WO2007115398A1

WO2007115398A1 - Treatment of elevated c-reactive protein levels

Info

Publication number: WO2007115398A1
Application number: PCT/CA2007/000565
Authority: WO
Inventors: Arkady Mandel; Anthony Ernest Bolton
Original assignee: Vasogen Ireland Limited
Priority date: 2006-04-10
Filing date: 2007-04-05
Publication date: 2007-10-18

Abstract

There is provided a method of diagnosing mammalian patients at risk of developing cardiovascular disease, and providing prophylaxis or treatment of patients so diagnosed, which comprises measuring serum CRP levels in patients, selecting patients exhibiting elevated CRP levels, and administering to such patients an aliquot of autologous blood which has been treated ex vivo with at least one stressor selected from the group consisting of an oxidizing agent, an electromagnetic emission and elevated temperature.

Description

TREATMENT OF ELEVATED C-REACTIVE PROTEIN LEVELS

FIELD OF THE INVENTION

This invention relates to medical treatments, compositions for use in medical treatments, diagnosis of medical conditions and assessment of suitability of patients to receive selected medical treatments and compositions More specifically, it relates to medical treatments which involve regulation of C-reactive protein and reduction of known risk factors for cardiovascular disease and other conditions in the mammalian body, and diagnoses and assessments involving C- reactive protein measurements

BACKGROUND OF THE INVENTION

C-reactive protein is produced by the liver in response to inflammation, and is an acute phase protein produced in response to major trauma etc Increased blood levels of C-reactive protein are present in many infections and inflammatory diseases, and measurements of CRP in blood have been used for many years to assist the diagnosis of such conditions A highly sensitivity assay for serum CRP has recently become widely available, and, using this more sensitive assay system, it has been shown that mildly elevated serum levels of CRP correlate with increased risk of developing a number of severe pathological events (Ridker PM et al Ore (1998) 97(20) 2007-11 , Biasucci LM et al Circ 1999) 99(7) 855-60, Ridker PM et al N Engl J Med (2000) 342 (12) 836-43, Ridker PM et al N Engl J Med (2005) 352(1 ) 20-8, Danesh J et al N Engl J Med (2004) 350(14) 1387 97), including heart attacks, stroke and other cardiovascular events CRP levels in blood are routinely measured using a high sensitivity assay when assessing a patient's cardiovascular condition Tests of CRP levels in serum are used to classify patients as at low risk, medium risk or high risk of developing cardiovascular disease or cardiovascular events in the future A serum CRP level from 1 0 - 3 0 mg'L indicates a patient at medium risk, a serum CRP level higher than 3 0 mg/L CRP indicates a patient at high risk of developing future cardiovascular disease or cardiovascular events Patients with serum CRP levels less than 1 0 mg/L are considered to be of low risk of cardiovascular events (Pearson TA, Mensah GA, Alexander RW, et al Markers of inflammation and cardiovascular disease application to clinical and public health practice A statement for healthcare professionals from the Centers for Disease Control and Prevention and the American Heart Association Circulation 2003, 107 499-51 1 )

Elevated serum CRP levels are considered to be the strongest and most significant predictor of the risk of future cardiovascular events yet identified (Ridker PM et al, N Engl J Med 2000,342 836-43) As lowering of certain other cardiovascular risk factors, eg blood cholesterol, has been shown to reduce the risk of patients developing cardiovascular disease, it is now widely accepted that lowering of serum CRP levels is also beneficial in lowering the risk that such a palient will suffer from cardiovascular disease, cardiovascular events, thrombosis or other conditions associated with elevated serum CRP levels in the future A substantial body of evidence has established that CF P is not just a passive reflection of systemic inflammation but actually plays a central role in the inflammatory processes involved in atherosclerosis (Jialal I, Devaraj S, Venugopal SK C-reactive protein risk marker or mediator in atherothrombosis? Hypertension 2004 Jul,44(1 ) 6-1 1 ), and in thrombosis There are also other known risk factors for cardiovascular disease or disorders, which are used by medical professionals to gauge the risk of a person developing a cardiovascular disorder High blood pressure increases the risk of developing heart disease, heart attack and stroke High cholesterol (especially the low density lipoprotein, LDL) will contribute to the formation of plaque on artery walls which contributes to atherosclerosis, thus increasing the risk of cardiovascular disorders Diabetes is also a risk factor for cardiovascular disorders (Texas Heart Institute website www texasheartinstitute org/HIC/Topics/Hsmart/riskfact cfm)

