows the ratio of histone acetyltransferase (HAT) and HDAC activities in the nuclear tractions in the VPA group compared to the animals without VPA pretreatment (NS group).

Figure 9 is a comparison of the effects of valproic acid on the expression of HSP70 and SOD based on RT-PCR results.

Figure 10 shows serum HSP70 levels by ELISA and tissue levels by IHC assays.

Detailed Description

The invention encompasses a method of treating a subject suffering from hemorrhagic shock comprising administering to said subject an effective amount of one or more agents selected from the group consisting of hydroxamic acids and carboxylic acids with greater than five carbon atoms and esters thereof. In some embodiments of the invention, the carboxylic acids with greater than five carbon atoms have about eight to about thirty carbon atoms. In other embodiments, the hemorrhagic shock is caused by penetrating or blunt trauma.

The invention also encompasses a method of protecting a subject against tissue injury associated with a surgical procedure utilizing a cardiopulmonary bypass machine comprising administering to said subject an effective amount of one or more agents capable of inhibiting deacetylation of one or more proteins. In some embodiments, the one or more proteins is a histone protein and include, but are not limited to, H2A, H2B, H3 and H4 histone proteins. Thus, in one embodiment of the invention, the agents are histone deacetylase inhibitors.

Treatment of Hemorrhagic Shock

As used herein, hemorrhagic shock is associated with the substantial loss of blood and divided into four categories depending on its severity. Class I hemorrhage corresponds to a less than 15% blood volume loss and generally is well tolerated. Blood donors fall into this category. Treatment is oral rehydration or judicious use of IV fluids. These patients do have a diminished intravascular volume, but generally compensate well enough to have no classic physical signs of shock. Class II hemorrhage is a 15% to 30% volume loss. These patients generally will have tachycardia, anxiety and a lowered urine output. These are the first signs of shock. In these cases, prompt control of bleeding and resuscitation with IV crystalloid solutions are essential. Restoration to a completely normal blood pressure usually can wait until definitive control of bleeding has been achieved. If complete hemostasis cannot be achieved in the field, then controlled hypotension with a mean arterial blood pressure (MAP) of 50 to 60 mmHg may be best for the patient. Rapid transport also is needed for these patients.

Class III hemorrhage is about 30% to 40% blood loss. These patients will have a decreased blood pressure, tachycardia, minimal urine output, and confusion. Such patients are unable to compensate for their volume loss. Class III and IV hemorrhagic shock are also known as decompensated shock. Control of bleeding and rapid resuscitation are essential to prevent later multiple organ dysfunction and death. This group of patients requires blood transfusion and may require surgical intervention to stop the source of bleeding. Class IV hemorrhage is a greater than 40% blood loss and is rapidly fatal in all patient age groups. These patients will be profoundly hypotensive, have cool extremities, minimal or no urine output, and be minimally responsive to external stimuli. The site of bleeding may be obvious in the case of penetrating trauma, but it is often less easily found in cases of blunt trauma. Bleeding in the extremities can be difficult to assess in the field because it may stay in the subfascial planes. For example, a fractured femur can cause a greater than one liter blood loss without obvious external hemorrhage. Intrathoracic bleeding will cause dullness on percussion and auscultation. This is easily found with a chest x-ray when the patient is in-hospital. The peritoneal cavity is a frequent site of occult bleeding. Several liters of blood may accumulate in the abdomen before any clinically notable distention. The retroperitoneal space may also mask large volumes of blood; this is particularly common after pelvic fractures. Because many bleeding sites may be difficult to assess in the initial field examination, physical signs of shock are of paramount importance. Cool, pale skin coupled with confusion or anxiety should alert a medical professional to the possibility of clinical shock. Urine output may be of help in assessing these patients, but this usually requires catheterization of the bladder to measure urine output over time. A more easily obtainable field assay is the urine-specific gravity, which is elevated if the shock state has been present for some time. As stated above, hemorrhagic shock is caused by the loss of both circulating blood volume and oxygen-carrying capacity. The most common clinical etiologies are penetrating and blunt trauma, gastrointestinal bleeding, and obstetrical bleeding. Although many clinical causes of shock exist, the basic cellular derangement in all types involves an imbalance of oxygen dynamics. Whenever cellular oxygen demand outweighs supply, both the cell and the organism are in a state of shock. On a multicellular level, the definition of shock becomes more difficult because not all tissues and organs will experience the same amount of oxygen imbalance for a given clinical disturbance. Well-described responses to acute loss of circulating volume exist. These responses act to systematically divert circulating volume away from non-vital organ systems so

that blood volume may be conserved for vital organ function. Acute hemorrhage causes a decreased cardiac output and decreased pulse pressure. These changes are sensed by baroreceptors in the aortic arch and atrium. With a decrease in the circulating volume, neural reflexes cause an increased sympathetic outflow to the heart and other organs. The response is an increase in heart rate, vasoconstriction, and redistribution of blood flow away from certain nonvital organs such as the skin, gastrointestinal tract, and kidneys. In addition to these global changes, many organ-specific responses occur which lead to cellular injury or death.

