CA2765479A1 - Treatment of inflammatory disorders, cardiovascular diseases and acute ischemic brain stroke with ozone - Google Patents

Abstract

Methods of treating mammalian subjects suffering from, or believed to be suffering from, acute ischemic brain stroke, cardiovascular disease, inflammatory disease or any of the conditions or symptoms related to those diseases, are disclosed in which a biological fluid withdrawn from the subject is processed through an ozone delivery system to deliver a measured amount of ozone to the biological fluid to produce a treated fluid that has a quantifiable absorbed-dose of ozone which, upon re- introduction of the treated fluid containing the quantifiable absorbed-dose of ozone to the subject, effectively treats any number of described disease conditions and symptoms. Methods for manufacturing medicaments for treatment of acute ischemic brain stroke, cardiovascular disease, inflammatory disease or any of the conditions or symptoms related to those diseases, are also disclosed.

Description

TREATMENT OF INFLAMMATORY DISORDERS, CARDIOVASCULAR DISEASES
AND ACUTE ISCHEMIC BRAIN STROKE WITH OZONE
BACKGROUND
Field of the invention: This invention relates to therapeutic treatments for inflammatory disorders, cardiovascular diseases, acute ischemic brain stroke and related medical conditions and symptoms in mammals, and specifically relates to 1o therapeutic treatment of such disorders using methods that provide a biological fluid, taken from a mammalian subject, that has been treated with a measured amount of ozone to produce a biological fluid having a quantifiable absorbed dose of ozone which, upon reintroduction to the mammalian subject, provides therapeutic treatment of disease or related conditions and symptoms.
Statement of the Related Art: The references discussed herein are provided solely for the purpose of describing the field relating to the invention.
Nothing herein is to be construed as an admission that the inventors are not entitled to antedate a disclosure by virtue of prior invention. Furthermore, citation of any document herein is not an admission that the document is prior art, or considered material to patentability of any claim herein, and any statement regarding the content or date of any document is based on the information available to the applicant at the time of filing and does not constitute an affirmation or admission that the statement is correct.
Disease conditions, such as inflammatory disease, cardiovascular disease and ischemic brain stroke, and related symptoms and conditions, present challenges to human and animal subjects alike, and are some of the leading causes of death in many countries of the world. For example, stroke is the second leading cause of death worldwide and is responsible for 4.4 million (9 percent) of the total 50.5 million deaths each year. In the United States, stroke is the No. 3 cause of death behind heart disease, to which it is closely linked, and cancer. It affects more than 800,000 annually in the U.S., of which 600,000 are initial attacks and 200,000 are recurrent.
At current trends, this number is projected to jump to one million annually by the year 2050.
Stroke represents the leading cause of disability in the U.S. with more than 4 million people living with the after-effects of an attack.
Stroke is characterized by the sudden loss of circulation to an area of the brain, resulting in a corresponding loss of neurologic function. Also called a cerebrovascular accident or stroke syndrome, stroke is a nonspecific term encompassing a heterogeneous group of pathophysiologic causes, including thrombosis, embolism and hemorrhage. Strokes currently are classified as either hemorrhagic or ischemic. Acute

2 PCT/US2010/001785 ischemic stroke refers to strokes caused by thrombosis or embolism, and accounts for approximately 87% of all strokes.
Ischemic stroke is caused by a blockage in a blood vessel that stops the flow of blood and deprives the surrounding brain tissue of oxygen. In the absence of oxygen, the brain cells in the immediate area begin to die and release a cascade of toxic chemicals that threaten brain tissue in the surrounding area. This phenomenon is referred to as the ischemic penumbra.
Current statistics indicate that 7.6 percent of individuals that suffer an ischemic stroke die within 30 days of the episode. Moreover, approximately 25 percent of all .
individuals die within a year of their first stroke. Fourteen percent of stroke patients will suffer a stroke relapse within one year, and within five years, the rate of relapse escalates to 25 percent. Fifty percent of stroke victims that survive experience moderate to severe impairment requiring special care including nursing home care or other long-term care facility treatment. When the direct costs (care and treatment) and indirect costs (lost productivity) of strokes are considered together, the total cost of stroke to the United States is estimated in 2008 at $65 billion per year, 87%
of which is attributed to ischemic stroke.
On the macroscopic level, ischemic strokes most often are caused by extracranial embolism or intracranial thrombosis. On the cellular level, any process that disrupts blood flow to a portion of the brain unleashes an ischemic cascade, leading to the death of neurons and cerebral infarction.
Thrombotic stroke is caused by a thrombus (blood clot) that develops in an artery supplying blood to the brain. This is usually because of a repeated buildup of fatty deposits, calcium and clotting factors, such as fibrinogen and cholesterol, carried in the blood. The body perceives the buildup as an injury to the vessel wall and responds by forming blood clots. These blood clots get trapped onto the plaque on the vessel walls, eventually stopping blood flow.
There are two types of thrombotic stroke, based upon vessel diameter. Large vessel thrombosis, the most common form of thrombotic stroke, occurs in the brain's larger arteries. The impact and damage tends to be magnified because all the smaller vessels that the artery feeds are deprived of blood. In most cases, large vessel thrombosis is caused by a combination of long-term plaque buildup (atherosclerosis) followed by rapid blood clot formation. Small vessel disease, also referred to as lacunar infarction, occurs when blood flow is blocked to a very small arterial vessel.
It has been linked to high blood pressure (hypertension) and is an indicator of atherosclerotic disease. Thrombotic disease accounts for about 60 percent of acute ischemic strokes.
Of those, approximately 70 percent are large vessel thrombosis.

3 In embolic stroke, a clot forms outside of the brain, usually in the heart or large arteries of the upper chest and neck, and is transported through the bloodstream to the brain. There, it eventually reaches a blood vessel small enough to block its passage.
Emboli can be fat globules, air bubbles or, most commonly, pieces of an atherosclerotic plaque (i.e. lipid debris) that have detached from an artery wall. Many emboli are caused by a cardiac condition called atrial fibrillation causing blood to pool and form clots that can travel to the brain and cause a stroke. Cardiac sources of embolism account for 80 percent of embolic ischemic strokes.
Within seconds to minutes of the loss of perfusion to a portion of the brain, an ischemic cascade is initiated. Allowed to progress, it will cause a central area of irreversible infarction surrounded by an area of potentially reversible ischemic penumbra.
On the cellular level, the ischemic neuron becomes depolarized as ATP is depleted and membrane ion-transport systems fail. The resulting influx of calcium leads to the release of a number of neurotransmitters, including large quantities of glutamate, which in turn activates N-methyl-D-aspartate (NMDA) and other excitatory receptors on other neurons. These neurons then become depolarized, causing further calcium influx, further glutamate release, and local amplification of the initial ischemic insult. This massive calcium influx also activates various degradative enzymes, leading to the destruction of the cell membrane and other essential neuronal structures. This metabolic aberration creates an intracellular gradient responsible for intracellular accumulation of water (cytotoxic edema).
Within hours to days after a stroke, specific genes are activated, leading to the formation of cytokines and other factors that, in turn, cause further inflammation and microcirculatory compromise. Cerebral endothelial cells are more resistant to ischemia than are neuronal cells. About 2-4 hours after the onset of ischemia, the integrity of the blood-brain barrier becomes compromised, and plasma proteins are able to pass into the extracellular space. The intravascular water follows when reperfusion occurs (vasogenic edema). This process reportedly begins 6 hours after the onset of stroke 3o and reaches a maximum 2-4 days after the onset of stroke. Reperfusion can also be accompanied by hemorrhagic transformation of the infarct, which is usually related to the volume and site of the infarct.
This vascular inflammation may be due to an imbalance between pro-inflammatory (e.g. interferon-gamma, TNF-gamma, IL-6 and IL-12) and anti-inflammatory (e.g. interleukin-4 and IL-10) cytokines release by immune-modulatory T
cells associated within the infarct site and ischemic penumbra. In addition, there is evidence indicating that the vascular endothelium plays a major role in the regulation of

4 blood flow and is of importance in connection with cardiovascular disorders, including acute ischemic brain stroke. Dysfunctional endothelium has been suggested as a contributory factor in many ischemic disorders and may play a role in the demise of the ischemic penumbra. Ultimately, the ischemic penumbra is consumed by these progressive insults, coalescing with the infarcted core, often within hours of the onset of the stroke.
The central goal of therapy in acute ischemic stroke is to preserve the ischemic penumbra. This can be accomplished by limiting the severity of ischemic injury (i.e.
neuronal protection) or reducing the duration of ischemia (i.e. restoring blood flow to the 1o compromised area). The timing of restoring cerebral blood flow appears to be a critical factor in the preservation of the ischemic penumbra and may prove pivotal in neuronal protection as well.
Further, cardiovascular diseases are responsible for a significant number of deaths in most industrialized countries. One such disease is atherosclerosis, a disease of large and medium-sized muscular arteries and is characterized by endothelial dysfunction, vascular inflammation, and the buildup of lipids, cholesterol, calcium, and cellular debris within the intima of the vessel wall. This buildup results in plaque formation, vascular remodeling, acute and chronic luminal obstruction and abnormalities of blood flow, and also results in ischemia (diminished oxygen supply to organs and tissues) of target organs such as the heart, brain and other vital organs.
Prolonged or sudden ischemia may result in a clinical heart attack or stroke from which the patient may or may not recover.
The true frequency of atherosclerosis is difficult, if not impossible, to accurately determine because it is predominantly an asymptomatic condition. The process of atherosclerosis begins in childhood with the development of fatty streaks and advances with increasingly more complicated lesion formation throughout adult life.
In the United States, approximately 7.8 million myocardial infarctions occur annually, and more than 13.2 million Americans have chronic coronary artery disease.
Of persons older than 50 years, 30% have some evidence of carotid artery disease, 3o and cerebrovascular disease is responsible for over 160,000 deaths per year in the United States. More than 50 million people in the United States are candidates for some form of dietary and/or drug treatment to modify their lipid profile.
A complex and incompletely understood interaction exists between the critical cellular elements of the atherosclerotic lesion. These cellular elements include endothelial cells, smooth muscle cells, platelets, and leucocytes. Vasomotor function, the thrombogenicity of the blood vessel wall, the state of activation of the coagulation cascade, the fibrinolytic system, smooth muscle cell migration and proliferation, and

5 PCT/US2010/001785 cellular inflammation are complex and interrelated biological processes that contribute to atherogenesis and the clinical manifestations of atherosclerosis.
The mechanisms of atherogenesis remain uncertain. It is presently believed that early events include endothelial injury, which cause vascular inflammation and a fibroproliferative response ensues.
The earliest pathologic lesion of atherosclerosis is the fatty streak and is observed in the aorta and coronary arteries of most individuals by age 20 years. The fatty streak is the result of localized accumulation of serum lipoproteins within the intima of the vessel wall. The fatty streak may progress to form a fibrous plaque, and is the 1o result of progressive lipid accumulation and the migration and proliferation of smooth muscle cells. Activators of cell-division are produced by activated platelets, macrophages and dysfunctional endothelial cells that characterize early atherogenesis, vascular inflammation, and platelet-rich thrombosis at sites of endothelial disruption.
Vascular inflammation, believed to be a significant component in the etiology of atherosclerosis, may be due to an imbalance between pro-inflammatory (e.g.
interferon-gamma, TNF-gamma, IL-6, IL-8 and IL-12) and anti-inflammatory cytokine (e.g.
interleukin-4 and IL-10) release by immunomodulatory T cells associated with an atherosclerotic lesion. Similar imbalances have been implicated in other autoimmune diseases such as psoriasis, rheumatoid arthritis, scleroderma, lupus, diabetes mellitus, organ rejection, miscarriage, multiple sclerosis, inflammatory bowel disease as well as graft versus host disease.
There is also an emerging body of literature which indicates that the vascular endothelium plays a major role in the regulation of blood flow through the cardiovascular system and is of importance in connection with cardiovascular disorders.
A dysfunctional endothelium has been suggested as a contributory factor in many cardiovascular diseases such as atherosclerosis, peripheral arterial occlusive disease and many other circulatory disorders observed in mammalian patients. Recent evidence indicates that a relative deficiency in endothelium-derived nitric oxide, a vasodilator, further potentiates the proliferative stage of plaque maturation.
Growth of the fibrous plaque results in vascular remodeling, progressive luminal narrowing, blood-flow abnormalities, and compromised oxygen supply to target organs.
Human coronary arteries enlarge in response to plaque formation, and luminal stenosis may only occur once the plaque occupies greater than 40% of the area bounded by the internal elastic lamina.
The stripping or removal (i.e. denudation) of the overlying endothelium or rupture of the protective fibrous cap may result in exposure of the thrombogenic contents of the core of the plaque to the circulating blood. A plaque rupture may result in thrombus

6 PCT/US2010/001785 formation, partial or complete occlusion of the blood vessel, and progression of the atherosclerotic lesion due to organization of the thrombus and incorporation within the plaque.
The physical symptoms of atherosclerosis provide objective evidence of extracellular lipid deposition, stenosis or dilatation of large muscular arteries, or target organ ischemia or infarction, and include the physical symptoms discussed hereinafter.
Claudication, which is defined as reproducible ischemic muscle pain, is one of the most common manifestations of peripheral arterial occlusive disease caused by atherosclerosis. Claudication occurs during physical activity and is relieved after a short rest. Calf, thigh or buttock pain develops because of inadequate blood flow.
The most feared consequence of claudication is severe limb-threatening ischemia leading to amputation. However, studies of large patient groups with claudication reveal that amputation is uncommon. Intermittent claudication may be accompanied by pallor of the extremity and paresthesias (abnormal sensation, such as tingling or burning of touch without stimulus). Intermittent claudication typically causes pain that occurs with physical activity. Determining how much physical activity is needed before the onset of pain is crucial. Typically, vascular surgeons relate the onset of pain to a particular walking distance in terms of street blocks (e.g. two-block claudication). This helps to quantify patients with some standard measure of walking distance before and after therapy. Other important aspects of claudication pain are that the pain is reproducible within the same muscle groups and that it ceases with a resting period of 2-5 minutes.
Location of the pain is determined by the anatomical location of the arterial lesions.
Reduced blood flow that may be caused by either cholesterol embolism or arterial stenosis is frequently associated with muscular symptomology in an extremity or muscle group distal to the embolism or vascular constriction. Numbness and tingling, muscular spasm, weakness and loss of movement are common reportable events.
Reduced flow of blood resulting in oxygen deprivation to an organ or tissue (ischemia) is commonly associated with both atheroembolism (cholesterol embolism) and arterial stenosis. This is frequently associated with a measurable decrease the temperature of 3o an extremity distal to the site of the embolism or vascular narrowing.
A decrease or loss of pulse due to reduced blood flow in instances of atheroembolism and arterial stenosis is a quantifiable parameter and frequently associated with loss of pallor in an extremity. The primary factor in hypertension is an increase in peripheral resistance resulting from vasoconstriction of peripheral blood vessels secondary to arterial stenosis. Another major factor underlying weight gain is lipid deposition secondary to the accumulation of excessive triglycerides or the inhibition in the clearance of triglycerides.

7 Clinical events relating to cardiovascular disease include progressive luminal narrowing of an artery due to expansion of a fibrous plaque, which results in impairment of flow when more than 50-70% of the lumen diameter is obstructed. This impairment in flow results in symptoms of inadequate blood supply to a target organ in the event there is an increase in metabolic activity and therefore oxygen demand. Stable angina pectoris, intermittent claudication, and mesenteric angina are examples of the clinical consequences of this condition.
Rupture of a plaque or denudation of the endothelium overlying a fibrous plaque may result in exposure of the highly thrombogenic subendothelium and lipid core. This exposure may result in thrombus formation, which may partially or completely occlude flow in the involved artery. Unstable angina pectoris, myocardial infarction, transient ischemic attack, and stroke are examples of the clinical manifestations of partial or complete acute occlusion of an artery.
Atheroembolism, also known as cholesterol embolism, refers to the occlusion of small- and medium-caliber arteries (100-200 pm in diameter) by cholesterol crystals. It may present with symptoms of digital necrosis, hypertension, gastrointestinal bleeding, myocardial infarction, retinal ischemia, cerebral infarction, and renal failure. Physical signs include Livedo reticularis (a persistent purplish network-patterned discoloration of the skin caused by dilation of capillaries and venules due to stasis or changes in underlying blood vessels), gangrene, cyanosis, and ulceration. The presence of pedal pulses in the setting of peripheral ischemia suggests microvascular disease.
Angina pectoris is characterized by retrosternal chest discomfort that typically radiates to the left arm and may be associated with dyspnea. Angina pectoris is exacerbated by exertion and relieved by rest or nitrate therapy. Unstable angina pectoris describes a pattern of increasing frequency or intensity of episodes of angina pectoris and includes pain at rest. A prolonged episode of angina pectoris that may be associated with diaphoresis is suggestive of myocardial infarction.
Cerebrovascular disease designates any abnormality of the brain resulting from a pathologic process of the blood vessels, e.g. occlusion of the lumen by a thrombus or 3o embolus, rupture of a vessel, any lesion or altered permeability of the vessel wall and increased viscosity or other change in quality of blood. Disorders of the cerebral circulation include any disease of the vascular system that causes ischemia or infarction of the brain or spontaneous hemorrhage into the brain or subarachnoid space.
A cerebrovascular accident (CVA) or stroke is the sudden death of brain cells due to impaired blood flow resulting in abnormal brain function. Blood flow to the brain can be disrupted by either a blockage (clogging of arteries within the brain, carotid

8 PCT/US2010/001785 arterial occlusion, embolism) or rupture of an artery (cerebral hemorrhage or subarachnoid hemorrhage) to the brain. A transient ischemic attack (TIA) is a short-lived episode (less than 24 hours) of temporary impairment of the brain that is caused by a loss of blood supply. A TIA causes a loss of function in the area of the body that is controlled by the portion of the brain affected.
Causative factors involved in cerebrovascular disease includes cerebral infarction and ischemia which is caused by sudden occlusion of an artery supplying the brain, or, less often, by low flow distal to an already occluded or highly stenosed artery.
Occlusion or stenosis can be the result of disease of the arterial wall or embolism from 1o the heart. Infarction originates as a result of an impediment to normal perfusion that usually is caused by atherosclerosis and coexisting thrombosis. Atheroembolism (atheroma) occurs when a particle of a thrombus originating from a proximal source (arterial, cardiac or transcardiac) travels through the vascular system and leads to a distal occlusion.
A corollary and additional factor in cerebrovascular disease is the incidence of intracranial small vessel disease (microatheroma). The small penetrating arteries of the brain are not supported by a good collateral circulation and occlusion of one of these arteries is rather likely to cause infarction, often in a small, restricted area of the brain.
Inflammatory vascular disease of the arterial (or venous) wall may provoke enough cellular proliferation, necrosis and fibrosis to occlude the lumen, precipitate thrombosis and then embolism, or promote aneurysm formation, dissection and even rupture of the vessel. These vasculitic disorders may present with, or be complicated during their course by, ischemic stroke, intracranial hemorrhage, intracranial venous thrombosis and most often a generalized ischemic encephalopathy.
Physical signs of cerebrovascular disease include diminished carotid pulses, carotid artery bruits, and focal neurological deficits. Peripheral arterial occlusive disease (PAOD) typically manifests as intermittent claudication, impotence, and non-healing ulceration and infection of the extremities. PAOD is most common with the distal superficial femoral artery (located just above the knee joint), which corresponds to claudication in the calf muscle area (the muscle group just distal to the arterial disease).
When atherosclerosis is distributed throughout the aortoiliac area, thigh and buttock muscle claudication predominates. Physical signs include decreased peripheral pulses, peripheral arterial bruits (an unexpected audible swishing sound or murmur heard over an artery or vascular channel which indicates increased turbulence often caused by a partial obstruction), pallor, peripheral cyanosis, gangrene, ulceration.
Visceral ischemia may be occult or symptomatic prior to symptoms and signs of target organ failure.