The present invention includes methods for treating and preventing damage to tissues and organs resulting from hemorrhagic shock. The methods of the invention encompass procedures that employ an effective amount of an agent selected from the group consisting of hydroxamic acids and carboxylic acids with greater than five carbon atoms and esters thereof. In some embodiments, the carboxylic acids with greater than five carbon atoms have about eight to about thirty carbon atoms, hi another embodiment, said agent is a capable of inhibiting deacylation of one or more protein. In another embodiment, the agent is a histone deacetylase inhibitor, with the proviso that said agent it is not /3-hydroxybutyrate. In another embodiment, the carboxylic acids with greater than five carbon atoms are selected from the group consisting of valproic acid, phenylbutrate, and pharmaceutically acceptable salts thereof. In another embodiment, the carboxylic acids with greater than five carbon atoms are valproic acid or analogs thereof, such as e.g., 2M2P. In another embodiment, the hydroxamic acids are selected from the group consisting of suberoylanilide hydroxamic acid, trichostatin A and pharmaceutically acceptable salts thereof.

The methods of the invention for treating or preventing hemorrhagic shock and/or related symptoms generally employ an effective amount of an agent selected from the group consisting of hydroxamic acids and carboxylic acids with greater than five carbon atoms and esters thereof. These agents (hereinafter known as agents for the treatment of hemorrhagic shock) will be therapeutically effective and well tolerated among mammalian subjects, in useful and commercially feasible dosage amounts as indicated below, and without unacceptable adverse side effects. In more detailed embodiments, the compounds, compositions and methods of the invention are therapeutically effective to alleviate one or more hemorrhage-associated conditions and/or related symptoms identified herein, including any combination of these conditions and/or related symptoms, without unacceptable adverse side effects. In certain embodiments, the therapeutic methods and compositions of the invention effectively treat and/or prevent a hemorrhage-associated condition or symptom, while avoiding or reducing one or more

side effects associated with a current alternate drug treatment. In this context, the methods and compositions of the invention for treating the disorder and/or related symptom(s) will often yield a reduction or elimination of one or more side effect(s) observed with alternate drug or non-drug treatments for hemorrhagic shock, including, but not limited to, increased mortality, memory loss or other cognitive impairment, low blood pressure, problems with end-organ functions, among other side effects.

The amount of the agent selected from the group consisting of hydroxamic acids and carboxylic acids with greater than five carbon atoms and esters thereof used for the treatment of hemorrhagic shock which will be effective in the treatment of a person suffering from hemorrhagic shock can be determined by standard research techniques. For example, the dosage of the composition which will be effective in the treatment of hemorrhagic shock can be determined by administering the composition to an animal model such as, e.g., the animal models disclosed herein or known to those skilled in the art. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. In one embodiment, that agent is administered in an amount of about 150 mg/kg to about 600 mg/kg. In an alternate embodiment, that agent is administered in an amount of about 10 mg/kg to about 300 mg/kg.

Selection of the preferred effective dose can be determined (e.g., via clinical trials) by a skilled artisan based upon the consideration of several factors which will be known to one of ordinary skill in the art. Such factors include the disease to be treated or prevented, the symptoms involved, the patient's body mass, the patient's immune status and other factors known by the skilled artisan to reflect the accuracy of administered pharmaceutical compositions.

The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the injury, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

In addition, the timing when to administer the agent selected from the group consisting of hydroxamic acids and carboxylic acids with greater than five carbon atoms and esters thereof used for the treatment of hemorrhagic shock can also be determined by clinical trials. For example the agents can be administered immediately following to about 72 hours following the onset of hemorrhagic shock. In addition, more than one administration of the agents of the invention is also contemplated.

Uses Associated With Cardiopulmonary Bypass Surgery

A cardiopulmonary bypass machine, also known as a heart-lung machine, takes over for the heart by replacing the heart's pumping action. This means that surgeries which require the heart to be still for the operation, which is necessary in some surgeries (i.e., open-heart surgery), takes over the work of the heart. The heart-lung machine carries blood from the upper-right chamber of the heart (the right atrium) to a special reservoir called an oxygenator. Inside the oxygenator, oxygen bubbles up through the blood and enters the red blood cells. This causes the blood to turn from dark (oxygen-poor) to bright red (oxygen-rich). Then, a filter removes the air bubbles from the oxygen-rich blood, and the blood travels through a plastic tube to the body's main blood conduit (the aorta). From the aorta, the blood moves throughout the rest of the body. The heart-lung machine can take over the work of the heart and lungs for hours. Trained technicians called perfusion technologists (blood flow specialists) make sure that the heart-lung machine does its job properly during the surgery. Even so, surgeons still try to limit the time that patients must spend connected to the machine to prevent tissue injury that may occur. The cardiopulmonary bypass is associated with various insults to normal physiology of different tissues which can lead to tissue injury. Such tissue injury include anticoagulation, hemodilution, hypothermia, chemical cardiac arrest, increased endogenous cathecolamine and vasopressin release, electrolyte disturbances, platelet activation, aggregation and destruction, and activation of complement and other plasma protein systems. These multiple interacting factors represent a number of potential routes for myocardial dysfunction or injury.

The invention therefore also encompasses a method of protecting a subject against tissue injury associated with a surgical procedure utilizing a cardiopulmonary bypass machine comprising administering to said subject an effective amount of one or more agents capable of inhibiting deacetylation of one or more proteins. In some embodiments, the one or more proteins is a histone protein and includes, but are not limited to, H2A, H2B, H3 and H4 histone proteins. Thus, in one embodiment of the invention, the agents are histone deacetylase inhibitors. In another embodiment, one or more agents is selected from the group consisting of valproic acid, sodium butyrate, trichostatin A, suberoylanilide hydroxamic acid, phenylbutyrate, beta-hydroxybutyrate and pharmaceutically acceptable salts thereof. In another embodiment, the histone deacetylase inhibitor is not jβ-hydroxybutyrate.

The methods and compositions of the invention for treating or preventing cellular injury associated with a patient attached to a cardiopulmonary bypass machine and/or related symptoms generally employ an effective amount of a histone deacetylase inhibitor (HDACI). In

more detailed embodiments, the compounds, compositions and methods of the invention are therapeutically effective to alleviate one or more tissue injury associated with cardiopulmonary bypass machine and/or related symptoms identified herein, including any combination of these conditions and/or related symptoms, without unacceptable adverse side effects. In certain embodiments, the therapeutic methods and compositions of the invention effectively treat and/or prevent a hemorrhage-associated condition or symptom, while avoiding or reducing one or more side effects associated with a current alternate drug treatment. In this context, the methods and compositions of the invention for treating the disorder and/or related symρtom(s) will often yield a reduction or elimination of one or more side effect(s) observed with alternate drug or non-drug treatments for cellular injury associated with a patient attached to a cardiopulmonary bypass machine, including, but not limited to, increased mortality, memory loss or other cognitive impairment, low blood pressure, problems with end-organ functions, among other side effects.

The amount of a HDACI used for the treatment of cellular injury associated with a patient attached to a cardiopulmonary bypass machine that will be effective can be determined by standard research techniques. For example, the dosage of the composition that will be effective in the prevention cellular injury associated with a patient attached to a cardiopulmonary bypass machine can be determined by administering the composition to an animal model such as, e.g., the animal models disclosed herein or known to those skilled in the art. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges.

Selection of the preferred effective dose can be determined (e.g., via clinical trials) by a skilled artisan based upon the consideration of several factors which will be known to one of ordinary skill in the art. Such factors include the disease to be treated or prevented, the symptoms involved, the patient's body mass, the patient's immune status and other factors known by the skilled artisan to reflect the accuracy of administered pharmaceutical compositions.

The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the injury, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. In addition, the timing of when to administer the HDACI used for the treatment of cellular injury associated with a patient attached to a cardiopulmonary bypass machine can also

be determined by clinical trials. For example, the agents can be administered just prior to the surgery or hours or days before the surgery, hi addition, more than one administration of the agent and more that one agent are also contemplated.

Histone Deacetylase Inhibitors Chromatin is a highly specialized structure composed of tightly compacted chromosomal DNA. Gene expression within the nucleus is controlled, in part, by a host of protein complexes that continuously pack and unpack the chromosomal DNA from the inaccessible, tightly packed nucleosomal particles to the accessible, unwound nucleosomal particles. Regulation of gene expression is mediated by several mechanisms such as DNA methylation, ATP-dependent chromatin remodeling, and post-translational modifications of histones, which include the dynamic acetylation and deacetylation of epsilon-amino groups of lysine residues present in the tail of core histones. One of the known mechanisms of this packing and unpacking process involves the acetylation and deacetylation of the histone proteins comprising the nucleosomal core. A single nucleosomal core particle contains an octamer of histone proteins H2A, H2B, H3, and H4. Acetylated histone proteins confer accessibility of the DNA template to the transcriptional machinery for expression. Several known steroid receptor coactivators in the pi 60 family, such as SRC-I and AIBl, possess histone acetyltransferase (HAT) activity which is thought to be critical for function. Histone deacetylases (HDACs) are chromatin remodeling factors that deacetylate histone proteins and thus, may act as transcriptional repressors. HDACs are classified by their sequence homology to the yeast

HDACs. The enzymes responsible for reversible acetylation/deacetylation processes are histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. HATs act as transcriptional coactivators, and HDACs are part of transcriptional corepressor complexes. Inhibitors of histone deacetylase induce hyperacetylation of histones that modulate chromatin structure and gene expression. These inhibitors also induce growth arrest, cell differentiation, and apoptosis of tumor cells. Thus, controlled balance between histone acetylation and deacetylation is essential for normal cell functions.

The invention thus encompasses a method of protecting a subject against tissue injury associated with a surgical procedure utilizing a cardiopulmonary bypass machine comprising administering to said subject an effective amount of one or more agents capable of inhibiting deacetylation of one or more proteins, hi some embodiments, the tissue injury is not associated with focal ischemia but rather a total physiological ischemia in multiple tissues and organs. In some embodiments, the one or more proteins is a histone protein and include, but are not limited