9 PCT/US2010/001785 Mesenteric angina is characterized by epigastric or periumbilical postprandial pain and may be associated with hematemesis, melena, diarrhea, nutritional deficiencies, and weight loss. Abdominal aortic aneurysm typically is asymptomatic prior to the dramatic and often fatal symptoms and signs of rupture, although patients may describe a pulsatile abdominal mass. Physical signs include pulsatile abdominal mass, peripheral embolism and circulatory collapse.
Dyslipidemia is a disorder of lipoprotein metabolism, including lipoprotein overproduction or deficiency. Dyslipidemias may be manifested by elevation of the total cholesterol, low-density lipoprotein (LDL) cholesterol and the triglyceride concentrations, and a decrease in the high-density lipoprotein (HDL) cholesterol concentration in the blood.
Congestive Heart Failure (CHF), most frequently resulting from coronary artery disease or hypertension, and occurs when the heart can no longer meet the metabolic demands of the body at normal physiologic venous pressures. As the demands on the heart outstrip the normal range of physiologic compensatory mechanisms, signs of CHF
occur. These signs include tachycardia, venous congestion, high catecholamine levels and, ultimately, insufficient cardiac output. Chronic inflammation is recognized as an underlying pathology contributing to the development and progression of chronic heart failure.
Raynaud's disease refers to a disorder in which the fingers or toes (digits) suddenly experience decreased blood circulation. Raynaud's disease can be classified as either primary (or idiopathic) and secondary (also called Raynaud's phenomenon).
Primary Raynaud's disease is milder, and causes fewer complications. Secondary Raynaud's disease is more complicated, severe, and more likely to progress. A
number of medical conditions predispose a person to secondary Raynaud's disease, including scleroderma, which is a serious disease of the connective tissue in which tissues of the skin, heart, esophagus, kidney and lung become thickened, hard and constricted.
About 30% of patients who develop scleroderma will first develop Raynaud's disease.
Other medical conditions predisposing a person to secondary Raynaud's disease include connective tissue diseases, such as systemic lupus erythematosus, rheumatoid arthritis, dermatomyositis and polymyositis, and diseases which result in blockages of arteries (i.e. atherosclerosis). Both primary and secondary types of Raynaud's symptoms are believed to be due to over-reactive arterioles (small arteries).
While cold normally causes the muscle which makes up the walls of arteries to contract, in Raynaud's disease the degree is extreme, and blood flow to the area is severely restricted.

10 PCT/US2010/001785 The relationship between dietary lipid, serum cholesterol and atherosclerosis has long been recognized. In many epidemiological studies it has been shown that a single measurement of serum cholesterol has proved to be a significant predictor of the occurrence of coronary heart disease. Thus diet is the basic element of all therapy for hyperlipidemia (excessive amount of fat in plasma). However, the use of diet as a primary mode of therapy requires a major effort on the part of physicians, nutritionists, dietitians and other health professionals. If dietary modification is unsuccessful, drug therapy is an alternative. Several drugs, used singly or in combination, are available.
However, there is no direct evidence that any cholesterol-lowering drug can be safely io administered over an extended period.
A combination of both drug and diet may be required to reduce the concentration of plasma lipids. Hypolipidemic drugs are therefore used as a supplement to dietary control. Many drugs are effective in reducing blood lipids, but none work in all types of hyperlipidemia and they all have undesirable side effects. There is no conclusive 1s evidence that hypolipidemic drugs can cause regression of atherosclerosis.
Thus, despite progress in achieving the lowering of plasma cholesterol to prevent heart disease by diet, drug therapies, surgical revascularization procedures and angioplasty, atherosclerosis remains the major cause of death in Western countries.
In view of the above, new approaches are being sought to reduce the frequency 20 of clinical sequelae secondary to the myriad of diseases and disorders broadly characterized as cardiovascular diseases.
In addition to the disease conditions described above, including the inflammatory conditions relating to acute ischemic brain stroke and cardiovascular disease, other inflammatory diseases and related symptoms and conditions present 25 serious medical dilemmas. For example, autoimmune diseases are generally believed to be caused by the failure of the immune system to discriminate between antigens of foreign invading organisms (non-self) and tissues native to its own body (self). When this failure to discriminate between self and non-self occurs and the immune system reacts against self antigens, an autoimmune disorder may arise. Autoimmune diseases, 30 or diseases having an autoimmune component include rheumatoid arthritis, multiple sclerosis, systemic lupus erythromatosis (SLE), scleroderma, diabetes, inflammatory bowel disease, psoriasis, pemphigus, atherosclerosis (wherein the vasculature is regarded as a specific organ) and chronic heart failure.
Rheumatoid arthritis is an example of a common human autoimmune 35 disease, affecting about 1% of the population. This disease is characterized by chronic inflammation of the synovial joints which may lead to progressive destruction of cartilage and bone. Pemphigus is a group of autoimmune diseases characterized by

11 the formation of watery blisters on the skin. It is an intraepidermal blistering disease characterized clinically by superficial blisters and erosions of the skin and/or mucous membranes, especially the mouth. Anti-inflammatory agents such as corticosteroids are frequently used to inhibit the inflammatory process by inhibiting specific cytokine production. Systemic lupus erythromatosis (SLE) is an inflammation of the connective tissues and can afflict every organ system. Ninety percent of patients experience joint inflammation similar to rheumatoid arthritis. Treatment includes anti-inflammatory drugs to control arthritic symptoms and topical corticosteroids for skin. Oral steroids, such as prednisone, are used for treatment of systemic symptoms. Scleroderma is a symptom of a group of diseases that involve the abnormal growth of connective tissue, which supports the skin and internal organs. The rheumatic component of scleroderma is characterized by inflammation and/or pain in the muscles, joints, or fibrous tissue.
Diabetes has been increasingly recognized as a disease with low-grade systemic inflammation. This mild inflammatory state is closely related to obesity and insulin resistance wherein adipocytes, especially in the obese, secrete a number of pro-inflammatory cytokines. Psoriasis is the result of highly reactive early cellular inflammation. Psoriasis simultaneously has a rapidly proliferating epidermis, a vigorous acute inflammatory reaction, an accelerated rate of dermal breakdown and repair, and vascular and fibroblast proliferation. Inflammatory bowel disease describes two autoimmune disorders of the small intestine; Crohn's disease and ulcerative colitis.
Treatment includes the use of anti-inflammatory drugs, including corticosteroids for acute episodes of these diseases.
As noted above, atherosclerosis involves an ongoing inflammatory response, which has a fundamental role in mediating all stages of the disease from initiation through progression and, ultimately, the thrombotic complications of atherosclerosis.
Elevation in markers of inflammation predicts outcomes of patients with acute coronary syndromes. Low-grade chronic inflammation, as indicated by levels of the inflammatory marker C-reactive protein, prospectively defines risk of atherosclerotic complications.
Chronic heart failure is a debilitating condition in which the heart's ability to function as 3o a pump is impaired, most frequently as a result of coronary artery disease or hypertension. Chronic inflammation is recognized as contributing to the development and progression of heart failure.
Alloimmune diseases are referred to herein as disorders such as graft versus host disease and tissue transplant rejection, in which an immune response against or by foreign, transplanted tissue can lead to serious complications or can be fatal. In the treatment of these disorders, it is desired to prevent the body from reacting against non-self antigens. Corticosteroids are frequently used to decrease inflammation by

12 PCT/US2010/001785 suppressing migration of polymorphonuclear leukocytes and reversing increased capillary permeability.
Inflammatory cytokines are implicated in inflammation-related disorders of the brain, namely the neuroinflammatory, neurodegenerative and neurological disorders such as Alzheimer's disease, senile dementia, multiple sclerosis, depression, Down's syndrome, Huntington's disease, peripheral neuropathies, spinal cord diseases, neuropathic joint diseases, chronic inflammatory demyelinating disease (CIPD), neuropathies including mononeuropathy, polyneuropathy, symmetrical distal sensory neuropathy, cystic fibrosis, neuromuscular junction disorders, myasthenias and io Parkinson's disease.
Certain neurological brain disorders such as Down's syndrome, epilepsy, brain trauma and Huntington's disease (chorea) are currently understood to involve inflammation of brain cells as a significant component of the underlying pathology of the disorder. Other neurological disorders which have a significant inflammatory component include Guillain-Barr syndrome (GBS), chronic inflammatory demyelinating polyneuropathy (CIDP), myasthenia gravis (MG), dermatomyositis, polymyositis, inclusion body myositis, ischemic stroke, neurosarcoidosis, vascular dementia, vasospasm, subarachnoid hemorrhage, adrenal leukocytic dystrophy (storage disorders), inclusion body dermatomyostis, minimal cognitive impairment and Duchenne muscular dystrophy.
Chronic inflammatory demyelinating polyneuropathy (CIDP) is a neurological disorder characterized by slowly progressive weakness and sensory dysfunction of the legs and arms. The disorder, which is sometimes called chronic relapsing polyneuropathy, is caused by damage to the myelin sheath of the peripheral nerves.
Primary symptoms include slowly progressive muscle weakness and sensory dysfunction affecting the upper and lower extremities. CIDP is closely related to the more common, acute demyelinating neuropathy known as Guillain-Barr syndrome (GBS). CIPD is considered the chronic counterpart of the acute disease GBS.
CIDP is distinguished from GBS, chiefly by clinical course and prognosis. Guillain-Barr Syndrome (GBS) is an acute predominately motor polyneuropathy with spontaneous recovery that may lead to severe quadriparesis and requires artificial ventilation in 20-30% of patients. The most common disease that underlies this syndrome has been classified as acute inflammatory demyelinating polyneuropathy (AIDP).
Autoimmune myasthenia gravis (MG) is a disorder of neuromuscular transmission leading to fluctuating weakness and abnormal fatigueability.
Weakness is attributed to the blockade of acetylcholine receptors at the neuromuscular endplate by circulating autoantibodies, followed by local complement activation and destruction of

13 acetylcholine receptors. The causes of the inflammatory muscle diseases dermatomyositis, polymyositis and inclusion body myositis (IBM) are unknown, but immune mechanisms are strongly implicated. Although clinically and immunopathologically distinct, these diseases share three dominant histological features: inflammation, fibrosis and loss of muscle fibers.
Sarcoidosis is a multisystem chronic disorder with unknown cause and a worldwide distribution. Neurosarcoidosis is a complication of sarcoidosis involving inflammation and abnormal deposits in the tissues of the nervous system.
Sudden, transient facial palsy is common with involvement of cranial nerve VII. Other 1o manifestations include aseptic meningitis, hydrocephalus, parenchymatous disease of the central nervous system, peripheral neuropathy and myopathy. Intracranial sarcoid may mimic various forms of meningitis, including carcinomatous and intracranial mass lesions such as meningioma, lymphoma and glioma, based on neuroradiological imaging.
Vascular dementia (VaD) is the general term for dementia caused by organic lesions of vascular origin, such as cerebral infarction, intracerebral hemorrhage or ischemic changes in subcortical white matter. It is the most frequent cause of dementia after Alzheimer's dementia accounting for about 20% of cases and 50% in subjects over 80 years. An inflammatory component has been indicated in a variety of underlying diseases under the umbrella of VaD.
Cerebral vasospasm is delayed onset cerebral artery narrowing in response to blood clots left in the subarachnoid space after spontaneous aneurysmal subarachnoid hemorrhage (SAH). It is angiographically characterized as the persistent luminal narrowing of the major extraparenchymal cerebral arteries and affects the cerebral microcirculation and causes decreased cerebral blood flow (CBF) and delayed ischemic neurological deficits. Production of pro-inflammatory cytokines in the cerebrospinal fluid following SAH has also been demonstrated.
Duchenne muscular dystrophy (DMD) is one of the most common, inherited, lethal disorders in childhood. It is an X-linked neuromuscular disease that affects 1 in 3500 males. Progressive muscle weakness begins between 2 and 5 years of age and ultimately leads to premature death by respiratory or cardiac failure during the middle to late twenties. DMD patients lack the protein dystrophin which is an essential link in the complex of proteins that connect the cytoskeleton to the extracellular matrix.
Evidence suggests that these patients exhibit immune cells similar to those found in inflammatory disease such as polymyositis. Current research further indicates that T cells may play a role in the pathology of dystrophin deficiency as well as an autoimmune component.

14 PCT/US2010/001785 Multiple sclerosis, an autoimmune disease of the central nervous system expresses an inflammatory component that is treated with corticosteroids to reduce inflammation.
As noted previously, ischemic stroke is caused by a blockage in a blood vessel that stops the flow of blood and deprives the surrounding brain tissue of oxygen.
Within seconds to minutes of the loss of perfusion to a portion of the brain, an ischemic cascade is initiated. Allowed to progress, it will cause a central area of irreversible infarction surrounded by an area of potentially reversible ischemic penumbra.
Metabolic aberrations create an intracellular gradient responsible for intracellular 1o accumulation of water (cytotoxic edema). This is followed by the formation of pro-inflammatory cytokines and other factors that, in turn, cause further inflammation and microcirculatory compromise resulting in vasogenic edema. In addition, there is evidence indicating that the vascular endothelium plays a major role in the regulation of blood flow and is of importance in connection with cardiovascular disorders including inflammatory diseases. A dysfunctional endothelium may be a contributory factor in the demise of the ischemic penumbra.
Edema is a condition characterized by abnormally large fluid volume in the circulatory system or in tissues between the body's cells (interstitial spaces) which can cause mild to severe swelling in one or more parts of the body. Factors that can upset the balance of fluid in the body to cause edema, including: immobility of the lower limbs, medications (steroids, hormone replacements, non-steroidal anti-inflammatory drugs (NSAIDs), intake of salt, menstruation and pregnancy. Medical conditions that may cause edema include: heart failure, kidney disease, thyroid or liver disease, malnutrition, thrombosis, infection, lymphedema and solid tumors. Symptoms vary depending on the cause of edema. In general, weight gain, puffy eyelids, and swelling of the legs may occur as a result of excess fluid volume. Pulse rate and blood pressure may be elevated. Edema-related conditions include traumatic brain injury, which is associated with a variety of physiological and cellular phenomena such as ischemia, increased permeability of the blood-brain barrier, necrosis and motor and memory 3o dysfunction. Ischemia caused by the initial brain injury induces a cascade of secondary events which ultimately lead to cellular death. Experimental models for closed head injury have demonstrated induction of pro-inflammatory cytokine release which in conjunction with damage to endothelial cells results in disruption of the blood brain barrier integrity.
Spinal cord injury initiates a cascade of biochemical and cellular events that includes an inflammatory immune system response. Immediately after the injury, a major reduction in blood flow to the site occurs. Cells that line the still-intact blood vessels in the spinal cord begin to swell, which continues to reduce blood flow to the injured area. Influx of fluid and immune cells (neutrophils, T cells, macrophages and monocytes) past the compromised blood brain barriers causes inflammation, which is exacerbated by pro-inflammatory cytokine release by a variety of neuroglial cells and 5 astrocytes furthering damage to the injured spinal cord.
Soft tissue injury is an acute connective tissue injury that may involve muscle, ligament, tendon, capsular and cartilaginous structures. In a sprain, strain, bruise or crush, the local network of blood vessels is damaged, and the oxygenated blood can no longer reach the affected tissue, resulting in cellular damage. Increases in temperature, 1o redness, pain and swelling (localized edema) characterize the initial inflammatory phase. Inflammatory swelling starts to develop approximately two hours after the injury and may last for days or weeks. Immediate management includes control of the acute inflammatory response.
A variety of imaging techniques are available to assess the degree of edema

15 surrounding an infarct site and blood flow to the ischemic penumbra in ischemic brain stroke patients. Examples include: Computerized Axial Tomography (CT scan), Doppler sonography, and Magnetic Resonance Imaging. At present, the most common method of assessing endothelium-mediated vasorelaxation is brachial arterial (BA) imaging, which involves taking high resolution ultrasound images to determine the diameter of the BA before and after several minutes of arterial occlusion. The change in arterial diameter is a measure of flow-mediated vasorelaxation (FMVR).
Other methods of vasorelaxation measurement include inducing an artificial pulse at the superficial radial artery via a linear actuator. An ultrasonic Doppler stethoscope detects the pulse 10-20 cm upstream from the initial pulse. The delay between pulse application and detection provides the pulse transit time (PTT).
PTT is measured before and after five minutes of BA occlusion and reactive hyperemia.
As the blood flow increases after occlusion, the endothelial cells that line the inner wall of the artery sense the increased friction and chemical composition of the blood and release relaxing agents into the artery's smooth muscle. The healthier the vascular system, the 3o better the endothelial layer functions and the greater the difference will be between the pre-and post-occlusion measurements.
Measures of patient inflammation may include physical assessment of joint stiffness, elevated temperature and reported pain. Laboratory measures of inflammation may include elevation in leukocyte count including differential, coagulation system measurement, inflammatory cytokine (including IL-6 and IL-8) elevation, and increases in C-reactive protein (including high sensitivity CRP) and procalcitonin levels.

16 PCT/US2010/001785 APOPTOSIS
Apoptosis specifically refers to an energy-dependent, asynchronous, genetically controlled process by which unnecessary or damaged single cells self-destruct when apoptosis genes are activated (Martin, SJ 1993; Earnshaw, WC 1995). There are three distinct phases of apoptosis. Initially, the cell shrinks and detaches from neighboring cells. The nucleus is broken down with changes in DNA including strand breakage (karyorhexis) and condensation of nuclear chromatin (pyknosis). In the second phase, nuclear fragments and organelles condense and are ultimately packaged in membrane-bound vesicles, exocytosed and ingested by surrounding cells. In final phase, membrane integrity is finally lost and permeability to dyes (i.e. trypan blue) occurs. The absence of inflammation differentiates apoptosis from necrosis when phagocytized by macrophages and epithelial cells (Kam, PCA 2000).
In contrast, necrotic cell death is a pathological process caused by overwhelming noxious stimuli (Lennon, SV 1991). Synchronously occurring in multiple cells, it is characterized by cell swelling or "oncosis," resulting in cytoplasmic and nuclear swelling and an early loss of membrane integrity. Bleb formation (blister-like, fluid filled structures) of the plasma membrane occurs, in which ultimate rupture may occur causing an influx of neutrophils and macrophages in the surrounding tissue, and leading to generalized inflammation (Majno, G 1995).
Four main groups of stimuli for apoptosis have been reported: ionizing radiation and alkylating anticancer drugs causing DNA damage, receptor mechanism modulation (i.e. glucocorticoids, tumor necrosis factor-a, nerve growth factor or interleukin-3), enhancers of apoptotic pathways (i.e. phosphatases and kinase inhibitors), and agents that cause direct cell membrane damage and include heat, ultraviolet light and oxidizing agents (i.e. superoxide anions, hydroxyl radicals and hydrogen peroxide) (Kam, PCA
2000).
In addition to the oxidizing agents, many chemical and physical treatments capable of inducing apoptosis are also known to evoke oxidative stress (Buttke, M
1994, Chandra, J 2000). Ionizing and ultraviolet radiation both generate reactive oxygen intermediates such as hydrogen peroxide and hydroxyl free radicals. Low doses of hydrogen peroxide (10-100 pM) induces apoptosis in a number of cell types directly establishing oxidative stress as a mediator of apoptosis. However, high doses of this oxidant induce necrosis, consistent with the concept that the severity of the insult determines the form of cell death (apoptosis vs. necrosis) that occurs. A free radical is not a prerequisite for inducing apoptosis; doxorubucin, cisplatin and ether-linked lipids are anti-neoplastics that induce apoptosis and oxidative damage.

17 Alternatively, oxidative stress can be induced by decreasing the ability of a cell to scavenge or quench reactive oxygen intermediates (ROI ) (Buttke, M 1994).
Drugs (i.e.
butathionine sulfoxamine) that reduce intracellular glutathione (GSH) render cells more susceptible to oxidative stress-induced apoptosis. Cell studies report a direct relationship between extracellular catalase levels and sensitivity to hydrogen peroxide-induced apoptosis. Apoptosis induced through tumor necrosis factor-a stimulation has been demonstrated to be associated with an increase in intracellular ROI.
However, this apoptosis has been inhibited by the addition of a number of antioxidants;
thioredoxin, a free radical scavenger, and N-acetylcysteine, an antioxidant and GSH
precursor.
There is growing evidence that apoptotic neutrophils have an active role to play in the regulation and resolution of inflammation following phagocytosis by macrophages and dendritic cells. A hallmark of phagocytic removal of necrotic neutrophils by macrophages is an inflammatory response including the release of proinflammatory cytokines (Vignola, AM 1998, Beutler, B 1988, Moss, ST 2000, Fadok VA, 2001).
In contrast, apoptotic neutrophil clearance is not accompanied by an inflammatory response; phagocytosis of these apoptotic cells has been shown to inhibit macrophage production of pro-inflammatory cytokines (GM-CSF, IL-10, IL-8, TNF-a, TxB2, and LTC4) with a concomitant activation of anti-inflammatory cytokine production (TGF-(31, PGE2 and PAF)(Fadok, VA 1988, Cvetanovic, M 2004). This phenomenon of suppression of proinflammatory cytokine production by macrophages has been extended to include phagocytosis of apoptotic lymphocytes (Fadok, VA 2001).
In addition to macrophages, down regulation of proinflammatory cytokine release in response to apoptotic cells has also been demonstrated by non-phagocytizing cells including human fibroblasts, smooth muscle, vascular endothelial, neuronal and mammary epithelial cells (Fadok, VA 1988, 2000; McDonald, PP 1999, Cvetanovic M, 2006). Apoptotic neutrophils in contact with activated monocytes elicit an immunosuppressive cytokine response, with enhanced IL-10 and TGF-R production and only minimal TNF-a and IL-1P cytokine production (Byrne, A 2002). Byrne et al.
concluded that the interaction between activated monocytes and apoptotic neutrophils may create a unique response, which changes an activated monocyte from being a promoter of the inflammatory cascade into a cell primed to deactivate itself and other cellular targets.
Techniques to identify and quantify apoptosis, and distinguish this event from necrosis, may include staining with nuclear stains allowing visualization of nuclear chromatin clumping (i.e. Hoeschst 33258 and acridine orange) (Earnshaw, WC
1995).
Accurate identification of apoptosis is achieved with methods that specifically target the

18 characteristic DNA cleavages. Agarose gel electrophoresis of extracted DNA
fragments yields a characteristic 'ladder' pattern which can be used as a marker for apoptosis (Bortner, CD 1995). A lesser extent of DNA degradation produces hexameric structures called 'rosettes' where necrotic cells leave a nondescript smear (Pritchard, DM 1996). Terminal transferase deoxyuridine nick-end labeling of DNA break points (TUNEL method), which labels uridine residues of the nuclear DNA fragments, can also be used to quantify apoptosis (Gavrieli, Y 1992).
Several signature events in the process of apoptosis may also be quantified by flow cytometry. These include dissipation of the mitochondrial membrane potential 1o which is an early apoptotic event, externalization of phosphotidylserine through capture with annexin V, loss of plasma membrane integrity and nuclear chromatin condensation (distinguishing live, apoptotic and necrotic cells), and activation of caspase enzymes (early stage feature of apoptosis) (Technical Bulletin - InVitrogen 2004).
Vascular endothelial cells, including human umbilical vein endothelial cells (HUVECs), are known to release potent vasodilators, including nitric oxide (NO) and prostacyclins. Treatment of HUVECs with ozonated serum, an oxidative stressor, results in a significant and steady increase in NO production. Moreover, during twenty-four (24) hour HUVEC incubation with ozonated serum, inhibition of E-selectin release (a proinflammatory mediator) and no effect on endothelin-1 production (a potent vasoconstrictor) has been reported (Valacchi, G 2000). Valacchi et al. has suggested that reinfusion of ozonated blood into patients, by enhancing release of NO, may induce vasodilation in ischemic areas and reduce hypoxia.
C-Reactive Protein (CRP) CRP is a product of inflammation the synthesis of which by the liver is stimulated by cytokines in response to an inflammatory stimulus. CRP activates the classic complement pathway and participates in the opsonization of ligands for phagocytosis.
Initially suggested as solely a biomarker and powerful predictor of cardiovascular risk, CRP now appears to be a mediator of atherogenesis. CRP has a direct effect on promoting atherosclerotic processes and endothelial cell activation. CRP
potently down 3o regulates endothelial nitric oxide synthase (eNOS) transcription and destabilizes eNOS
mRNA, which decreases both basal and stimulated nitric oxide (NO) release.
In a synchronous fashion, CRP has been shown to stimulate endothelin-1 (potent vasoconstrictor) and interleukin-6 release (pro-inflammatory cytokine), upregulate adhesion molecules, and stimulate monocyte chemotactic protein-1 while facilitating macrophage LDL uptake. More recently, CRP has been shown to facilitate endothelial cell apoptosis and inhibit angiogenesis, as well as potentially upregulate

19 PCT/US2010/001785 nuclear factor kappa- B, a key nuclear factor that facilitates the transcription of numerous pro-atherosclerotic genes.
The direct pro-atherogenic effects of CRP extend beyond the endothelium to the vascular smooth muscle where it directly upregulates angiotensin type 1 receptors and stimulates vascular smooth muscle migration, proliferation, neointimal formation and reactive oxygen species production. CRP has several deleterious effects (e.g., reduced survival, differentiation, function, apoptosis, and endothelial progenitor cell-eNOS
mRNA expression) on endothelial progenitor cells which are important in neovascularization including induction of blood flow recovery in ischemic limbs and 1o increase in myocardial viability after infarction.
A variety of imaging techniques are available to assess the degree of edema surrounding the infarct site and blood flow to the ischemic penumbra in ischemic brain stroke patients. Examples of such imaging techniques are discussed below.
Emergent non-contrast head CT scanning is mandatory for rapidly distinguishing ischemic from hemorrhagic infarction and for defining the anatomic distribution of stroke. Most patients who have had onset of ischemic stroke symptoms within 6 hours initially will have normal findings on CT scan. After 6-12 hours, sufficient edema is recruited into the stroke area to produce a regional hypodensity on CT scan. A
large hypodense area present on CT scan within the first 3 hours of symptom onset should prompt careful re-questioning regarding the time of stroke symptom onset.
In Transcranial Doppler TCD, a probe is placed over areas on the head to detect blood velocity and pressure in certain arteries at various depths in the brain. In the early hours after occlusive stroke, TCD allows the assessment of the location and extent of occlusions or atheromatous plaques in extracranial carotid and large intracranial vessels, including the middle cerebral and basilar arteries.
Despite initial screening by CT to distinguish ischemic from hemorrhagic stroke, MRI has demonstrated greater accuracy in the identification of the acute infarction and greater predictive accuracy in the degree of lesion volume of the ischemic penumbra.
One method of MRI analysis, diffusion weighted imaging (DWI), reflects the microscopic random motion of water molecules and is highly sensitive to early changes immediately following stroke onset.
For example, in the hyperacute phase of the ischemic stroke (0-24 hr), MRI is able to detect ischemic changes within minutes of onset. A few hours after stroke onset, MRI analysis can detect early signature events ascribed to cytotoxic edema.
After 8 hours, MRI signals are interpreted as to discern changes associated with cytotoxic and vasogenic edema. Enhanced sensitivity to subtle changes in the acute (1-7 days), subacute (7-21 days) and chronic phases (>21 days) of the ischemic stroke process by

20 PCT/US2010/001785 MRI has led to its increased use in the diagnosis and management of acute ischemic stroke.
Perfusion-weighted imaging (PWI) is an MRI technique that yields information about the perfusion status of the brain. It can be used to estimate cerebral blood volume. Coupled with arterial input the relative cerebral blood flow can be calculated.
DWI and PWI together have been shown to be highly sensitive to the early phases (up to 48 hours) after the onset of stroke. In conjunction, they provide information about location and extent of infarction within minutes of onset; when performed in series, they can provide information about the pattern of evolution of the ischemic lesion.
The physical symptoms of an acute ischemic stroke provide objective evidence of brain ischemia, including the initial infarction and resulting edema, due to an obstructed or reduced blood flow. Contributory factors may include vascular inflammation and a dysfunctional endothelium. Some of the more common symptoms of stroke include loss of (or abnormal) sensations in an arm, leg or one side of the body, weakness or paralysis of an arm or leg or one side of the body (including asymmetrical facial expressions - facial palsy), partial loss of vision [gaze paresis (slight or partial paralysis) or hemianopia (blindness in one half of the visual field of one or both eyes)] and hearing. Additional physical symptoms include, double vision (diplopia), dizziness (including syncope), slurred speech (dysarthria -difficulty in articulating words, caused by impairment of the muscles used in speech), problems thinking of or saying the right word (aphasia - partial or total loss of the ability to articulate ideas or comprehend spoken or written language), inability to recognize parts of the body (hemi-inattention, extinction or anosognosia - failure to recognize paralysis), and imbalance and falling (including limb ataxia - loss of the ability to coordinate muscular movement).
There are a number of standard instruments that have been designed for patient assessment in stroke. Outcome measures are typically selected on the basis of their reliability, familiarity to the neurologic community, and adaptability for use in patients who have had a stroke. An example of four stroke assessment tools that meet these criteria include the following described methods. The Barthel index (measures of disability/activities of daily living) is a reliable and valid measure of the ability to perform activities of daily living such as eating, bathing, walking, and using the toilet. Patients able to perform all activities with complete independence are given a score of 100. The modified Rankin scale (global disability scale) is a simplified overall assessment of function in which a score of 0 indicates the absence of symptoms and a score of 5, severe disability. The Glasgow outcome scale (level-of-consciousness scale) is a global assessment of function in which a score of 1 indicates a good recovery;
a score

21 PCT/US2010/001785 of 2, moderate disability; a score of 3, severe disability; a score of 4, survival but in a vegetative state; and a score of 5, death.
The National Institutes of Health Stroke Scale (NIHSS; stroke deficit scale), a serial measure of neurologic deficit, is a 42-point scale that quantifies neurologic deficits in 11 categories. For example, a mild facial paralysis is given a score of 1, and complete right hemiplegia with aphasia, gaze deviation, visual-field deficit, dysarthria, and sensory loss is given a score of 25. Normal function without neurologic deficit is scored as zero.
Other scales used to evaluate stroke patients may include: pre-hospital stroke assessment tools [i.e. Cincinnati Stroke Scale and Los Angeles Prehospital Stroke Screen (LAPSS)]; acute assessment scales [i.e. Canadian Neurological Scale (CNS), Glasgow Coma Scale (GCS), Hempispheric Stroke Scale, Hunt & Hess Scale, Mathew Stroke Scale, Mini-Mental State Examination (MMSE), Orgogozo Stroke Scale, Oxfordshire Community Stroke Project Classification (Bamford) and Scandinavian Stroke Scale]; functional assessment tools [i.e. Berg Balance Scale, Stroke Impact Scale (SIS), Stroke Specific Quality of Life Measure (SS-QOL)]; and, outcome assessment tools [i.e. American Heart Association Stroke Outcome Classification (AHA
SOC), Functional Independence Measurement (FIM), and Health Survey SF-36 & SF-12 ].
There are a number of treatments that are currently used to ameliorate the effects of stroke, and to prevent future strokes, including the therapies.
Currently, tissue Plasminogen Activator (tPA) is the only thrombolytic agent (also known as a fibrinolytic or 'clot-busting' drug) approved by the Food and Drug Administration (FDA) for treating acute ischemic stroke. There are two ways to administer tPA, intravenously or intra-arterially directly to the clot site. Despite an increased incidence of intracerebral hemorrhage, an improvement in clinical outcome at three months was found in patients treated with intravenous t-PA within three hours of the onset of acute ischemic stroke.
Aside from the severe restriction that an ischemic stroke patient can only receive tPA within a strict four and one half hour window from incident onset, patients receiving Vitamin-K antagonist therapy (i.e. warfarin), exhibit severely elevated blood pressure or blood sugar, exhibit a low platelet count, suffer from end-stage liver or kidney disorders, or have undergone recent surgery, are precluded from thrombolytic treatment.
Currently, tPA therapy is appropriate for about 5 percent to 10 percent of stroke patients.
Attempts to widen the therapeutic window until six hours for tPA
administration have evidenced no clear benefit of tPA therapy; a time period when a substantial number of patients present for evaluation. Therapeutic failure may have occurred

22 because some patients treated 4.5 to 6 hours after symptom onset have already sustained severe, irreversible brain injury and others have already undergone spontaneous recanalization of their occluded arteries. Treatment of these patients is unlikely to produce beneficial effects and may result in harm secondary to brain hemorrhage.
A majority of patients arrive at the hospital too late to qualify for intervention with tPA or have some other contraindications that effectively prohibit the use of the drug.
An endovascular procedure involving the use of a cork-screw shaped device is the first FDA approved mechanical device for the treatment of ischemic stroke. This device is lo used on the end of a catheter to physically pull out all or part of a clot.
The major limitation to the retriever device is that the clot must be visible and accessible in order for the physician to guide the catheter to the location of the clot. The Penumbra SystemTM, comprising an aspiration platform containing multiple devices that are size-matched to the specific neurovascular anatomy allowing clots to be gently aspirated out of intracranial vessels, was approved by the FDA in 2008 for post stroke revascularization. Cerebral artery size, advanced surgical and imaging techniques, and vessel perforation significantly limit the adoption of these mechanical clot disruption technologies.
Administration of anticoagulants can play a role in preventing ischemic stroke and its recurrence. They are drugs used to prevent clot formation or to prevent a clot that has formed from enlarging. They cannot, however, dissolve clots that already have formed. Anticoagulant drugs fall into three categories: inhibitors of clotting factor synthesis (i.e. warfarin), inhibitors of thrombin, and antiplatelet drugs (i.e. aspirin, Clopidogrel, Eptifibatide, dipyridamole, and Ticlopidine).
In view of the limitations that are presented with currently practiced therapies, new approaches are being sought to reduce the frequency and severity of clinical sequelae secondary to acute ischemic brain stroke.
Historically, ozone has been used as a disinfectant or sterilizing agent in a wide variety of applications. These include fluid-based technologies, such as purification of potable water, sterilization of fluids in the semi-conductor industry, disinfection of wastewater and sewage, and inactivation of pathogens in biological fluids.
Ozone has also been used in the past as a topical medicinal treatment, as a systemic therapeutic and as a treatment of various fluids that were subsequently used to treat a variety of diseases. Specifically, there have been numerous attempts utilizing a variety of ozone-based technologies to treat various medical conditions, including those described herein.