to, H2A, H2B, H3 and H4 histone proteins. Thus, in one embodiment of the invention, the agents of the invention are histone deacetylase inhibitors, hi exemplary embodiments, agents of the invention comprise valproic acid (VPA) (and structural analogs thereof, such as, e.g., 2- methlyl-2-pentenoic acid (2M2P)), trichostatinA (TSA), oxamflatin, MS 27-275, hybrid polar compounds and their derivatives (including but not limited to suberic bishydroxamic acid, m- carboxycinnamic acid bishydroxamide, and suberoylanilide hydroxamic acid (SAHA)), biaryl hydroxamates, short-chain fatty acids and their derivatives (including but not limited to phenylacetate, phenyl butyrate, beta-hydroxybutyrate (BHB)), trichostatin and derivatives, amino-epoxy-oxodecanoic acid containing cyclotetrapeptides and their derivatives (including but not limited to trapoxin A, HC toxin, K-trap, apicidin), FR 901228, benzamides. The agents of the invention may also comprise pharmaceutically acceptable salts of the previously mentioned compound, where applicable. In another embodiment, the surgical procedure utilizing a cardiopulmonary bypass machine comprises, cardiac bypass surgery, atrial or ventricle valve replacement or heart transplant surgery.

Formulations

HDACI or agents for the treatment of hemorrhagic shock for use within the methods and compositions of the invention can be formulated with a pharmaceutically acceptable carrier and/or various excipients, vehicles, stabilizers, buffers, preservatives, etc. Operable compounds within these aspects of the invention can be readily selected from among the various existing and developed candidate compounds described herein using well-known methods, including the various animal models described below. These and other methods can be used to select, identify, and determine optimal dosages and combinations of the compounds described herein. Within the therapeutic methods and compositions of the invention, HDACI or agents for the treatment of hemorrhagic shock is selected for use in a composition or method for treating or preventing cellular injury and/or related symptom(s) will be formulated for therapeutic use in an "effective" or "therapeutic" amount or dose. These terms collectively describe either an effective amount or dose of a compound as described herein that is sufficient to elicit a desired pharmacological or therapeutic effect in a mammalian subject typically resulting in a measurable reduction in an occurrence, frequency, or severity of a disorder, and/or of one or more symptom(s) associated with a disorder, in the subject. In certain embodiments, when a compound of the invention is administered to treat a disorder, for example hemorrhagic shock, an effective amount of the compound will be an amount sufficient in vivo to delay or eliminate onset of one or more symptoms associated with the disorder, for example mortality, morbidity

and/or cellular injury. Therapeutically-effective formulations and dosages can alternatively be determined by an administering formulation/dosage that yields a decrease in the occurrence, frequency or severity of one or more symptoms of cellular injury, for example by a decline in the frequency or intensity of one or more symptoni(s). An effective amount of HDACI or agents for the treatment of hemorrhagic shock in this context will typically yield a detectable, therapeutic reduction in the nature or severity, occurrence, frequency, and/or duration of one or more symptom(s) associated with the targeted cellular injury. Therapeutically effective amounts, and dosage regimens, of the HDACI compositions of the invention, including pharmaceutically effective salts, solvates, hydrates, polymorphs or prodrugs thereof, will be readily determinable by those of ordinary skill in the art, often based on routine clinical or patient-specific factors.

Suitable routes of administration for the HDACI or agents for the treatment of hemorrhagic shock include, but are not limited to, oral, buccal, sublingual, anal, nasal, inhalable, topical, transdermal, mucosal, injectable, slow release, controlled release, although various other known delivery routes, devices and methods can likewise be employed. Useful injectable delivery methods include, but are not limited to, intravenous, intramuscular, intraperitoneal, intraspinal, intrathecal, intracerebroventricular, intra-arterial, and subcutaneous injection. To practice coordinated treatment methods HDACI or agents for the treatment of hemorrhagic shock is administered simultaneously or sequentially, in a coordinated treatment protocol with one or more of the secondary or adjunctive therapeutic agents or methods described above. The coordinated administration may be done simultaneously or sequentially in either order, and there may be a time period where only one or both (or all) active therapeutic agents, individually and/or collectively, exert their biological activities.

Pharmaceutical dosage forms of HDACI or agents for the treatment of hemorrhagic shock within the instant invention may further include one or more excipients or additives, including, without limitation, binders, fillers, lubricants, emulsifiers, suspending agents, sweeteners, flavorings, preservatives, buffers, wetting agents, disintegrants, effervescent agents and other conventional excipients and additives. The compositions of the invention for treating cellular injury can include any one or combination of the following: a pharmaceutically acceptable carrier or excipient; other medicinal agent(s); pharmaceutical agent(s); adjuvants; buffers; preservatives; diluents; and various other pharmaceutical additives and agents known to those skilled in the art. These additional formulation additives and agents will often be

biologically inactive and can be administered to patients without causing deleterious side effects or interactions with the active agent.

If desired, HDACI or agents for the treatment of hemorrhagic shock of the invention can be administered in a controlled release form by use of a slow release carrier, such as a hydrophilic, slow release polymer. Exemplary controlled release agents in this context include, but are not limited to, hydroxypropyl methyl cellulose, having a viscosity in the range of about 100 cps to about 100,000 cps.