23 Previous technologies were incapable of measuring and differentiating between the amount of ozone that was delivered and the amount of ozone actually absorbed and used. This meant previous medicinal technologies used in patient treatment were incapable of measuring, reporting or differentiating the amount of ozone delivered from the amount of ozone that was actually absorbed by any material being treated.
This problem made regulatory approval as a therapeutic unlikely.
In the treatment of disease conditions, previous technologies were also incapable of measuring, reporting or differentiating the amount of ozone delivered from the amount that was actually absorbed by the fluid and utilized by the patient. The 1o inability to measure the amount of ozone absorbed may result in excessive absorption resulting in unacceptable levels of cellular necrosis in the leukocyte fraction of the treated blood, which, when reinfused, may result in promotion of an inflammatory response. Furthermore, any technology considered to treat disease conditions utilizing blood ex vivo with ozone may have to be able to maintain the biological integrity of the fluid for its subsequent intended therapeutic use.
In addition, early approaches of mixing ozone with fluids employed gas-fluid contacting devices that were engineered with poor mass transfer efficiency of gas to fluids. Later, more efficient gas-fluid contacting devices were developed, but these devices used construction materials that were not ozone inert and therefore, reacted with and absorbed ozone. This resulted in absorption of ozone by the construction materials making it impossible to determine the amount of ozone delivered to and absorbed by the fluid. Furthermore, ozone absorption by construction materials likely caused oxidation and the subsequent release of contaminants or deleterious byproducts of oxidation into the fluid.
Experimental research confirms the problem of ozone absorption by construction materials. An ozone/oxygen admixture at 1200 ppmv was passaged through a commercially available membrane oxygenator. For a period in excess of two hours, a majority of the ozone delivered to the device was absorbed by the construction materials. This data strongly suggests commercially-available membrane gas-fluid contacting devices, made from ozone reactive materials, cannot be used with ozone, and supports the necessity for developing novel ozone-inert gas-fluid contacting devices.
In addition, prior methods do not quantify the amount of ozone that does not react with the biological fluid. The inability to measure residual-ozone has led to inaccurate and imprecise determination of the amount of ozone actually absorbed and utilized by the fluid.

24 PCT/US2010/001785 Prior technologies also include inefficient methods of mixing ozone with fluids, yielding irregular exposure. For example, relatively large amounts of ozone may be exposed to some of the fluid and less to other portions. The result of this inefficient mixing causes a wide variation in the amount of ozone exposed to the fluid.
This wide variation in ozone exposure may cause diverse biochemical events, including unacceptable levels of cellular necrosis in various portions of the fluid leading to untoward and irreproducible results.
Prior techniques also failed to recognize that fluids of varying composition display different absorption phenomena. The range of values for extracellular 1o antioxidants in blood, including ascorbic acid (0.4 - 1.5 mg/dL), uric acid (2.1 - 8.5 mg/dL), bilirubin (0 - 1.0 mg/dL) and Vitamin A (30 - 65 pg/dL) and other oxidizable substrates such as cholesterol (140-240 mg/dL), LDL-cholesterol (100-159 mg/dL), HDL-cholesterol (33-83 mg/dL) and triglycerides (45-200 mg/dL), may alter the amount of ozone necessary to be delivered to the fluid and subsequently absorbed and utilized to achieve a desired clinical effect.

BRIEF SUMMARY OF THE INVENTION
In accordance with the present invention, methods are provided for therapeutic treatment of various disease conditions, including acute ischemic brain stroke, cardiovascular disease, inflammatory disease, and related symptoms and conditions of these diseases. Methods of the present invention generally comprise extracorporeal treatment of blood, blood fractionate, or other biological fluids, taken from a mammalian subject or patient and exposing such fluids to a precise, measured amount of ozone to produce a treated biological fluid that has a quantifiable absorbed dose of ozone. The methods of the invention further include reinfusion of the treated fluid having the quantified absorbed dose of ozone into the mammalian subject to provide and elicit therapeutic effects which treat the disease, condition or symptoms of the disclosed diseases, as well as other diseases.
The methods of the present invention further provide for the manufacture of substances or compositions that are useful in the therapeutic treatment of various disease conditions, including acute ischemic brain stroke, cardiovascular disease, inflammatory disease, and related symptoms and conditions of these diseases.
The methods of the present invention further provide for the use of such substances and compositions in the manufacture of medicaments or other administrable substances for the therapeutic treatment of various disease conditions, including acute ischemic brain stroke, cardiovascular disease, inflammatory disease, and related symptoms and conditions of these diseases.
Treatment of patients who are or have experienced acute ischemic brain stroke produces other therapeutic effects, including measured patient improvement 5 from or in paralysis, motor weakness, loss of sensation, ocular and auditory functions, stroke-free survival, severity of recurrent stroke, cognitive function, verbal communication, re-attainment of independence, and improvement in overall survival.
The present invention is directed to providing methods in treating blood with ozone extracorporeally to generate leukocyte apoptosis, without excessive necrosis, 10 sufficient to reduce edema associated with the ischemic penumbra, increase blood flow to the area surrounding the infarct, which may include ischemic tissue, and the ischemic penumbra, promote relaxation of the vascular endothelium and reduce inflammation once the treated blood is reinfused.
The present invention is further directed to methods of treating blood with 15 ozone extracorporeally which, once reinfused, causes reduction in CRP
sufficient to elicit clinical benefit.
The present invention is further directed to providing methods for the treatment of blood, blood fractionate or other fluid, and the use of this treated blood, blood fractionate or other fluid in the treatment of acute ischemic brain stroke in a 20 mammalian patient by administration to the patient of such treated blood, blood fractionate or other fluid.
The present invention further comprises extracorporeally subjecting an aliquot of a mammalian patient's blood, or the separated cellular fractions of the blood, or mixtures of the separated cells, including platelets, to a measured amount of ozone

25 such that the fluid absorbs a quantifiable absorbed-dose of ozone. On re-introduction of this autologous aliquot to the patient's body, the blood, blood fractionate or other fluid with a quantifiable absorbed-dose of ozone may affect improvement of any condition caused by stroke.
The present invention is further directed to methods for treatment of acute ischemic brain stroke, including a methods of delivery of a measured amount of ozone and subsequent absorption of a quantifiable absorbed-dose of ozone by blood or derivatives thereof extracorporeally, to cause or promote sufficient leukocyte apoptosis necessary to elicit clinical benefit when reinfused autologously into a patient.
The present invention is further directed to methods that induce apoptosis in the leukocyte fraction of blood, or a blood derivative, which has absorbed a quantifiable absorbed-dose of ozone to reduce inflammation when reinfused autologously into a patient suffering from stroke.

26 The present invention is further directed to inducing apoptosis in the leukocyte fraction of blood or blood derivative which has absorbed a quantifiable absorbed-dose of ozone without causing excessive necrosis, to reduce inflammation when reinfused autologously into a patient suffering from stroke.
The present invention is further directed to treating acute ischemic stroke by using blood or a blood derivative which has absorbed a quantifiable absorbed-dose of ozone preventing excessive necrosis which may be pro-inflammatory when reinfused autologously into a patient.
The present invention is directed to providing methods of treatment which 1o reduce inflammation in patients suffering from acute ischemic stroke by a method comprising connecting a subject to a device for withdrawing blood, withdrawing blood and delivering a measured amount of ozone to the blood under conditions which may induce sufficient leukocyte apoptosis without excessive necrosis, wherein the treated blood is subsequently re-infused into the subject.
The methods of the present invention induce sufficient leukocyte apoptosis without excessive necrosis, which may be evaluated by a number of diagnostic methods including light microscopy with nuclear stains, electrophoretic analysis of DNA
fragmentation, TUNEL analysis and multiparameter flow cytometry.
The methods of the present invention induce apoptosis in the leukocyte fraction of blood, or a blood derivative which has absorbed a quantifiable absorbed-dose of ozone to reduce edema in the ischemic penumbra of stroke patients when reinfused autologously.
The present methods are directed to inducing apoptosis in the leukocyte fraction of blood or blood derivative which has absorbed a quantifiable absorbed-dose of ozone to reduce edema in the area surrounding the infarct which may include ischemic tissue and the ischemic penumbra of stroke patients when reinfused autologously.
The methods of the present invention are directed to reducing edema in the ischemic penumbra in patients suffering from acute ischemic stroke and/or reducing 3o edema in the area surrounding the infarct, which may include ischemic tissue and the ischemic penumbra, in patients suffering from acute ischemic stroke, by a method comprising connecting a subject to a device for withdrawing blood, withdrawing blood and delivering a measured amount of ozone to the blood under conditions which may induce sufficient leukocyte apoptosis without excessive necrosis wherein the treated blood is subsequently re-infused into the subject.
The present invention is further directed to inducing apoptosis in the leukocyte fraction of blood or blood derivative which has absorbed a quantifiable

27 PCT/US2010/001785 absorbed-dose of ozone to improve blood flow to the area surrounding the infarct which may include ischemic tissue and the ischemic penumbra of stroke patients when reinfused autologously.
The present invention is further directed to inducing apoptosis in the leukocyte fraction of blood or blood derivative which has absorbed a quantifiable absorbed-dose of ozone without causing excessive necrosis, to improve blood flow to the area surrounding the infarct which may include ischemic tissue and the ischemic penumbra of stroke patients when reinfused autologously.
The methods of the present invention are further directed to improving blood to flow to the area surrounding the infarct, which may include ischemic tissue and the ischemic penumbra, in patients suffering from acute ischemic stroke, by a method comprising connecting a subject to a device for withdrawing blood, withdrawing blood and delivering a measured amount of ozone to the blood under conditions which may induce sufficient leukocyte apoptosis without excessive necrosis wherein the treated blood is subsequently re-infused into the subject.
The methods of the present invention are directed to inducing apoptosis in the leukocyte fraction of blood or blood derivative which has absorbed a quantifiable absorbed-dose of ozone without causing excessive necrosis, to relax the vascular endothelium of stroke patients when reinfused autologously.
The methods of the present invention are directed to relaxing the vascular endothelium in patients suffering from acute ischemic stroke, and/or reducing the edema in the ischemic penumbra of acute ischemic brain stroke patients, and/or reducing the edema in the area surrounding the infarct which may include ischemic tissue and the ischemic penumbra of acute ischemic brain stroke patients, by a method comprising connecting a subject to a device for withdrawing blood, withdrawing blood and delivering a measured amount of ozone to the blood under conditions which may induce sufficient leukocyte apoptosis without excessive necrosis, and under conditions which may maintain the biological integrity of the blood. The treated blood is subsequently re-infused into the subject.
The present invention comprises method that reduce the edema in the ischemic penumbra of acute ischemic brain stroke patients, and/or reduce the edema in the area surrounding the infarct which may include ischemic tissue and the ischemic penumbra of acute ischemic brain stroke patients, as evaluated by a variety of diagnostic tools including MRI and Doppler imaging techniques.
The present invention is directed to reducing inflammation to cause a reduction in edema in the ischemic penumbra of acute ischemic brain stroke patients, and to reduce inflammation causing a reduction in edema in the area surrounding the

28 PCT/US2010/001785 infarct, which may include ischemic tissue and the ischemic penumbra of acute ischemic brain stroke patients.
The effects of blood, blood fractionate or other fluid which has absorbed a quantifiable absorbed-dose of ozone, when re-infused into a mammalian patient's body may include effects that reduce inflammation.
Reduction of inflammation may occur though a reduction in pro-inflammatory cytokines (e.g. interferon-gamma, TNF-gamma, IL-6 and IL-12) and/or an increase in anti-inflammatory cytokines (e.g. interleukin-4 and IL-10) released by immunomodulatory T cells. The effect of reducing inflammation may result in any 1o number of clinical benefits including improving endothelial function including endothelial cellular repair or replacement. These results may lead to a reduction in edema in the ischemic penumbra.
Reduction of inflammation may occur though a reduction in pro-inflammatory cytokines (e.g. interferon-gamma, TNF-gamma, IL-6 and IL-12) and/or an increase in anti-inflammatory cytokines (e.g. interleukin-4 and IL-10) released by immunomodulatory T cells. The effect of reducing inflammation may result in any number of clinical benefits including improving endothelial function including endothelial cellular repair or replacement. These results may lead to a reduction in edema in the area surrounding the infarct which may include ischemic tissue and the ischemic penumbra.
The methods of the present invention provide for treatment of acute ischemic brain stroke, including a method of delivery of a measured amount of ozone and subsequent absorption of a quantifiable absorbed-dose of ozone by blood or derivatives thereof extracorporeally which, when reinfused autologously into a patient, may cause a reduction in CRP.
The methods of the present invention provide for treatment of acute ischemic brain stroke, including a method of delivery of a measured amount of ozone and subsequent absorption of a quantifiable absorbed-dose of ozone by blood or derivatives thereof extracorporeally which, when reinfused autologously into a patient, may cause a 3o reduction in CRP sufficient to elicit clinical benefit. Clinical benefits may include reduction of inflammation, increasing blood flow through vasodilation and increasing blood flow through neovascularization.
The methods of the present invention are directed to increasing blood flow to the area surrounding the infarct which may include ischemic tissue and the ischemic penumbra of acute ischemic brain stroke patients, the method comprising connecting a subject to a device for withdrawing blood, withdrawing blood and delivering a measured

29 amount of ozone to the blood under conditions which may maintain the biological integrity of the blood. The treated blood is subsequently re-infused into the subject.
The methods of the present invention are directed to increasing blood flow to the area surrounding the infarct which may include ischemic tissue and the ischemic penumbra of acute ischemic brain stroke patients as evaluated by a variety of diagnostic tools including MRI and Doppler imaging techniques.
The methods of the present invention are further directed to reducing inflammation causing an increase in blood flow to the area surrounding the infarct which may include ischemic tissue and the ischemic penumbra of acute ischemic brain stroke patients.
The methods of the present invention further provide for a treatment of blood, blood fractionate or other fluid, and the administration of this treated fluid in the treatment of acute ischemic brain stroke is to relax the vascular endothelium.
The present method is based upon extracorporeally subjecting an aliquot of a mammalian patient's blood, or the separated cellular fractions of the blood, or mixtures of the separated cells, including platelets, to a measured amount of ozone such that it absorbs a quantifiable absorbed-dose of ozone. On re-introduction of this autologous aliquot to the patient's body, the blood, blood fractionate or other fluid with a quantifiable absorbed-dose of ozone may have certain beneficial effects. One of these effects is to relax the endothelium. This relaxation may result from an increase in vasodilation (i.e. promotion of vasodilators or inhibition of vasoconstrictors) improving endothelial function including endothelial cellular repair or replacement, and improving blood flow yielding enhanced oxygenation. Thus, the methods of the present invention are further directed to promoting relaxation in the vascular endothelium, thereby causing a reduction in edema in the ischemic penumbra of acute ischemic brain stroke patients. Relaxation of the vascular endothelium may result from a reduction in edema in the area surrounding the infarct, which may include ischemic tissue and the ischemic penumbra of acute ischemic brain stroke patients.
The effects of blood, blood fractionate or other fluid which has absorbed a 3o quantifiable absorbed-dose of ozone, when re-infused into a mammalian patient's body may include a relaxation of the vascular endothelium. These effects may result in an increase in vasodilation (i.e. promotion of vasodilators or inhibition of vasoconstrictors), improving endothelial function including endothelial cellular repair or replacement.
These results may lead to a reduction in the edema in the ischemic penumbra of patients suffering from acute ischemic brain stroke, as well as in the area surrounding the infarct, which may include ischemic tissue and the ischemic penumbra of patients suffering from acute ischemic brain stroke.