HDACI or agents for the treatment of hemorrhagic shock and related compositions of the invention will often be formulated and administered in an oral dosage form, optionally in combination with a carrier or other additive(s). Suitable carriers common to pharmaceutical formulation technology include, but are not limited to, microcrystalline cellulose, lactose, sucrose, fructose, glucose dextrose, or other sugars, di-basic calcium phosphate, calcium sulfate, cellulose, methylcellulose, cellulose derivatives, kaolin, mannitol, lactitol, maltitol, xylitol, sorbitol, or other sugar alcohols, dry starch, dextrin, maltodextrin or other polysaccharides, inositol, or mixtures thereof. Exemplary unit oral dosage forms for use in this invention include tablets, which may be prepared by any conventional method of preparing pharmaceutical oral unit dosage forms can be utilized in preparing oral unit dosage forms. Oral unit dosage forms, such as tablets, may contain one or more conventional additional formulation ingredients, including, but not limited to, release modifying agents, glidants, compression aides, disintegrants, lubricants, binders, flavors, flavor enhancers, sweeteners and/or preservatives.

Suitable lubricants include stearic acid, magnesium stearate, talc, calcium stearate, hydrogenated vegetable oils, sodium benzoate, leucine carbowax, magnesium lauryl sulfate, colloidal silicon dioxide, and glyceryl monostearate. Suitable glidants include colloidal silica, fumed silicon dioxide, silica, talc, fumed silica, gypsum, and glyceryl monostearate. Substances which may be used for coating include hydroxypropyl cellulose, titanium oxide, talc, sweeteners and colorants. The aforementioned effervescent agents and disintegrants are useful in the formulation of rapidly disintegrating tablets known to those skilled in the art. These typically disintegrate in the mouth in less than one minute, and often in less than thirty seconds. By effervescent agent is meant a couple, typically an organic acid and a carbonate or bicarbonate. Additional compositions of the invention comprise HDACI or agents for the treatment of hemorrhagic shock prepared and administered in any of a variety of inhalation or nasal delivery forms known in the art. Devices capable of depositing aerosolized HDACI or agents for the treatment of hemorrhagic shock include formulations in the sinus cavity or alveoli of a

patient include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like. Methods and compositions suitable for pulmonary delivery of drugs for systemic effect are well known in the art. Suitable formulations, wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, may include aqueous or oily solutions of HDACI or agents for the treatment of hemorrhagic shock and any additional active or inactive ingredient(s).

Intranasal delivery permits the passage of such a compound to the blood stream directly after administering an effective amount of the compound to the nose, without requiring the product to be deposited in the lung. In addition, intranasal delivery can achieve direct, or enhanced, delivery of the active compound to the central nervous system. For intranasal and pulmonary administration, a liquid aerosol formulation will often contain HDACI or agents for the treatment of hemorrhagic shock as described herein combined with a dispersing agent and/or a physiologically acceptable diluent. Alternative, dry powder aerosol formulations may contain a finely divided solid form of the subject compound and a dispersing agent allowing for the ready dispersal of the dry powder particles. With either liquid or dry powder aerosol formulations, the formulation must be aerosolized into small, liquid or solid particles in order to ensure that the aerosolized dose reaches the mucous membranes of the nasal passages or the lung. The term "aerosol particle" is used herein to describe a liquid or solid particle suitable of a sufficiently small particle diameter, e.g., in a range of from about 2 to 5 ^' microns, for nasal or pulmonary distribution to targeted mucous or alveolar membranes. Other considerations include the construction of the delivery device, additional components in the formulation, and particle characteristics. These aspects of nasal or pulmonary administration of drugs are well known in the art, and manipulation of formulations, aerosolization means, and construction of delivery devices, is within the level of ordinary skill in the art. Yet additional compositions and methods of the invention are provided for topical administration of HDACI or agents for the treatment of hemorrhagic shock. Topical compositions may comprise HDACI and any other active or inactive component(s) incorporated in a dermatological or mucosal acceptable carrier, including in the form of aerosol sprays, powders, dermal patches, sticks, granules, creams, pastes, gels, lotions, syrups, ointments, impregnated sponges, cotton applicators, or as a solution or suspension in an aqueous liquid, non-aqueous liquid, oil-in-water emulsion, or water-in-oil liquid emulsion. These topical compositions may feature HDACI or agents for the treatment of hemorrhagic shock dissolved or dispersed in a portion of water or other solvent or liquid to be incorporated in the topical

composition or delivery device. Transdermal administration may be enhanced by the addition of a dermal penetration enhancer known to those skilled in the art. Formulations suitable for such dosage forms incorporate excipients commonly utilized therein, particularly means, e.g., structure or matrix, for sustaining the absorption of drug over an extended period of time, for example 24 hours.

Yet additional formulations HDACI or agents for the treatment of hemorrhagic shock for treating or preventing cellular injuries are provided for parenteral administration, including aqueous and non-aqueous sterile injection solutions which may optionally contain anti-oxidants, buffers, bacteriostats and/or solutes which render the foπnulation isotonic with the blood of the mammalian subject; and aqueous and non-aqueous sterile suspensions which may include suspending agents and/or thickening agents. The formulations may be presented in unit-dose or multi-dose containers. These and other formulations of the invention may also include polymers for extended release following parenteral administration. Extemporaneous injection solutions, emulsions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described. Exemplary unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as described herein above, or an appropriate fraction thereof, of the active ingredient(s).

In other detailed embodiments, HDACI or agents for the treatment of hemorrhagic shock compositions may be encapsulated for delivery in microcapsules, microparticles, or microspheres, prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and polymethylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. The pharmaceutical agents and formulations of the current invention will typically be sterile or readily sterilizable, biologically inert, and easily administered.

The following examples illustrate certain embodiments of the present invention, and are not to be construed as limiting the present disclosure. The evidence provided in these examples demonstrates that HDACI or agents for the treatment of hemorrhagic shock as described herein are effective in the treatment and prevention of hemorrhagic shock and related symptoms in mammals.

Examples

The following methods are applicable to all of the Examples, which follow. For test animals, inbred male Wistar-Kyoto rats were used to increase data reliability/reproducibility and decrease the number of animals used. The rats were allowed to recover from shipping stress and observed for at least one week in the animal quarters before surgical manipulations to ensure a good state of health. Free access to food and water was allowed until the day of the experiment.

For surgical procedures, rats were anesthetized using 2% isoflurane anesthesia administered through an open nose chamber. The rats were kept in a supine position during the experiment and allowed to breathe spontaneously. The right inguinal area was shaved, and a small (less than two cm) inguinal incision made. The femoral vessels were isolated, and both the artery and the vein cannulated using polyethylene tubing (PE50) primed with heparin (100 units/ml). The femoral artery catheter was attached to a pressure transducer that allowed instantaneous measurement of the blood pressure (BP), heart rate (HR) and mean arterial pressure (MAP). Cannulation was done in less than thirty minutes. After cannulation, the concentration of isoflurane was reduced to 0.5 to 1% for the duration of the experiment. For the hemorrhagic shock protocol, total Blood Volume (TBV) of each rat was calculated using the formula TBV (ml) = 0.06x body weight (BW, grams) + 0.77. Blood was removed form rats through the femoral artery catheter using an infusion/withdrawal pump (Harvard Apparatus). To simulate the initial brisk arterial bleeding in traumatic injuries, 40% percent of the TBV was withdrawn in ten minutes. The continuous venous bleeding during delayed transport and treatment period was simulated by withdrawing 20% of the TBV from the femoral vein catheter over the next 170 minutes using the same pump. The total blood removed from a given rat was 60% of TBV over three hours. Controlled blood removal ceased at 3 hours, and anesthesia was maintained at a level of 0.5% isoflurane. The rats were observed until they expired (defined as zero readings on the physiologic monitor and apnea for greater than one minute). Blood pressure (BP), heart rate (HR) and MAP were taken at baseline, 10 (after the 40% TBV bleed), 60, 120 and 180 minutes.

In pilot experiments, thirteen rats were used to develop the hemorrhage model. All animals died from the hemorrhagic shock with the average death time of 250 minutes. The earliest death was recorded at 139 minutes while the latest was at 345 minutes. The Mean Arterial Pressure (MAP) dropped drastically after the first ten minutes of shock, slowly increasing to less than half of the baseline value at 60 and 120 minutes, and then gradually

decreasing until the time of death. It was observed that all the rats died within 5 to 10 minutes when MAP reading was at 5 mmHg. This was an important guide in the timing of tissue collection. Twenty-seven rats were randomly divided into two groups, the VPA group (n=12) and the control group, normal saline (NS) (n=15). The average weight of the VPA group was 258 grams, and the control NS group was 239 grams. The rats in the VPA group were injected subcutaneously with valproic acid or other test agents at 300 mg/kg given twice, one injection 24 hours prior to shock induction and another injection immediately before shock. VPA was dissolved in normal saline (NS), and fresh solutions were prepared daily. The rats in the NS group were injected subcutaneously with normal saline with the volume calculated using the same dosage rate as the VPA group. Both groups were subjected to the hemorrhagic protocol described. Blood samples were taken at baseline, 60, 120, and 180 minutes for the measurement of the arterial blood gases, blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and lactate. The volume of these blood samples collected was included in the total blood loss. Six rats were subjected to the same surgical manipulations as the experimental groups, but not hemorrhaged, and served as the reference group, sham.

Immediately before death (as guided by the hemorrhagic model), a midline sternal incision was made to expose the heart. Fresh tissue was harvested by dissecting the heart at the atrioventricular groove and promptly stored at -80 ⁰ C. Phosphate-Buffered Saline (PBS) was then slowly infused through the femoral artery as well as the femoral vein to drain the rat's remaining blood out through the heart. The heart opening was then sealed with an atraumatic clamp as 4% paraformaldehyde was slowly infused for thirty minutes through the femoral artery. The remaining heart tissue was then harvested and postfixed in the same fixative overnight and transferred to fresh PBS before embedding into paraffin. For the analysis of protein acetylation, heart tissue dissected from sham, VPA and saline-treated (NS) rats were evaluated for total nuclear protein acetylation, acetylated nonhistone protein (tubulin), total core histone H2A, H2B, H3 and H4. The core histones were subsequently evaluated for histones acetylated at different lysine sites by Western blot analysis. Briefly, 20 μg of nuclear protein, fraction F3 (for total acetylated proteins and histone) and cytoskeletal (for acetylated tubulin) were separated by 4 to 20% Tris-Glycine gel, and transferred to polyvinylidene difluoride (PVDF) membranes. After being blocked with SuperBlock blocking buffer, the membranes were probed with specific primary antibodies. Initially F3 fractions of heart tissues were probed with anti- acetylated-lysine polyclonal