The methods of the present invention are further directed to relaxing the vascular endothelium causing a reduction in edema in the ischemic penumbra of acute ischemic brain stroke patients, and/or relaxing the vascular endothelium causing a reduction in edema in the area surrounding the infarct which may include ischemic 5 tissue and the ischemic penumbra of acute ischemic brain stroke patients, as evaluated by a variety of diagnostic tools including MRI and Doppler imaging techniques.
The methods of the present invention are further directed to relaxing the vascular endothelium to cause an increase in blood flow to the area surrounding the infarct which may include ischemic tissue and the ischemic penumbra of acute ischemic 1o brain stroke patients.
The effects of blood, blood fractionate or other fluid which has absorbed a quantifiable absorbed-dose of ozone, when re-infused into a mammalian patient's body may include a relaxation of the vascular endothelium. These effects may result in an increase in vasodilation (i.e. promotion of vasodilators or inhibition of vasoconstrictors), 15 improving endothelial function including endothelial cellular repair or replacement.
These results may lead to an increase in blood flow to the area surrounding the infarct which may include ischemic tissue and the ischemic penumbra in patients suffering from acute ischemic brain stroke.
The methods of the present invention are, therefore, directed to relaxing the 20 vascular endothelium causing an increase in blood flow to the area surrounding the infarct which may include ischemic tissue and the ischemic penumbra of acute ischemic brain stroke patients as evaluated by a variety of diagnostic tools including MRI and Doppler imaging techniques.
The methods of the present invention are directed to the re-introduction of an 25 autologous aliquot of a mammalian patient's blood, blood fractionate or other fluid which has absorbed a quantifiable absorbed-dose of ozone may be through a variety of routes including intravenous, intramuscular and subcutaneous, or any combination thereof.
The methods of the present invention provide a treatment of acute ischemic 3o brain stroke in a mammalian patient by treating blood by a discontinuous flow method.
The method comprising connecting a subject to a device for withdrawing blood, withdrawing blood and delivering a measured amount of ozone to the blood under conditions which may maintain the biological integrity of the blood wherein the treated blood is subsequently re-infused into the patient.
The methods of the present invention further provide a treatment of acute ischemic brain stroke in a mammalian patient by treating blood, or a fraction thereof, including plasma or serum, by a discontinuous flow method. The method comprising connecting a subject to a device for withdrawing blood, withdrawing blood containing blood cells from the subject, separating a non-cellular fraction from the blood and delivering a measured amount of ozone to the fraction, under conditions which may maintain the biological integrity of the blood fraction. The treated fraction is subsequently recombined with the blood cells and re-infused into the subject.
The methods of the present invention provide a treatment that causes improvement in paralysis in patients suffering from acute ischemic brain stroke, that causes improvement in paralysis in patients suffering from acute ischemic brain stroke as evaluated by stroke scale assessment tools, that causes improvement in paralysis in patients suffering from acute ischemic brain stroke as evaluated by stroke scale assessment tools, and wherein clinical effectiveness is measured through statistical comparison with untreated stroke patients.
The methods of the present invention further provide a treatment that causes improvement in motor weakness in patients suffering from acute ischemic brain stroke, as may be evaluated by stroke scale assessment tools and/or wherein clinical effectiveness is measured through statistical comparison with untreated stroke patients.
The methods of the present invention further provide a treatment that causes improvement in loss of sensation in patients suffering from acute ischemic brain stroke, as may be evaluated by stroke scale assessment tools, and wherein clinical effectiveness may be measured through statistical comparison with untreated stroke patients.
The methods of the present invention provide a treatment that causes improvement in ocular and auditory functions in patients suffering from acute ischemic brain stroke, as may be evaluated by stroke scale assessment tools, and wherein clinical effectiveness may be measured through statistical comparison with untreated stroke patients.
The methods of the present invention provide a treatment that causes reduction in the severity of recurrent stroke in patients suffering from acute ischemic brain stroke, as may be evaluated by stroke scale assessment tools, and wherein clinical effectiveness may be measured through statistical comparison with untreated stroke patients.
The methods of the present invention further provide a treatment that causes improvement in cognitive function in patients suffering from acute ischemic brain stroke, as may be evaluated by stroke scale assessment tools, and wherein clinical effectiveness may be measured through statistical comparison with untreated stroke patients.

The methods of the present invention provide a treatment that causes improvement in verbal communication in patients suffering from acute ischemic brain stroke, as may be evaluated by stroke scale assessment tools, and wherein clinical effectiveness may be measured through statistical comparison with untreated stroke patients.
The methods of the present invention may further provide a treatment that causes re-attainment of independence in patients suffering from acute ischemic brain stroke as evaluated by stroke scale assessment tools and wherein clinical effectiveness is measured through statistical comparison with untreated stroke patients.
The methods of the present invention may further provide a treatment that causes improvement in the rate of stroke-free and/or overall survival in patients suffering from acute ischemic brain stroke, as evaluated through statistical comparison with untreated stroke patients, as may be evaluated through statistical comparison with untreated stroke patients.
The methods of the present invention may further provide a treatment of acute ischemic brain stroke wherein there is a shift from a pro-inflammatory state to an .anti-inflammatory state of the vascular endothelium.
The methods of the present invention may further provide for relaxation of the vascular endothelium through the release of anti-inflammatory cytokines including interleukin-4 and interleukin-10 and TGF-gamma. The methods of the present invention may further provide for the relaxation of the vascular endothelium through the inhibition of pro-inflammatory cytokines including interferon-gamma, TNF-gamma, IL-1, IL-6 and IL-12.
The methods of the present invention may further provide a treatment for acute ischemic brain stroke by inhibiting vasoconstriction of the vascular endothelium.
The methods of the present invention may further provide a treatment of acute ischemic brain stroke by promoting vasodilation of the vascular endothelium.
The methods of the present invention may further provide a treatment for acute ischemic brain stroke by causing the release of endothelium-derived relaxing factor, nitric oxide, prostacyclin or other related vasodilatory compounds.
The methods of the present invention may provide a treatment for acute ischemic brain stroke wherein there is an increase in oxygen delivered to the ischemic area. The methods of the present invention may further provide a treatment of acute ischemic brain stroke by promoting angiogenesis in the ischemic area.

BRIEF DESCRIPTION OF DRAWING

To further clarify the present invention, treatment systems of the present invention using an ozone delivery system are illustrated in the appended drawing, which schematically illustrate what is currently considered the best mode for carrying out the invention;
FIG. 1 illustrates, in a schematic diagram, alternate methods of carrying out treatment of a fluid from a patient, comprising a continuous loop format and, alternatively, a discontinuous flow method.

DETAILED DESCRIPTION OF THE EMBODIMENTS OF THE INVENTION
lo Definitions As used herein, "comprising," "including," "containing," "characterized by,"
and grammatical equivalents thereof are inclusive or open-ended terms that do not exclude additional, unrecited elements or method steps, but also include the more restrictive terms "consisting of' and "consisting essentially of."
As used herein, and in the appended claims, the singular forms, for example, "a", "an", and "the," include the plural, unless the context clearly dictates otherwise. For example, reference to "a gas-fluid contacting device" includes a plurality of such gas-fluid contacting devices, and reference, for example, to a "protein" is a reference to a plurality of similar proteins, and equivalents thereof.
An "ozone/oxygen admixture" refers to a concentration of ozone in an oxygen carrier gas. Various units of concentration utilized by those skilled in the art include micrograms of ozone per milliliter of oxygen, parts (ozone) per million (oxygen) by weight ('ppm') and parts per million by volume ('ppmv'). As a unit of concentration for ozone in oxygen, ppmv is defined as the molar ratio between ozone and oxygen.
One ppmv ozone is equal to 0.00214 micrograms of ozone per milliliter of oxygen.
Additionally, one ppm ozone equals 0.00143 micrograms of ozone per milliliter of oxygen. In terms of percentage ozone by weight, 1% ozone equals 14.3 micrograms of ozone per milliliter of oxygen. All units of concentration and their equivalents are calculated at standard temperature and pressure (i.e. 25 C at 1 atmosphere).
"Delivered-ozone" is the amount of ozone contained within a volume of an ozone/oxygen admixture that is delivered to a fluid, and is synonymous with the delivery of a measured amount of ozone.
"Absorbed-ozone" is the amount of delivered-ozone that is actually absorbed and utilized by an amount of fluid, and is synonymous with a quantifiable absorbed dose of ozone.

"Residual-ozone" is the amount of delivered-ozone that is not absorbed such that:
Residual-ozone = delivered ozone - absorbed-dose of ozone An "interface" is defined as the contact between a fluid and an ozone/oxygen admixture.
"Interface-time" is defined as the time that a fluid resides within a gas-fluid contacting device and is interfaced with an ozone/oxygen admixture.
"Interface surface area" is defined as the dimensions of the surface within a gas-fluid contacting device over which a fluid flows and contacts an ozone/oxygen lo admixture.
"Elapsed-time" is the time that a fluid circulates throughout an ozone delivery system, including passage through one or more gas-fluid contacting devices, connecting tubing and an optional reservoir.
"Ozone-inert materials" are defined as construction materials that do not react with ozone in a manner that introduces contaminants or deleterious byproducts of oxidation of the construction materials into a fluid, and materials that do not absorb ozone.
"Non-reactive" is defined as not readily interacting with other elements or compounds to form new chemical compounds.
"Measured-data" is defined as information collected from various measuring components (such as an inlet ozone concentration monitor, exit ozone concentration monitor, gas flow meter, fluid pump, data acquisition device, humidity sensor, temperature sensor, pressure sensor, absorbed oxygen sensor) throughout the system.
"Calculated-data" is defined as the mathematical treatment of measured-data by a data acquisition device.
"Absorption of ozone by a biological fluid" is defined as the phenomenon wherein ozone reacts with the fluid being treated by a variety of mechanisms, including oxidation. Regardless of the mechanism involved, the reaction occurs instantaneously, and the products of this reaction include oxidative products, of which lipid peroxides are an example.
A "biological fluid" is defined as a composition originating from a biological organism of any type. Examples of biological fluids include blood, blood products and other fluids, such as saliva, urine, feces, semen, milk, tissue, tissue samples, homogenized tissue samples, gelatin and any other substance having its origin in a biological organism. Biological fluids may also include synthetic materials incorporating a substance having its origin in a biological organism, such as a vaccine preparation containing alum and a virus (the virus being the substance having its origin in a biological organism), cell culture media, cell cultures, viral cultures, and other cultures derived from a biological organism.
A "blood product" is defined as including blood fractionates and therapeutic protein compositions containing proteins derived from blood. Fluids containing 5 biologically active proteins other than those derived from blood may also be treated by the method.
"In vivo" use of a material or compound is defined as the introduction of a material or compound into a living human, mammal or vertebrate.
"In vitro" use of a material or compound is defined as the use of the material or 1o compound outside a living human, mammal or vertebrate, where neither the material nor compound is intended for reintroduction into a living human, mammal or vertebrate.
An example of an in vitro use would be the analysis of a component of a blood sample using laboratory equipment.
"Ex vivo" use of a process is defined as using a process for treatment of a 15 biological material such as a blood product outside of a living human, mammal, or vertebrate. For example, removing blood from a human and subjecting that blood to a method to treat a cardiovascular disease is defined as an ex vivo use of that method if the blood is intended for reintroduction into that human or another human.
Reintroduction of the human blood into that human or another human would be an in 20 vivo use of the blood, as opposed to an ex vivo use of the method.
"Extracorporeal" is defined as a state wherein blood or blood fractionate is treated outside (ex vivo) of the body, for example, in the delivery of a measured amount of ozone to a sample of patient's blood.
"Synthetic media" is defined as an aqueous synthetic blood or blood product 25 storage media.
A "pharmaceutically-acceptable carrier" or "pharmaceutically-acceptable vehicle" is defined as any liquid including water, saline, a gel, salve, solvent, diluent, fluid ointment base, liposome, micelle or giant micelle, which is suitable for use in contact with a living animal or human tissue without causing adverse physiological 3o responses, and which does not interact with the other components of the composition in a deleterious manner.
"Biologically active" is defined as capable of effecting a change in the living organism or component thereof.
The "biological integrity of a biological fluid" is a quality or state of a fluid that, 35 subsequent to the method of treating for cardiovascular diseases described herein, sufficiently maintains its functionality upon re-infusion into a mammalian patient.

"Cardiovascular diseases" are defined as those diseases which may include atherosclerosis, arteriosclerosis, peripheral arterial occlusive disease, cerebrovascular disease, hypertension, Raynaud's disease, dyslipidemia and congestive heart failure.
"Edema" is defined as a condition of abnormally large fluid volume in the circulatory system or in tissues between the body's cells (interstitial spaces).
"Inflammatory diseases" are defined as diseases that are characterized by activation of the immune system to abnormal levels characterized by inflamed tissue, characterized by pain, swelling, redness and heat.
"Neurodegenerative diseases" are defined as disorders caused by the deterioration of certain nerve cells causing them to function abnormally, eventually bringing about their death.
"Autoimmune diseases" are defined as diseases believed to be caused by the failure of the immune system to discriminate between antigens of foreign invading organisms (non-self) and tissues native to its own body (self).
"Alloimmune diseases" are defined as diseases that result from an immune response against or by foreign, transplanted tissue.
"C-reactive protein" is defined as a liver-synthesized, acute phase reactant protein regarded as a marker of acute inflammation capable of activating the classical compliment pathway and opsonizing ligands for phagocytosis.

The present invention provides methods for therapeutic treatment of acute ischemic brain stroke, cardiovascular diseases, inflammatory diseases, and the conditions and symptoms related to those diseases, mediated by the delivery of a measured amount of ozone to a sample of a patient's blood, blood fractionate or other fluid, extracorporeally, through the use of an ozone delivery system. A
quantifiable absorbed-dose of ozone absorbed by the fluid is subsequently re-infused into the same patient. This autologous blood sample which contains a quantifiable absorbed-dose of ozone may affect reduction in edema, improvement in impaired blood flow, reduction in atherosclerotic plaques, regression in atherosclerotic plaque formation, reduction of the ischemic penumbra, relaxation of the vascular endothelium, reduction-of inflammation, and reduction in lipids and lipid deposits. The method may therefore be useful in the treatment of cardiovascular diseases including atherosclerosis, peripheral arterial occlusive disease, congestive heart failure, hypertension, cerebrovascular disease, dyslipidemia and vasospastic disorders, including Raynaud's disease, in a mammalian patient. Positive treatment outcomes may be measured by various methods of quantification or qualification, and include a measurable reduction in cholesterol, triglycerides and other lipids, reduction in the frequency, severity and duration of episodes of intermittent claudication, reduction in extremity numbness, weakness and loss of movement, improvement in extremity pallor, temperature and pulse, reduction in elevated blood pressure, and weight loss.
The methods of the present invention further provide effect treatment for and improvement in any condition caused by acute ischemic brain stroke including reduction in edema associated with the ischemic penumbra, improvement in impaired blood flow to the area surrounding the infarct which may include ischemic tissue and the ischemic penumbra, relaxation of the vascular endothelium, and reduction of inflammation. Positive treatment outcomes may be measured and may include improvement in paralysis, visual and auditory skills, cognitive function, re-attainment of independence, stroke-free survival, relapse frequency and severity, and overall post-stroke survival.
The methods of the present invention also provide effective treatment of and improvement in reduction of inflammation, relaxation of the vascular endothelium, reduction in edema and increased blood flow which are conditions related to inflammatory disease. Other diseases targeted as potential candidates for therapeutic treatment by the methods disclosed in the present invention include rheumatoid arthritis, multiple sclerosis, systemic lupus erythromatosis (SLE), scleroderma, diabetes, inflammatory bowel disease, psoriasis, pemphigus, atherosclerosis, chronic heart failure, graft versus host reactions including tissue transplant rejection, Alzheimer's disease, ischemic brain stroke, senile dementia, depression, Down's syndrome, Huntington's disease, peripheral neuropathies, spinal cord diseases, neuropathic joint diseases, chronic inflammatory demyelinating disease (CIPD) and neuropathies, including mononeuropathy, polyneuropathy, symmetrical distal sensory neuropathy, cystic fibrosis, neuromuscular junction disorders, myasthenias, Parkinson's disease, traumatic brain injury, spinal cord injury and soft tissue injuries.
Methods of the present invention are directed to providing therapeutic treatment of cardiovascular diseases, inflammatory diseases and acute ischemic brain stroke, and related conditions and symptoms, by providing a method of delivering a measured amount of ozone to, and subsequent absorption of a quantifiable absorbed-dose of ozone by, blood or derivatives thereof extracorporeally, which may cause sufficient leukocyte apoptosis without excessive necrosis necessary to elicit clinical benefit when reinfused autologously into a patient.
The present invention also provides methods for delivery of a measured amount of ozone and subsequent absorption of a quantifiable absorbed-dose of ozone by blood or derivatives thereof, extracorporeally, where, following reinfusion autologously into a patient, may cause a reduction in CRP sufficient to elicit clinical benefit. The disclosed methods may also affect relaxation of vascular endothelium, and may involve release of vasodilators, including nitric oxide and prostacyclins sufficient to elicit clinical benefit.
In one embodiment of the invention, methods are provided for treatment of blood, blood fractionate or other fluid, and use of that treated blood, blood fractionate or other fluid in the treatment of the diseases described herein, and for the manufacture of medicaments in the treatment of the diseases described herein, which may include, for example, atherosclerotic and dysfunctional endothelial associated disorders, relaxation of vascular endothelium and may involve release of vasodilators including nitric oxide and prostacyclins, sufficient to elicit clinical or therapeutic benefit. The medicaments may also be effective in therapeutic improvement in inflammatory disease-related conditions including reduction of inflammation, relaxation of the vascular endothelium, reduction in edema and increased blood flow.
The present methods, in some embodiments, may comprise extracorporeally .15 subjecting an aliquot of a mammalian patient's blood, or the separated cellular fractions of the blood, or mixtures of the separated cells, including platelets, to a measured amount of ozone such that the treated fluid absorbs a quantifiable absorbed-dose of ozone. On reintroduction of this autologous aliquot to the patient's body, the blood, blood fractionate or other fluid with a quantifiable absorbed-dose of ozone provides certain beneficial and therapeutic effects. These effects may result, for example, in the reduction and/or inhibition of atherosclerotic plaque formation, deposition or plaque rupture, and stimulation of the activity of a functionally deficient endothelium, reduction in inflammation and increased blood flow.
The effects of blood, blood fractionate or other fluid that has absorbed a quantifiable absorbed-dose of ozone, when reinfused into a mammalian patient's body, may include changes in lipid metabolism and enhancement of the immune system through stimulation of leukocytes (i.e. cell-cell interaction or cytokine release) throughout the peripheral blood of the patient. This may, for example, lead to a reduction in atherosclerotic plaque formation and deposition, a reduction in lipids and lipid deposits, a relaxation of the vascular endothelium, reduction in edema, and reduction of inflammation. These effects may result in a reversal in progressive luminal narrowing in arteries, a reduction in the rupture or denudation of plaques, and an increase in vasodilation (i.e. promotion of vasodilators or inhibition of vasoconstrictors), thereby decreasing the incidence of atheroembolism (cholesterol embolism), improving endothelial function including endothelial cellular repair or replacement, and improving blood flow yielding enhanced oxygenation.

Clinical signs of these effects may include reductions in episodes and severity of intermittent claudication, reduction of the ischemic penumbra, a reduction in elevated blood pressure, reduction in extremity weakness and pain, normalization of extremity temperature and an improvement in pallor. In addition, weight loss may be a result of this treatment approach.
Regarding disorders involving atherosclerotic plaque formation, deposition and plaque rupture, the present methods provide for therapeutic treatment and prophylaxis of a wide variety of such mammalian disorders, including cardiovascular diseases, such as atherosclerosis, peripheral arterial occlusive disease, to cerebrovascular disease (stroke and transient ischemic attack), myocardial infarction, angina, hypertension, retinal ischemia, renal failure, abdominal aortic aneurysm, and hyperlipidemia.
For those disorders involving endothelial dysfunction, the present methods provide for therapeutic treatment and prophylaxis of a wide variety of such mammalian 1s disorders including cardiovascular diseases, such as atherosclerosis, peripheral arterial occlusive disease, congestive heart failure, cerebrovascular disease (stroke), myocardial infarction, angina, hypertension, vasospastic disorders such as Raynaud's disease, cardiac syndrome X, and migraine.
The therapeutic effect of blood, or a derivative thereof, which has absorbed a 20 quantifiable absorbed-dose of ozone, may be the induction of sufficient leukocyte apoptosis without excessive necrosis necessary to elicit an anti-inflammatory response when reinfused autologously into a patient. The induction of apoptosis without excessive necrosis in the leukocyte fraction of the blood that has been treated may be evaluated by a number of diagnostic methods including light microscopy with nuclear 25 stains, electrophoretic analysis of DNA fragmentation, TUNEL analysis and multiparameter flow cytometry.
An effect of blood, or blood derivative thereof, which has absorbed a quantifiable absorbed-dose of ozone, may result in the reduction of CRP when reinfused autologously into a patient and may elicit clinical benefit, including an anti-

30 inflammatory response, neovascularization and vasodilation.
The effects of blood, blood fractionate or other fluid which has absorbed a quantifiable absorbed-dose of ozone, when reinfused into a mammalian patient's body, may include effects that reduce edema.
In addition, the effects of blood, blood fractionate or other fluid which has 35 absorbed a quantifiable absorbed-dose of ozone, when re-infused into a mammalian patient's body may include effects that increase blood flow to ischemic tissue. This reduction can be evaluated by a variety of diagnostic tools including MRI and Doppler imaging techniques.
Furthermore, the effects of blood, blood fractionate or other fluid which has absorbed a quantifiable absorbed-dose of ozone, when re-infused into a mammalian 5 patient's body, may include effects that include reduction of inflammation.
Reduction of inflammation may occur though a reduction in pro-inflammatory cytokines (e.g.
interferon-gamma, TNF-gamma, IL-6, IL-8 and IL-12) and/or an increase in anti-inflammatory cytokines (e.g. interleukin-4 and IL-10) released by immunomodulatory T
cells. The effect of reducing inflammation may result in any number of clinical benefits 1o including improvement in blood flow yielding enhanced oxygenation.
The effect of treated blood or blood derivative thereof with ozone by the present method to induce apoptotic leukocytes without excessive necrosis, when re-infused into a mammalian patient's body may include effects that include reduction of inflammation. Reduction of inflammation may occur though a reduction in pro-15 inflammatory cytokines (e.g. interferon-gamma, TNF-gamma, IL-6, IL-8 and IL-12) and/or an increase in anti-inflammatory cytokines (e.g. interleukin-4 and IL-10) released by immunomodulatory cells. The effect of reducing inflammation may result in any number of clinical benefits in the treatment of cardiovascular diseases, including improvement in blood flow yielding enhanced oxygenation.
20 Further embodiments of the methods of the present invention comprise extracorporeally subjecting an aliquot of a mammalian patient's blood, or the separated cellular fractions of the blood, or mixtures of the separated cells, including platelets, to a measured amount of ozone such that it absorbs a quantifiable absorbed-dose of ozone and. On re-introduction of this autologous aliquot to the patient's body, the blood, blood 25 fractionate or other fluid with a quantifiable absorbed-dose of ozone may have therapeutic effects on treating any condition caused by stroke, including the reduction in edema in ischemic penumbra, improvement in impaired blood flow to the area surrounding the infarct which may include ischemic tissue and the ischemic penumbra, relaxation of the vascular endothelium and reduction of inflammation.
30 Further, the effect of blood, or blood derivatives thereof, which has absorbed a quantifiable absorbed-dose of ozone, may be the induction of sufficient leukocyte apoptosis without excessive necrosis necessary to elicit an anti-inflammatory response when reinfused autologously into a patient. The induction of apoptosis without excessive necrosis in the leukocyte fraction of the blood treated may be evaluated, in 35 accordance with the methods of the present invention, by a number of diagnostic methods including light microscopy with nuclear stains, electrophoretic analysis of DNA
fragmentation, TUNEL analysis and multiparameter flow cytometry.

An effect of blood or blood derivative thereof which has absorbed a quantifiable absorbed-dose of ozone, may result in the reduction of CRP when reinfused autologously into a patient and elicit clinical benefit including an anti-inflammatory response, neovascularization and vasodilation. The effects of blood, blood fractionate or other fluid which has absorbed a quantifiable absorbed-dose of ozone, when reinfused into a mammalian patient's body may include effects that reduce the edema in the ischemic penumbra in acute ischemic brain stroke patients.
This reduction can be evaluated by a variety of diagnostic tools including MRI and Doppler imaging techniques. In addition, the effects of blood, blood fractionate or other fluid 1o which has absorbed a quantifiable absorbed-dose of ozone, when re-infused into a mammalian patient's body may include effects that increase blood flow to the area surrounding the infarct which may include ischemic tissue and the ischemic penumbra in patients suffering from acute ischemic stroke. This reduction can be evaluated by a variety of diagnostic tools including MRI and Doppler imaging techniques.
Moreover, the effects of blood, blood fractionate or other fluid which has absorbed a quantifiable absorbed-dose of ozone, when re-infused into a mammalian patient's body may include effects that relax the vascular endothelium in patients suffering from acute ischemic stroke. This relaxation may result from an increase in vasodilation (i.e. promotion of vasodilators or inhibition of vasoconstrictors) improving endothelial function including endothelial cellular repair or replacement, and improving blood flow yielding enhanced oxygenation. The effects of blood, blood fractionate or other fluid which has absorbed a quantifiable absorbed-dose of ozone, when re-infused into a mammalian patient's body may include effects that include reduction of inflammation. Reduction of inflammation may occur though a reduction in pro-inflammatory cytokines (e.g. interferon-gamma, TNF-gamma, IL-6 and IL-12) and/or an increase in anti-inflammatory cytokines (e.g. interleukin-4 and IL-10) released by immunomodulatory T cells. The effect of reducing inflammation may result in any number of clinical benefits including improvement in blood flow yielding enhanced oxygenation.
The effect of treated blood or blood derivative thereof with ozone by the present method to induce apoptotic leukocytes without excessive necrosis, when re-infused into a mammalian patient's body may include effects that include reduction of inflammation. Reduction of inflammation may occur though a reduction in pro-inflammatory cytokines (e.g. interferon-gamma, TNF-gamma, IL-6 and IL-12) and/or an increase in anti-inflammatory cytokines (e.g. interleukin-4 and IL-10) released by immunomodulatory cells. The effect reducing inflammation may result in any number of clinical benefits in the treatment of acute ischemic stroke including improvement in blood flow yielding enhanced oxygenation.
The methods of the present invention, comprising extracorporeally subjecting an aliquot of a mammalian patient's blood, or the separated cellular fractions of the blood, or mixtures of the separated cells, including platelets, to a measured amount of ozone such that it absorbs a quantifiable absorbed-dose of ozone, may be used in the treatment of, or used in the manufacture of medicaments for the treatment of any condition caused by stroke, including paralysis, motor weakness, visual and auditory skills, cognitive function, re-attainment of independence, stroke-free survival, relapse 1o frequency and severity, and overall survival. The therapeutic effects of the present methods may further affect improvement in any condition caused by stroke including improvement in paralysis, motor weakness, visual and auditory skills, cognitive function, re-attainment of independence, stroke-free survival, relapse frequency and severity, and overall survival which may be evaluated by stroke scale assessment tools.
The improvement in any condition caused by stroke mediated by the methods of the present invention may, in accordance with the methods described herein, be evaluated by stroke scale assessment tools, and clinical effectiveness may be measured through statistical comparison with untreated stroke patients.
Further embodiments of the methods of the present invention may comprise subjecting an aliquot of a mammalian patient's blood, or the separated cellular fractions of the blood, or mixtures of the separated cells, including platelets, to a measured amount of ozone such that the fluid absorbs a quantifiable absorbed-dose of ozone, and on reintroduction of this autologous aliquot to the patient's body, the blood, blood fractionate, or other biological fluid, with a quantified absorbed-dose of ozone, therapeutic and beneficial effects result in the improvement in inflammatory disease-related conditions, including reduction of inflammation, relaxation of the vascular endothelium, reduction in edema and increased blood flow.
The therapeutic effect of blood, blood fractionate, or other biological fluid which has absorbed a quantifiable absorbed-dose of ozone, may be the induction of sufficient leukocyte apoptosis, without excessive necrosis, necessary to elicit an anti-inflammatory response when reinfused autologously into a patient. The induction of apoptosis without excessive necrosis in the leukocyte fraction of the blood, blood fractionate, or other biological fluid that has been treated in accordance with the methods described herein may be evaluated by a number of diagnostic methods including light microscopy with nuclear stains, electrophoretic analysis of DNA
fragmentation, TUNEL analysis and multiparameter flow cytometry.

A therapeutic effect of blood, blood fractionate, or other biological fluid which has absorbed a quantifiable absorbed-dose of ozone, may result in the reduction of CRP when reinfused autologously into a patient and elicit clinical benefit including an anti-inflammatory response, neovascularization and vasodilation. The effects of blood, blood fractionate or other fluid which has absorbed a quantifiable absorbed-dose of ozone, when re-infused into a mammalian patient's body may include effects that reduce edema. In addition, the effects of blood, blood fractionate or other fluid which has absorbed a quantifiable absorbed-dose of ozone, when re-infused into a mammalian patient's body, may include effects that increase blood flow to ischemic lo tissue.
In accordance with the methods of the present invention, the effective promotion of reduced edema brought about in patients with inflammatory diseases may be evaluated by a variety of diagnostic tools including CT, MRI and Doppler imaging techniques. Additionally, it is a further element of the present methods to evaluate the effect of increased blood flow brought about through the methods described herein by use of a variety of diagnostic tools including MRI and Doppler imaging techniques.
The effects of blood, blood fractionate or other fluid which has absorbed a quantifiable absorbed-dose of ozone, when re-infused into a mammalian patient's body may include effects that include reduction of inflammation. Reduction of inflammation may occur though a reduction in pro-inflammatory cytokines (e.g. interferon-gamma, TNF-gamma, IL-6, IL-8 and IL-12) and/or an increase in anti-inflammatory cytokines (e.g. interleukin-4 and IL-10) released by immunomodulatory T cells. The effect of reducing inflammation may result in any number of clinical benefits including improvement in blood flow yielding enhanced oxygenation.
The effect of treated blood or blood derivative thereof with ozone by the present method to induce apoptotic leukocytes without excessive necrosis, when re-infused into a mammalian patient's body may include effects that include reduced inflammation. Reduction of inflammation may occur though a reduction in pro-.
inflammatory cytokines (e.g. interferon-gamma, TNF-gamma, IL-6, IL-8 and IL-12) 3o and/or an increase in anti-inflammatory cytokines (e.g. interleukin-4 and IL-10) released by immunomodulatory cells. Reduction of inflammation may result in any number of clinical benefits including improvement in blood flow yielding enhanced oxygenation.
Diagnostic markers to measure reduction of inflammation may include reduction in joint stiffness, reduction in temperature and reported pain, normalization of leukocyte count including differential, coagulation system measurement, inflammatory cytokines, C-reactive protein (including high sensitivity CRP) and procalcitonin levels.

Further therapeutic effects mediated by the methods of the invention include relaxation of vascular endothelium. This relaxation may be the result of vasodilation and involve release of nitric oxide and prostacyclins, or inhibition of vasoconstrictors leading to improvement in endothelial function including endothelial cellular repair or replacement.
Vasorelaxation may be clinically beneficial in the treatment of inflammatory conditions including improvement in blood flow yielding enhanced oxygenation. Thus, in accordance with the methods of the present invention, the evaluation of an amount or degree of vasorelaxation brought about by the therapeutic treatment methods can be measured by a variety of diagnostic methods including ultrasonography based flow-1o mediated vasorelaxation (FMVR) and pulse transit time (PTT).
In accordance with the various methods of the present invention to treat a mammalian subject suffering from, or believed to be suffering from a disease condition, reintroduction of treated blood, blood fractionate or other fluid autologously to a mammalian subject may be accomplished through a variety of routes, including intravenous, intramuscular and subcutaneous routes, or any combination thereof.
An ozone delivery system utilized in the treatment of acute ischemic brain stroke, cardiovascular diseases, inflammatory disease and conditions and symptoms thereof, delivers a measured amount of an ozone/oxygen admixture and is able to measure, control, report and differentiate between the delivered-ozone and absorbed-2o dose of ozone. The system provides a controllable, measurable, accurate and reproducible amount of ozone that is delivered to a controllable, measurable, accurate and reproducible amount of a biological fluid, and controls the rate of ozone absorption by the fluid resulting in a quantifiable absorbed-dose of ozone used in the treatment of cardiovascular diseases. The system may accomplish this by using a manufacturing component, control components, measuring components, a reporting component and calculating component (such as an ozone generator, gas flow meter, fluid pump, variable pitch platform, data acquisition device, inlet ozone concentration monitor, and exit ozone concentration monitor) that cooperate to manufacture and deliver a measured, controlled, accurate and reproducible amount of ozone, i.e., the delivered-ozone, to a fluid through the use of one or more gas-fluid contacting devices that provides for the interface between the ozone/oxygen admixture and fluid. Using control components, measuring components, a reporting component and calculating component (such as a gas flow meter, fluid pump, variable pitch platform, data acquisition device, inlet ozone concentration monitor and exit ozone concentration monitor) that cooperate, the system may instantly differentiate the delivered-ozone from the absorbed-dose of ozone.