antibody (1 : 1000). This antibody detects all proteins containing acetylated lysine residues. Therefore, it is possible to determine whether the change in degree of protein acetylation was fraction-specific and treatment-specific. Immunoblots were then stripped with Restore Western Blot Stripping Buffer at 37 ⁰ C for thirty minutes and reblotted for total anti-histone H2A, H2B, H3, and H4 antibodies to measure total core histone content. Finally, to determine the specific patterns of core histone acetylation, the nuclear fractions were probed with multiple antibodies generated against histones acetylated on different lysine residues: acetyl histone H2A-lys9, acetyl histone H2B-lys20, acetyl histone H3-lys9, 14, 18, 23, and acetyl histone H4-lys5, 8, 12, 16. The membranes were then incubated with corresponding secondary antibodies. The signals were developed by using Supersignal West Pico Chemiluminescent Substrate. The images were visualized on film by exposure to X-rays. The films were then digitized and quantified with the GEL DOClOOO/2000 Quantity One program (Bio-Rad) software. The HAT (histone acetyltransferase) and HDAC (histone deactylase) activities in the nuclear fraction from heart tissues were measured by HAT Assay Kit and HDAC Colorimetric Activity Assay Kit (Biovision) according to the manufacturer's instructions.

Example 1: Effect of valproic acid.

Rats were administered valproic acid (VPA, 300 mg/kg, subcutaneously (SC)) 24 hours before the surgery. A remarkable observation is that VPA pretreatment results in significant (85%) increase in survival of severely hypotensive rats (Figure 1). Compared with the longest surviving vehicle-injected animal, VPA-pretreated animals with the longest survival time demonstrated almost five-fold increase in survival duration. Similar extension of life in a shock victim would certainly help to rescue a patient with severe trauma at times when rescue logistics are complicated, such as in rural areas or a battlefield scenario. The ability of VPA to alleviate the mortality associated with a lethal hemorrhage is associated with its HDAC inhibiting activities. As demonstrated in Figure 2, which depicts the therapeutic range of VPA, the dose employed in our study was within pharmacological, toxicity-free limits (10 to 300 mg/kg) for valproate. However, the dose was higher than that (100 to 120 mg/kg) required for valproate antiepileptic activity. Valproate, similar to other drugs that act by more than one mechanism, can elicit different effects at different doses. VPA's IC ₅₀ for HDAC inhibition is higher than VPA therapeutic plasma concentration range associated with antiepileptic activity. VPA demonstrates profound HDACI-dependent anti-neoplastic activities only at high concentrations (500 mg/kg/day). VPA also increased the amount of acetylated nuclear proteins (Figure 3). Moreover, the pattern of acetylation of

individual histones was similar to the one attributable to HDAC Class I and II inhibition specifically (Figure 4). Pro-survival effects of VPA correlate with its HDAC-inhibiting activities (Figure 5). The structural analog of VPA, 2-methlyl-2-pentenoic acid (2M2P), was shown to have the same range of anti-epileptic action, but is significantly less efficacious in inhibiting HDAC {see Figure 5).

Both VPA and SAHA affect mortality and morbidity associated with hemorrhage when administered post-insult, as additives to resuscitation fluids, as well. Resuscitation with fluids containing BHB has an effect on tissue and organs, alleviating the signs of I/R-induced apoptosis. VPA, SAHA and BHB administered as components of "standard-of-care" post-shock fluid therapy, administered fluid volume that is equal three times (3x) of the lost blood volume, induce acetylation of nuclear histones and influence HDAC activity in tissues. HDACI, exemplified by VPA, are efficacious as part of limited volume resuscitation. The opportunity to perform successful limited-volume resuscitation (Ix, 0.5 ^χ , 0.25x of the volume of lost blood) is very attractive for logistical reasons. VPA additive administered as a part of 0.5x resuscitation regime produces 40% increase in survival following lethal hemorrhage. VPA-supplemented fluid treatment delivered post-insult and in a very short exposure time restricted to the period of resuscitation procedure is potent enough to induce the hyperacetylation of tissue proteins. Moreover, this treatment results in alleviation of the serum levels of the markers of tissue injury associated with hemorrhage and issue injury {see Figure 6).

Example 2: Pre-exposure to valproic acid enhances the duration of survival.

Animals were pretreated with valproic acid (VPA group) or vehicle, normal saline (NS group), hemorrhaged and monitored until death. A remarkable observation was that VPA pretreatment significantly increased the survival of severely hypotensive rats {see Figure 7). The VPA group had an average time to death of 671.9 minutes (range of 135 to 1590 minutes) while the control group survived for an average of 248.7 minutes (range of 139 to 345 minutes). Despite the hypotensive state (MAP below 30 mm Hg), five out of twelve VPA-treated animals remained alive for over 12 hours post-hemorrhage, an almost three-fold increase in survival time. Two of the VPA-pretreated animals lived for more than 24 hours post-hemorrhage. Compared with the longest surviving NS -injected animal, VPA-pretreated animals with the longest survival time demonstrated almost five-fold increase in survival duration. Similar extension of life in a shock victim would certainly help to rescue a patient with severe trauma at times when rescue logistics are complicated, such as in rural areas or a battlefield scenario.