The system utilizes (a gas flow meter, fluid pump, variable pitch platform, data acquisition device, inlet ozone concentration monitor, and exit ozone concentration monitor) control components, measuring components, a reporting component and calculating component that cooperate and instantly report data that may include the delivered-ozone, residual-ozone, absorbed-dose of ozone, interface-time, elapsed-time and the amount and flow rate of the fluid delivered to the gas-contacting device.
A particularly suitable ozone delivery system that may be used in carrying out the methods of the present invention is disclosed in U.S. Patent No. 7,736,494 and co-pending application Serial No. 12/813,371, the contents of which are incorporated 1o herein in their entirety. The disclosed ozone delivery system is particularly and uniquely constructed such that all ozone-contacting surfaces of the device are made of ozone-inert material so that the amount of ozone that is actually absorbed by the biological fluid being treated is accurately determinable. That is, by virtue of being constructed with ozone-inert materials in all ozone-contacting surfaces, no ozone is absorbed by the device itself, and the determination of the amount of ozone absorbed by the biological fluid is not inaccurately reflected as a result of ozone being absorbed by any structure of the device The ozone delivery system utilizes measuring components, reporting components and calculating components (such as an inlet ozone concentration monitor, exit ozone concentration monitor, gas flow meter, fluid pump, data acquisition device) that cooperate together to determine certain calculated-data including the delivered-ozone, the residual-ozone and the absorbed-dose of ozone.
Delivered-ozone is an amount of ozone calculated by multiplying the measured volume of ozone/oxygen admixtures, as reported by gas flow meters, by the measured concentration of ozone within the ozone/oxygen admixture as it enters the gas-fluid contacting device, as reported by the inlet ozone concentration monitor. The measured volume of ozone/oxygen admixtures is calculated by multiplying the measured gas flow reported by gas flow meters, by the elapsed-time.
Residual-ozone is an amount of ozone calculated by multiplying the measured volume of ozone/oxygen admixtures, as reported by gas flow meters, by the measured concentration of ozone within the ozone/oxygen admixture exiting the gas-fluid contacting device, as reported by the exit ozone concentration monitor.
The measured volume of ozone/oxygen admixtures is calculated by multiplying the measured gas flow reported by gas flow meters, by the elapsed-time.
The quantifiable absorbed-dose of ozone is an amount of ozone calculated by subtracting the amount of residual-ozone from the amount of delivered-ozone. The quantifiable absorbed-dose of ozone in the methods of the invention may range from 1 to 10,000,000 micrograms per milliliter of fluid, and may be between 1 and 10,000 pg per milliliter of fluid.
All measured-data, including measured data from the gas flow meters, inlet and exit ozone concentration monitors, the fluid pump, temperature sensors, pressure sensors, absorbed oxygen sensor and humidity sensors are reported to a data acquisition device. The data acquisition device has instant, real-time reporting, calculating and data storing capabilities to process all measured data. The data acquisition device may use any measured data or any combination of measured data as variables to produce calculated-data. Examples of calculated-data may include 1o delivered-ozone, residual-ozone, absorbed-dose of ozone, absorbed-dose of ozone per unit volume of fluid, and the quantifiable absorbed-dose of ozone per unit volume of fluid per unit time.
An ozone delivery system particularly suitable to the present invention includes an ozone generator for the manufacture and control of a measured amount of an ozone/oxygen admixture and where the admixture volume contains the delivered-ozone. A commercially available ozone generator capable of producing ozone in a concentration range between 10 and 3,000,000 ppmv of ozone in an ozone/oxygen admixture may be employed. Ozone/oxygen admixture concentrations entering the gas-fluid contacting device are instantly and constantly measured in real time, through an inlet ozone concentration monitor that may utilize UV absorption as a detection methodology. A flow meter controls and measures the delivery of the delivered-ozone in an ozone/oxygen admixture to the gas-fluid contacting device at a specified admixture flow rate. Ozone/oxygen admixture flow rates are typically in the range between 0.1 and 5.0 liters per minute.
Measurement of the humidity of the ozone/oxygen admixture delivered to the gas-fluid contacting device may be included through the use of a humidity sensor. A
humidity sensor port may be provided in the ozone/oxygen admixture connecting tubing; however, it can be placed in a variety of locations. For example, the humidity sensor may be located in the connecting tubing prior to the admixture's entrance into gas-fluid contacting device.
Measurement of the temperature within the gas-fluid contacting device during the interface-time may be provided by inclusion of a temperature sensor port in the gas fluid contacting device through which a temperature sensor may be inserted.
The temperature at which ozone/oxygen admixtures interface fluids ranges from 4 C
to 100 C, and may be performed at ambient temperature, 25 C, for example. The temperature at which the interface occurs can be controlled by placing the gas-fluid contacting device, optional reservoir, and both gas and fluid connecting tubing in a temperature controlled environment and/or by the addition of heating or cooling elements to the gas-fluid contact device.
Measurement of the pressure within the gas-fluid contacting device during the interface-time is provided by inclusion of a pressure sensor port in the gas-fluid contacting device through which a pressure sensor may be inserted. The pressure at which an ozone/oxygen admixtures interfaces with a fluid ranges from ambient pressure to 50 psi and may be performed between ambient pressure and 3 psi, for example. A pressure sensor port may be provided in each gas-fluid contacting device to measure and report the pressure at which the interface occurs.
The concentration of the ozone/oxygen admixtures exiting the gas-fluid contacting device, and where the admixture volume contains the residual-ozone, are instantly and constantly measured in real time through an exit ozone concentration monitor that may utilize UV absorption as a detection methodology.
A fluid pump controls and measures the flow rate of the fluid delivered to the gas-fluid-contacting device at a specified fluid flow rate. Fluid flow rates through the gas-fluid contacting device typically will range from 1 ml to 100 liters per minute, and for example, may be between 1 ml to 10 liters per minute. The fluid is generally contained within a closed-loop design and may be circulated through the gas-fluid contacting device once or multiple times.
Measurement of the amount of oxygen absorbed into a fluid while it interfaces with the ozone/oxygen admixture within the gas-fluid contacting device may be provided through the use of an absorbed oxygen sensor. The sensor is inserted within the absorbed oxygen sensor port located in the tubing as it exits the gas-fluid contacting device. Measurement of absorbed oxygen may be recorded in various units, including ppm, milligrams/liter or percent saturation.
The ozone delivery system may also include a fluid access port for fluid removal. The port is generally located in the tubing member after the fluid exits through the fluid exit port of the gas-fluid contacting device and prior to an optional reservoir.
A data acquisition device, such as a DAQSTATION (Yokogawa), for 3o example, reports, stores and monitors data instantly and in real-time, and performs various calculations and statistical operations on data acquired. Data is transmitted to the data acquisition device through data cables, including data from ozone concentration monitors, flow meters, a humidity sensor, temperature sensors, pressure sensors, a fluid pump and an absorbed oxygen sensor.
Calculated-data in carrying out the methods of the present invention include delivered-ozone, residual-ozone, and the quantifiable absorbed-dose of ozone.
Measurement of the volume of the ozone/oxygen admixture delivered can be calculated though data provided from the flow meter and the time measurement capability of the data acquisition device. Measurement of the volume of fluid delivered to the gas-fluid contacting device can be calculated by the data acquisition device utilizing fluid flow rate data transmitted from the fluid pump.
The elapsed-time can be measured and controlled through the data acquisition device. The elapsed-time that the fluid circulates through the apparatus, including the gas-fluid contacting device, and is interfaced with an ozone/oxygen admixture can vary, generally for a duration of up to 120 hours. The interface-time may also be measured by the time measuring capacity of the data acquisition device. The 1o interface-time between a fluid and an ozone/oxygen admixture may be controlled through a composite of controls. These controls include the angle of the gas fluid contacting device, the fluid flow rate via the fluid pump, and the time controlling capacity of the data acquisition device. The interface-time may vary in duration of up to 720 minutes, and generally within duration of up to 120 minutes.
Controllable variables for an ozone delivery system may include delivered amounts and concentrations of ozone in the entering ozone/oxygen admixtures, fluid flow rates, admixture flow rates, temperature in the gas-fluid contacting device, interface-time between fluid and admixture, and the elapsed-time that the fluid may circulate through the apparatus and interface with an ozone/oxygen admixture.
Measurable variables may include ozone/oxygen admixture flow rates, amounts and concentrations of ozone in the entrance and exit ozone/oxygen admixtures, fluid flow rates, temperature and pressure in the gas-contacting device, humidity of the entrance admixture to the gas-fluid contacting device, absorbed oxygen by the fluid, interface-time and elapsed-time.
Data representing controllable variables and measurable variables acquired by the apparatus allows for a variety of calculations including delivered-ozone, residual-ozone, quantifiable absorbed-dose of ozone, quantifiable absorbed-dose of ozone per unit volume of fluid and the quantifiable absorbed-dose of ozone per unit volume of fluid per unit time.
FIG. 1 schematically illustrates an embodiment of the present invention where fluid that has been taken from a subject is extracorporeally interfaced with an ozone/oxygen admixture. In general, blood may be circulated in a discontinuous manner where a fluid (e.g., an aliquot of blood) has been removed from a patient and is introduced into an ozone delivery system through a common reservoir, and is recirculated in a closed loop format. Alternatively, fluid may be circulated in a continuous loop format in a venovenous extracorporeal exchange format. As an example, this continuous loop can be established through venous access of the antecubital veins of both right and left arms. Prior to establishing a discontinuous closed loop format, blood from the patient may be anticoagulated with citrate or any other suitable anticoagulant before being introduced in to the reservoir. For an extracorporeal continuous loop circuit, a patient may optionally be anticoagulated with heparin or any other suitable anticoagulant known to those skilled in the art.
For the gas flow, in either the discontinuous format or continuous loop system, oxygen flows from a pressurized cylinder (1-1), through a regulator (1-2), through a particle filter (1-3) to remove particulates, through a flow meter (1-4) where the oxygen and subsequent ozone/oxygen admixture flow rate is controlled and measured.
The oxygen proceeds through a pressure release valve (1-5), through an ozone generator (1-6) where the concentration of the ozone/oxygen admixture is manufactured and controlled and where the admixture volume includes the delivered-ozone. The ozone/oxygen admixture flows through an optional moisture trap (1-7), to reduce moisture.
The admixture proceeds through an inlet ozone concentration monitor (1-8) that measures and reports the inlet ozone concentration of the ozone/oxygen admixture that contains the delivered-ozone. This real-time measurement may be based on ozone's UV absorption characteristics as a detection methodology. The ozone/oxygen admixture then passes through a set of valves (1-9) used to isolate a gas-fluid contacting device for purging of gasses. The ozone/oxygen admixture may pass an optional humidity sensor (1-20) where humidity may be measured and recorded, and into a gas-fluid contacting device (1-10) where it interfaces with fluid. The interface-time between fluid and ozone/oxygen admixture may be controlled through adjustment of a variable pitch platform, a fluid pump and the time controlling capacity of the data acquisition device.
The interface-time may then be measured by the data acquisition device (1-17). Temperature (1-21) and pressure (1-22) may be measured by the use of optional temperature and pressure sensors, respectively, inserted into their respective ports.
The resultant ozone/oxygen admixture containing the residual-ozone exits the gas-fluid contacting device and flows through the exit purge valves (1-11), through a moisture trap (1-7), through an exit ozone concentration monitor (1-12), which may utilize a similar detection methodology as the inlet ozone concentration monitor (1-8), that measures and reports the exit ozone/oxygen admixture concentration. The exiting ozone/oxygen admixture then proceeds through a gas drier (1-13), through an ozone destructor (1-14) and a flow meter (1-19).
In the fluid flow for the discontinuous format, blood is introduced into the reservoir (1-30). In the continuous loop system, intravenous blood flows from the patient through tubing through a pressure gauge (1-27) which monitors the pressure of the blood flow exiting the patient. Generally, the pressure of the blood exiting the patient ranges from a negative pressure of 100 - 200 mm Hg, and may be between a negative pressure of 150 and 200 mm Hg, with a maximum cutoff pressure of minus 250 mm Hg. In either format, the blood flows through a fluid pump (1-15) and is optionally admixed with heparin or other suitable anticoagulant as provided by an optional heparin pump (1-16).
The blood then passes through the gas-fluid contacting device (1-10) where it interfaces with the ozone/oxygen admixture containing the delivered-ozone.
Ports for the insertion of sensors may be located in the gas-fluid contacting device for the measurement of temperature and pressure, respectively. After interfacing with the ozone/oxygen admixture, the fluid exits into tubing that may contain a port for an optional absorbed oxygen sensor (1-23) followed by a fluid access port (1-24).
The blood continues through an air/emboli trap (1-25) that removes any gaseous bubbles or emboli, and the blood then continues through a fluid pump (1-26).
In a discontinuous format, the blood is then directed back into the reservoir (1-30) any may continue in a recirculating mode, passaging as often as required. In the continuous loop format, the blood is directed into a pressure gauge (1-28) which monitors the pressure of the blood flow before returning the fluid to the patient.
Generally, the pressure of the blood entering the patient ranges from a pressure of 100 - 200 mm Hg, and may be between 150 and 200 mm Hg, with a maximum cutoff pressure of 250 mm Hg. The blood continues through a priming fluid access port (1-29) that allows for the removal of the priming fluid from the extracorporeal loop.
The blood is then re-infused directly into the patient.
A data acquisition device (1-17), such as a DAQSTATION (Yokogawa), for example, has time measurement capabilities, reports, stores and monitors data instantly and in real-time, and performs various calculations and statistical operations on data acquired. All data is transmitted to the data acquisition device through data cables (1-18), including: data from ozone concentration monitors (1-8) and (1-12), flow meters (1-4) and (1-19), humidity sensor (1-20), temperature sensor (1-21), pressure sensor (1-22), fluid pumps (1-15) and (1-26), pressure gauges (1-27) and (1-28), and absorbed oxygen sensor (1-23). The elapsed time, a composite of both the interface time and the period of time that the fluid circulates through the other elements of the apparatus can be measured and controlled through the data acquisition device (1-17).
Other possible configurations for an extracorporeal blood are included within the spirit of this disclosure.

One or more gas-fluid contacting devices may be included in an ozone delivery system to increase the surface area of a fluid to be treated allowing for an increase in the mass transfer efficiency of the ozone/oxygen admixture. Gas-fluid contacting devices may encompass the following properties: closed and isolated from the ambient atmosphere, gas inlet and outlet ports for the entry and exit of ozone/oxygen admixtures, fluid inlet and outlet ports for the entry and exit of a fluid, components (temperature sensor, pressure sensor and data acquisition device) for the measurement and reporting of temperature and pressure within a gas-fluid contacting device, generation of a thin film of the fluid as it flows within a gas-fluid contacting 1o device and construction from ozone-inert construction materials including, quartz, ceramic composite, borosilicate, stainless steel, PFA and PTFE.
Gas-fluid contacting devices include designs that encompass surfaces that may be horizontal or approaching a horizontal orientation. These surfaces may include ridges, indentations, undulations, etched surfaces or any other design that results in a contour change and furthermore, may include any pattern, regular or irregular, that may disrupt the flow, disperse the flow or cause turbulence. These surfaces may or may not contain holes through which a fluid passes through. The surface of the structural elements may have the same or different pitches. Designs of gas-fluid contacting devices may include those that involve one or more of the same shaped surfaces or any combination of different surfaces, assembled in any combination of ways to be encompassed within the device which may include cones, rods, tubes, flat and semi-flat surfaces, discs and spheres.
The interface between an ozone/oxygen admixture and a fluid may be accomplished by the use of a gas-fluid contact device that generates a thin film of the fluid that interfaces with the ozone-oxygen admixture as it flows through the device.
One of skill in the art will appreciate that generation of any interface that increases the surface area of the fluid and thereby maximizes the contact between a fluid and an admixture, may be used. Additional examples include the generation of an aerosol through atomization or nebulization.
The interface-time within a gas-fluid contacting device is measurable, controllable, calculable and reportable. Furthermore, the interface-time may be for duration of up to 720 minutes, generally however, for duration of up to 120 minutes.
Following the interface-time, the fluid exits the gas-fluid contacting device containing the quantifiable absorbed-dose of ozone. The elapsed-time, a composite of both the interface-time and the time for circulation of a fluid through other elements of an ozone delivery system is also measurable, controllable, calculable and reportable.
This elapsed-time is for duration of up to 120 hours.

The pressure at the interface between fluid and ozone/oxygen admixture within a gas-fluid contacting device may be measured. Measurement of pressure within the device may be accomplished through the use of a pressure sensor inserted at the pressure port of the gas-fluid contacting device. The pressure at which an ozone/oxygen admixture interfaces with a fluid ranges from ambient pressure to 50 psi and may be performed between ambient pressure and 3 psi.
The temperature within a gas-fluid contacting device may be controlled by housing the device such that the connecting tubing containing both gas and fluid and an optional reservoir are maintained in a controlled temperature environment. A
flow hood that provides for temperature regulation is an example of a controlled temperature environment. Alternatively, the addition of heating or cooling elements to the gas-fluid contact device may provide for the control of temperature. Measurement of temperature within the device may be accomplished through the use of a temperature sensor inserted at the temperature port of a gas-fluid contacting device. The temperature at which ozone/oxygen admixtures interface fluids ranges from 4 C
to ' 100 C, and may be performed at ambient temperature, 25 C, for example.
Gas-fluid contacting devices may be utilized individually or in conjunction with other such devices, whether they are similar or dissimilar in construction, design or orientation. In the event that multiple devices are utilized, either of the same design, or a combination of different gas-fluid contacting devices of different designs, these devices may be arranged one after the other in succession (in series), making a single device out of multiple individual contact devices.
In a series configuration of devices, a fluid flowing through the different contact devices flows in series, from the fluid exit port of one contact device to the fluid entrance port of the next, until passing through all the devices. The ozone/oxygen admixture may flow in a number of arrangements. In one example, the ozone/oxygen admixture flows through different contact devices in series, from the admixture exit port of one contact device to the admixture entrance port of the next. As an alternative example, the ozone/oxygen admixture may flow directly from the admixture source to the entrance port of each different contact device. Another alternative is a combination of the foregoing examples where the ozone/oxygen admixture flows from the exit port of some devices to the entrance port of other devices and in addition, to the entrance of some devices directly from the admixture source. In the event that multiple devices are utilized, the resultant fluid from the terminal device can either be collected or returned to the original device and recirculated.
When arranged in series with other contact devices, interface time between the fluid and ozone/oxygen admixture is controllable, and can be adjusted based on the individual pitch chosen for each device in series, or by adding additional devices to the series. The overall interface surface area will range from 0.01M2 for an individual device, and upwards based on the number of devices serially utilized.

An example of data measured and calculated by the ozone delivery system that utilizes a fluid target described herein is included in Table 1. Newborn Calf Serum commercially obtained was utilized as the target fluid. A variable pitch device with variable pitch platform, as disclosed in U.S. Patent No. 7,736,494, was employed as the gas-fluid contacting device. The following initial conditions were utilized;
300 ppmv ozone inlet concentration, 145 ml initial fluid volume, 1000 ml per minute gaseous flow rate, 189 ml per minute fluid flow rate counter current to the ozone/oxygen admixture flow. Incremental reductions in fluid volume are due to sampling of fluid through the fluid access port.

NEWBORN CALF SERUM
MEASURED VARIABLES
Average Inlet Ozone Average Exit Ozone Elapsed-time Fluid Volume Gas Flow Rate Fluid Flow Rate Concentration Concentration (5 min intervals) (milliliters) (liters/minute) (liters/minute) (ppmv) (ppmv) 5 145 0.998 0.189 305.2 38.2 10 143 0.972 0.189 361.5 40.4 141 1.000 0.189 312.7 20.6 139 1.000 0.189 314.0 37.3 CALCULATED VARIABLES
Average Differential Ozone Ozone-Absorbed per Absorbed-dose Elapsed-time Concentration Delivered-ozone Residual-ozone Interval of Ozone (minutes) (ppmv) (ug) (ug) (ug) (ug) 5 267.0 3.26E+03 4.08E+02 2.86E+03 2.86E+03 10 321.1 7.02E+03 8.28E+02 3.34E+03 6.20E+03 15 292.1 1.04E+04 1.06E+03 3.12E+03 9.32E+03 20 276.7 1.37E+04 1.46E+03 2.96E+03 1.23E+04 An additional example of data measured and calculated by the system described herein is in Table 2 below. Newborn Calf Serum commercially obtained was utilized as the target fluid. The variable pitch device with variable pitch platform, as disclosed in U.S. Patent No. 7,736,494, was employed as the gas-fluid contacting 20 device. The following initial conditions were utilized; 600 ppmv ozone inlet concentration, 137 ml initial fluid volume, 1000 ml per minute gaseous flow rate, 189 ml per minute fluid flow rate counter current to the ozone/oxygen admixture flow.
Incremental reductions in fluid volume are due to sampling of fluid through the fluid access port.

Table 2 NEWBORN CALF SERUM
MEASURED VARIABLES
Average Inlet Ozone Average Exit Ozone Elapsed- time Fluid Volume Gas Flow Rate Fluid Flow Rate Concentration Concentration (5 minute intervals) (milliliters) (liters/minute) (liters/minute) (ppmv) (ppmv) 137 1.000 0.189 604.2 72.0 5 135 1.000 0.189 609.6 63.5 5 133 1.000 0.189 606.6 70.8 5 131 1.000 0.189 605.3 71.7 CALCULATED VARIABLES

Average Differential Ozone Ozone Absorbed Absorbed-dose Elapsed-time Concentration Delivered-ozone Residual-ozone per Interval of ozone (minutes) (ppmv) (ug) (ug) (ug) (ug) 5 532.2 6.47E+03 7.70E+02 5.69E+03 5.69E+03 546.1 1.30E+04 1.45E+03 5.84E+03 1.15E+04 535.8 1.95E+04 2.21E+03 5.73E+03 1.73E+04 533.6 2.60E+04 2.98E+03 5.71 E+03 2.30E+04 Another example of data measured and calculated by the system described herein is in Table 3 below. Newborn Calf Serum commercially obtained was utilized as 5 the target fluid. The variable pitch device, as disclosed in U.S. Patent No.
7,736,494, was employed as the gas-fluid contacting device. The following initial conditions were utilized; 900 ppmv ozone inlet concentration, 145 ml initial fluid volume, 1000 ml per minute gaseous flow rate, 189 ml per minute fluid flow rate counter current to the ozone/oxygen admixture flow. Incremental reductions in fluid volume are due to 1o sampling of fluid through the fluid access port.

NEWBORN CALF SERUM
MEASURED VARIABLES

Average Inlet Ozone Average Exit Ozone Elapsed- time Fluid Volume Gas Flow Rate Fluid Flow Rate Concentration Concentration (5 minute intervals) (milliliters) (liters/minute) (liters/minute) (ppmv) (ppmv) 145 1.000 0.189 908.1 68.0 5 143 1.000 0.189 911.4 50.1 5 141 1.000 0.189 904.4 46.6 5 139 1.000 0.189 904.7 50.9 CALCULATED VARIABLES

Average Differential Ozone Ozone Absorbed Absorbed-dose Elapsed-time Concentration Delivered-ozone Residual-ozone per Interval of ozone (minutes) (ppmv) (ug) (ug) (ug) (ug) 5 840.1 9.72E+03 7.28E+02 8.99E+03 8.99E+03 861.3 1.95E+04 1.26E+03 9.22E+03 1.82E+04 857.8 2.92E+04 1.76E+03 9.18E+03 2.74E+04 853.8 3.88E+04 2.31E+03 9.13E+03 3.65E+04 In one embodiment of the invention, a method is provided to treat cardiovascular diseases, inflammatory diseases, acute ischemic brain stroke, and conditions or symptoms related to the diseases, in a mammal. The method involves subjecting an 5 amount of blood, blood fractionate or other biological fluid, ex vivo, to an amount of ozone delivered by an ozone delivery system, as previously described. The method may also provide for the maintenance of the biological integrity of the treated fluid. The method provides treatment conditions at temperatures compatible with maintaining the biological integrity of biological fluids.
10 For blood products, the biological integrity of plasma may be measured by the functionality of its protein components either in whole plasma or after separation into plasma fractions. The biological integrity of red blood cell and platelet preparations may be determined by the methods and criteria known by those skilled in the art and are similar to those used in establishing the suitability of storage and handling 15 protocols. In practical terms, the biological integrity of a biological fluid is a fluid that, subsequent to the method of treating diseases or conditions described herein, has sufficiently maintained its functionality upon re-infusion into a mammalian patient.

Fluid-contacting surfaces, including gas-fluid contacting devices constructed from ozone-inert material, may be treated with a human serum albumin (HSA) solution to prevent platelet adhesion, aggregation and other related platelet phenomena in the instances when a biological fluid to be treated contains platelets (i.e. whole blood, platelet concentrates). Generally, HSA solutions ranging between 1 and 10% may be employed. An HSA solution prepared in a biocompatible bacteriostatic buffer solution will be passaged throughout the gas-fluid contacting device. Subsequent to passage, the HSA solution will be drained from the device. The gas-fluid contacting device and all surfaces that are in contact with the biological fluid during the method described are 1o consequently primed for use with platelet-containing biological fluids.
The present methods provide treatment of blood, blood fractionate or other fluid with a quantifiable absorbed dose of ozone to produce treated fluid that are then useful in the therapeutic treatment of the various diseases, conditions or symptoms described herein in mammals by administration of the treated blood, blood fractionate or other fluid to the patient. Thus, the fluid manufactured in accordance with the methods described herein provide substances or compositions that may be characterized as medicaments for the treatment of the disease conditions described herein, or may be used in the manufacture of therapeutic medicaments for the treatment of the diseases, conditions and/or symptoms described herein.
In one or more embodiments of the invention, an aliquot of a mammalian patient's blood, or the separated cellular fractions of the blood, or mixtures of the separated cells, including platelets, is extracorporeally subjected to a measured amount of ozone such that it absorbs a quantifiable absorbed-dose of ozone. On reintroduction of this autologous aliquot to the patient's body, the blood, blood fractionate or other fluid that has been treated with a quantifiable absorbed-dose of ozone is used to promote and produce beneficial effects in the treatment of the diseases described herein.
Reintroduction of this treated autologous aliquot may be through a variety of routes including intravenous, intramuscular and subcutaneous, or any combination thereof.
In another embodiment of the invention, the methods are directed to causing sufficient leukocyte apoptosis necessary to elicit clinical benefit when reinfused autologously into a patient. The methods may further cause sufficient leukocyte apoptosis without excessive necrosis necessary to elicit clinical benefit when reinfused autologously into a patient.
In certain embodiments of the invention, the autologous reinfusion of a patient's own blood or other body fluids provide therapeutic treatment by causing a reduction in CRP sufficient to elicit clinical benefit.

The methods of the present invention may comprise connecting a subject to a device for withdrawing blood, withdrawing blood containing blood cells from the subject, separating a non-cellular fraction from the blood and delivering a measured amount of ozone to the fraction under conditions which maintain the biological integrity of the blood fraction. The treated fraction is subsequently recombined with the blood cells and re-infused into the subject.
In another embodiment of the invention, therapeutic treatments for cardiovascular diseases and vascular disorders associated with deficient endothelial function, such as vasospastic disorders, may elicit a reduction of inflammation when to reinfused autologously into a patient by reducing proinflammatory cytokines (e.g.
interferon-gamma, TNF-gamma, IL-6, IL-8 and IL-12) and/or an increase in anti-inflammatory cytokines (e.g. interleukin-4 and IL-10) released by immunomodulatory T
cells.
The therapeutic effects derived from the treatment and reinfusion of blood, blood fractionate or other fluid which have absorbed a quantifiable absorbed-dose of ozone in accordance with the methods described herein include changes in lipid metabolism and enhancement of the immune system through stimulation of leukocytes (i.e. cell-cell interaction or cytokine release) throughout the peripheral blood of the patient leading to reduction in atherosclerotic plaque formation and deposition. These effects may result in a reversal in progressive luminal narrowing in arteries, a reduction in the rupture or denudation of plaques decreasing the incidence of atheroembolism (cholesterol embolism), and improved blood flow yielding enhanced oxygenation.
Regarding disorders involving atherosclerotic plaque formation, deposition and rupture, the present methods provide therapeutic treatment and prophylaxis of a wide variety of such mammalian disorders including cardiovascular diseases, such as atherosclerosis, peripheral arterial occlusive disease, cerebrovascular disease (stroke and transient ischemic attack), myocardial infarction, angina, hypertension, retinal ischemia, renal failure, abdominal aortic aneurysm, and hyperlipidemia.
Further, treatment and reinfusion of biological*fluids in accordance with the methods described herein provide therapeutic treatment of cardiovascular disease, including vascular disorders associated with deficient endothelial function, such as vasospastic disorders. The present methods provide for therapeutic stimulation of the activity of a functionally deficient endothelium.
Other beneficial effects that may derive from the methods of the present invention, as described herein, include reduction of edema, which may be brought about by inducing leukocyte apoptosis without excessive necrosis. Additional benefits include reduction of CRP to elicit an anti-inflammatory response, and the promotion of blood flow to ischemic tissue. The effectiveness of blood flow to ischemic tissue brought about by the therapeutic methods of the present invention may be evaluated by a variety of diagnostic tools including MRI, CT perfusion and Doppler imaging techniques.
Therapeutic treatment and reinfusion of biological fluids in accordance with the methods described herein are further directed to enhancement of the immune system through stimulation of leukocytes (i.e. cell-cell interaction or cytokine release) throughout the peripheral blood of the patient leading to improved blood flow, increase in vasodilation (i.e. promotion of vasodilators or inhibition of vasoconstrictors), 1o improvement in endothelial function including endothelial cellular repair or replacement and enhanced oxygenation. The present methods provide for the therapeutic treatment and prophylaxis of a wide variety of such mammalian disorders, including cardiovascular diseases such as atherosclerosis, peripheral arterial occlusive disease, congestive heart failure, cerebrovascular disease (stroke), myocardial infarction, angina, hypertension, vasospastic disorders such as Raynaud's disease, cardiac syndrome X, and migraine.
The methods of the present invention may include treatment of blood or other biological fluids from a mammalian patient by a discontinuous flow method where blood is withdrawn from the patient using a device suitable for withdrawing blood, separating a non-cellular fraction from the blood and delivering a measured amount of ozone to the fraction under conditions which maintain the biological integrity of the blood fraction, recombining the blood cells with the blood and reinfusing the treated blood into the patient. In those aspects of the invention where the method of treatment involves a discontinuous approach, the volume of blood removed can range from 1 to 5000 ml, depending on patient size and blood volume. This discontinuous treatment approach may be performed once or multiple consecutive times during a single treatment.
Methods of the present invention also include removing blood directly from a subject and re-infusing it to the same subject or patient in a continuous loop configuration. The blood may circulate through the loop, which includes the gas-fluid contacting device, once or multiple times, wherein a measured amount of ozone is delivered to the blood under conditions which maintain the biological integrity of the blood. The treated blood is constantly being re-infused directly back into the same patient.
Methods of the present invention are further effective in providing therapeutic treatment of blood and biological fluids to reduce cholesterol, triglycerides and other lipids in a mammalian patient. Such methods comprise removing blood directly from a subject and reinfusing it to the same patient in a continuous loop configuration.

In those aspects of the invention where the method of treatment involves a continuous loop approach, the volume of blood treated can range between can vary between 10 ml and the total estimated circulating blood volume of a mammalian patient being treated multiple times. Generally, the blood volume treated will range between 10 ml and 10,000 ml and preferably range between 10 ml and 6,000 ml.
The time required for an individual treatment through the use of a continuous loop format is based on a number of factors including the desired number of passes through the loop, volume of the fluid treated, the flow rate at which the fluid is circulating, the interface time required between the fluid and the amount of delivered-ozone, and the amount of the quantifiable absorbed-dose of ozone required. The time for the treatment can range from 1 minute to 720 minutes and preferably range from 1 minute to 180 minutes.
The number and frequency of treatments can vary considerably based upon the clinical situation of a particular patient. Generally the number of treatments can range between an individual treatment and 200 treatments, to be provided on a daily, alternate day or other schedule based on the clinical evaluation of the patient and desired clinical outcomes. Upon completion of a number of treatments and evaluation by a health care practitioner, another course of treatments may be indicated.
Alternative applications of the present methods involve plasmapheresis, wherein the patient's plasma is selectively removed while the balance of the blood cells is immediately returned to the patient. A measured amount of ozone is delivered to the isolated plasma under conditions which may maintain the biological integrity of the plasma. The treated plasma is subsequently re-infused into the subject.
The methods of the present invention are described for treatment of conditions attendant to cardiovascular disease, inflammatory disease, acute ischemic brain stroke, and conditions and symptoms related thereto, comprising methods that employ ozone delivery devices that are constructed with all ozone-contacting surfaces being made or constructed of ozone-inert materials to assure accurate determination of the amount of ozone delivered to a fluid being treated, and to assure accurate determination of the 3o amount of ozone absorbed by the fluid. The ozone delivery structures and related methods to treat blood and other biological fluids with ozone, and the use of those fluids for therapeutic treatments as disclosed herein, may be varied from those described to adapt them to specific applications. Therefore, reference to specific constructions and methods of use are by way of example and not by way of limitation.

Claims (21)

1. A method of producing a therapeutic substance for the treatment of acute ischemic brain stroke, and related symptoms or conditions, comprising:
providing a biological fluid;
delivering to the biological fluid a measured amount of ozone to produce a therapeutic substance having a quantifiable absorbed-dose of ozone which, upon administration to a subject suffering from, or believed to be suffering from, acute ischemic brain stroke, effectively treats the symptoms and conditions related to the acute ischemic brain stroke.

2. A method of producing a therapeutic substance for the treatment of cardiovascular disease and related symptoms or conditions thereof, comprising:

providing a biological fluid;
delivering to the biological fluid a measured amount of ozone to produce a therapeutic substance having a quantifiable absorbed-dose of ozone which, upon administration to a subject suffering, or believed to be suffering, from cardiovascular disease, effective treats the symptoms and conditions related to the cardiovascular disease.

3. A method of producing a therapeutic substance for the treatment of inflammatory disease and related symptoms or conditions, comprising:
providing a biological fluid;
delivering to the biological fluid a measured amount of ozone to produce a therapeutic substance having a quantifiable absorbed-dose of ozone which, upon administration to a subject suffering, or believed to be suffering, from inflammatory disease, effectively treats the symptoms and conditions related to the inflammatory disease.

4. The method according to claims 1, 2 or 3, wherein the biological fluid is blood, a blood derivative or blood fractionate.

5. The method according to claims 1, 2 or 3, wherein said blood derivative is plasma.

6. The method according to claims 1, 2 or 3, wherein said blood fractionate comprises platelets.

7. The method according to claims 1, 2 or 3, wherein the biological fluid is withdrawn from a mammalian subject.

8. The method according to claims 1, 2 or 3, wherein said therapeutic substance having a quantifiable absorbed dose of ozone is suitable for autologous reintroduction to a donor subject.

9. The method according to claims 1, 2 or 3, wherein said therapeutic substance is made under conditions that maintain the biological integrity of the fluid.

10. The use of a therapeutic substance manufactured in accordance with any of the preceding claims for preparation of a therapeutic medicament that is administrable to a mammalian patient suffering from, or thought to be suffering from, acute ischemic brain stroke, cardiovascular disease, inflammatory disease, or related conditions or symptoms associated with any of those diseases.

11. A medicament for the treatment of acute ischemic brain stroke, and the symptoms or conditions related thereto, comprising a biological fluid containing a quantifiable absorbed-dose of ozone to provide efficacious therapeutic effect to a subject suffering, or believed to be suffering, from acute ischemic brain stroke and the symptoms or conditions related thereto upon administration of the medicament to the subject.

12. A medicament for the treatment of acute ischemic brain stroke, and the symptoms or conditions related thereto, comprising a biological fluid containing a quantifiable absorbed-dose of ozone to provide efficacious therapeutic effect to a subject suffering, or believed to be suffering, from acute ischemic brain stroke and the symptoms or conditions related thereto upon administration of the medicament to the subject.

13. A medicament for the treatment of inflammatory disease, and the symptoms or conditions related thereto, comprising a biological fluid containing a quantifiable absorbed-dose of ozone to provide efficacious therapeutic effect to a subject suffering, or believed to be suffering, from inflammatory disease and the symptoms or conditions related thereto upon administration of the medicament to the subject.

14. The medicament according to claims 11, 12 or 13, wherein the biological fluid is blood, a blood derivative or blood fractionate.

15. The medicament according to claims 11, 12 or 13, wherein the blood fractionate is comprised of platelets.

16. The medicament according to claims 11, 12 or 13, wherein the blood derivative is plasma.

17. The medicament according to claims 11, 12 or 13, wherein the biological fluid, blood, blood derivative or blood fractionate is harvested from a mammalian subject.

18. The medicament according to claims 17, wherein the fluid containing a quantifiable absorbed-dose of ozone is manufactured under conditions that maintain the biological integrity of the fluid.

19. The medicament according to claim 18, wherein the fluid containing a quantifiable absorbed-dose of ozone is effective in treating reduction in edema associated with an ischemic penumbra, improvement in impaired blood flow to an area surrounding an infarct that may include ischemic tissue and the ischemic penumbra, relaxation of vascular endothelium, reduction of inflammation, paralysis, visual and auditory deficits and cognitive function incident to acute ischemic brain stroke.

20. The medicament according to claim 18, wherein the fluid containing a quantifiable absorbed-dose of ozone is effective in treating conditions or symptoms relating to cardiovascular disease including reduction in atherosclerotic plaques, regression in atherosclerotic plaque formation, reduction of the ischemic penumbra, relaxation of the vascular endothelium, reduction of inflammation, reduction in lipids and lipid deposits, reduction in or improvement of atherosclerosis, peripheral arterial occlusive disease, congestive heart failure, hypertension, cerebrovascular disease, dyslipidemia and vasospastic disorders, including Raynaud's disease, reduction in cholesterol, triglycerides and other lipids, reduction in the frequency, severity and duration of episodes of intermittent claudication, reduction in extremity numbness, weakness and loss of movement, improvement in extremity pallor, temperature and pulse, reduction in elevated blood pressure, and weight loss.

21. The medicament according to claim 18, wherein the fluid containing a quantifiable absorbed-dose of ozone is effective in treating conditions or symptoms relating to inflammatory disease, including rheumatoid arthritis, multiple sclerosis, systemic lupus erythromatosis (SLE), scleroderma, diabetes, inflammatory bowel disease, psoriasis, pemphigus, atherosclerosis, chronic heart failure, graft versus host reactions including tissue transplant rejection, Alzheimer's disease, ischemic brain stroke, senile dementia, depression, Down's syndrome, Huntington's disease, peripheral neuropathies, spinal cord diseases, neuropathic joint diseases, chronic inflammatory demyelinating disease (CIPD) and neuropathies, including mononeuropathy, polyneuropathy, symmetrical distal sensory neuropathy, cystic fibrosis, neuromuscular junction disorders, myasthenias, Parkinson's disease, traumatic brain injury, spinal cord injury and soft tissue injuries.