Example 3: Hemodynamic and Biochemical Parameters.

The baseline MAP of the VPA group was slightly higher than that of the NS group. MAP was drastically decreased in both groups after the initial bleed of 40% of TBV. MAP increased to almost half the baseline values at sixty minutes, then gradually decreased until 180 minutes in both groups. There was no significant difference between the MAP of either group at any point in time up to 240 minutes. BUN and creatinine levels increased during hemorrhage in both groups but were significantly lower post-hemorrhage in the VPA group, suggesting renal protection (see Table 1). Although lactate levels at baseline were higher in the VPA group, they were stabilized at an average of 6.84 mmol/1, significantly lower than 7.84 mmol/1 in NS group. These differences in serum lactate and base deficit, which are markers of tissue ischemia, would predict a better prognosis in the VPA group upon resuscitation. Liver function markers AST, ALT and LDH tended to increase, but did not reach significance in the VPA group. Hepatocellular damage has been shown to occur with long-term use of valproic acid, but its side-effects in prophylaxis or short-term use are mild and tolerable. In addition, pH was lower for the VPA group at 120 and 180 minutes. ρθ ₂ was higher for NS group at 60 minutes, while sθ ₂ was higher for the VPA group at baseline.

Example 4: Valproic acid selectively increases the acetylation of nuclear histones.

To prove that at the studied dose VPA exhibits HDAC-inhibiting activity and has direct effects on acetylation patterns, acetylated proteins in cardiac nuclear fraction were studied. Nuclear proteins include histones, the nonhistone chromosomal proteins, and nonchromosomal nuclear proteins (associated with the nuclear envelope and nuclear matrix). Initial treatment of the nuclear fractions with rabbit anti-acetylated-lysine polyclonal antibody allowed for detection of every acetylated lysine site on all nuclear proteins. As shown in Figure 3, all detectable protein bands associated with acetylated lysines had molecular weights lower than 18 kD, corresponding to molecular weights of histones (Figure 3). For densitometric analysis, proteins were divided into three groups according to their molecular weights: 18 kD-1, 18 kD-2, and 18kD-3. VPA treatment significantly increased the expressions of 18 kD-1 and 18 kD-2 proteins compared to the NS group.

Example 5: Valproic acid treatment induces specific patterns of histone acetylation.

It was found that VPA treatment induced hyperacetylation of H2AK9, H3-K9, H3-K14, H3-K18, H4-K5, H4-K8, H4-K12 and H4-K16 proteins relative to the histones extracted from NS -treated rats (Figure 4). VPA pretreatment did not affect the expression of total H2A, H2B and H3 (Figure 4). The acetylation status of H2B-K20 and H3-K23 was unaffected. We also detected acetylated α-tubulin in the heart cytoskeletal fractions were also detected. VPA pretreatment also significantly increased the acetylation of a- tubulin (175.78 ± 19.53%, p<0.05 compared to NS). The effects of valproic acid on the HAT and HDAC activities were also investigated. The ratio of HAT and HDAC activities in the nuclear fractions in the VPA group was significantly increased compared to the animals without VPA pretreatment (NS group) (Figure 8). This indicates that the balance between activities of histone modifying enzymes was shifted, favoring HAT activity following exposure to VPA, a potent HDAC inhibitor.

Example 6: Effects of valproic acid on the expression of HSP70 and SOD.

VPA pretreatment enhanced the expression of SOD gene, but did not affect HSP70 gene expression based on RT-PCR results (Figure 9). In order to search for the possible downstream

effects of VPA-induced hyperacetylation, serum HSP70 levels were analyzed by ELISA and tissue levels by IHC assays (Figure 10). VPA treatment did not affect the circulating levels of HSP70. However, the levels of HSP70 in tissue from the VPA group were significantly higher compared to the animals with lethal hemorrhagic shock without VPA pretreatment.

Example 7: Effect of Suberoylanilide hydroxamic acid (SAHA).

SAHA in HOP, 0.25 mM (S-0.25) was tested as pharmacological additive to traditional fluid resuscitation. We demonstrated that addition of SAHA (0.25 mM) to resuscitation treatment induced full biological response: inhibition of HDAC activity in the heart and hyperacetylation of Iys5 and Iysl2 on H4, Iys9 and Iys23 on H3, and lys20 on H2B. SAHA treatment resulted in normalization of the levels of the tissue injury markers ALT and AST, suggesting a better prognosis and recovery. SAHA treatment induced a biological response following a very short exposure (45 minutes during resuscitation phase of experiment). This response was induced post-insult and did not require pre-treatment.

Example 8: Effect of Phenylbutyrate. 100 mg/kg of phenylbutyrate was administered as a single intravenous bolus post-shock, it attempt to reverse the shock damage and lethality. It was determined that 100 mg/kg phenylbutyrate prolongs animal death up to eight hours.

While the invention has been described and illustrated herein by references to various specific materials, procedures and examples, it is understood that the invention is not restricted to the particular combinations of material and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art. It is intended that the specification and examples be considered as exemplary, only, with the true scope and spirit of the invention being indicated by the following claims. All references, patents, and patent applications referred to in this application are herein incorporated by reference in their entirety.