Obesity and being overweight can also contribute to cardiovascular disorders as the extra weight or obesity can lead to increased total cholesterol levels, high blood pressure, and diabetes, as well as increasing the risk of suffering cardiovascular disorders Often, a peison's body mass index (BMI) is used to measure obesity, where the peison's mass in kilograms is divided by height in meters squared A pei son with a BMI of over 25 is considered to be overweight, while a BMI of over 30 is considered obese, according to the National Heart, Lung, and Blood Institute (Texas Heart Institute, supra) Thus a person with a BMI of 25 has a greater risk of developing cardiovascular disease or disorders

Smoking also increases the risk of heart disease and peripheral vascular disease Smoking increases heart rate, constricts major arteries, raises blood pressure, and can create irregularities in the tirring of heartbeats, thus causing the heart to work harder In addition, some of the chemical by-products of smoking can cause build-up of plεque in the arteries, and can affect cholesterol, and fibrinogen (a compound involved with clotting), thereby increasing the risk of the formation of blood clots which can lead to a heart attack (Texas Heart Institute, supra)

Stress is also considered as a contributing factor for heart disease Emotional stress, psychosomatic stress and physical stress can affect the heart by the elevation of blood pressure and heart rate As well, in times of stress, various hormones (and most often adrenaline) are released which can raise blood pressure or contribute to injury of the lining of arteries, thus possibly facilitating the formation of plaque in the artery Stress also increases the amount of blood clotting factors in circulation, thus increasing the possibility of clot formation, which can contribute to cardiovascular disorders

Excessive alcohol consumption can also contribute to cardiovascular disorders by playing a role high blood pressure, stroke, irregular heartbeats, and cardiomyopathy In addition, consumption of alcohol can increase body weight, which increased body weight can increase the risk of development of cardiovascular disorders (Texas Heart Institute, supra)

Hormone replacement therapy (HRT) for the treatment of menopause can contribute to the development of cardiovascular disease as women undergoing HRT may have an increased risk of blood clots, heart attack, and stroke (U S Food and Drug Administration Heart Health

Online, www fda qov/hearthealth/riskfactors/riskfactors html)

Both vascular smooth muscle cells and endothelial cells can produce CRP in response to stimulation by inflammatory cytokines (Calabro et al, 2003, Venugopal et al, 2005) In turn, CRP stimulates the release of nflammatory cytokines including TNF-alpha, IL-1 beta and IL6 from monocytes (Ballou & Lozaπski, 1992) This suggests the possibility of a localised positive feedback system at the level of the endothelium, with inflammatory cytokines released by monocytes stimulating the endothelium to produce CRP, which will stimulate further release of inflammatory cytokines by monocytes

CRP has a direct pro-inflammatory effect on human endothelial cells CRP induces the expression of the macrophage chemokines IL-8 (Devaraj et al, 2003b) and MCP-1 by endothelial cells (Torzewski et al, 2000, Pasceri et al, 2001 ) In addition to macrophage attraction, CRP also significantly induces the up-regulation of expression of adhesion molecules, including ICAM-1 (by -10 fold), VCAM-1 and E-selectin (Pasceri et al, 2000), and thus facilitates the diapedesis of macrophages into the arterial wall, an important event in the development of atherosclerotic plaque In pathological terms, this interaction extends to highly specific binding to oxidised LDL (Chang et al, 2002) -enhancing its uptake by macrophages (Zwaka et al, 2001 ), a process resulting in foam cell formation Uptake of CRP by macrophages leads to the induction of reactive oxygen species (ROS) production and release (Tebo & Mortensen, 1991 ) - ROS are implicated in the pathogenesis of a number of cardiovascular diseases including atherosclerosis CRP has been demonstrated to stimulate the expression of metalloproteinase MMP-1 by macrophages (Williams et al, 2004) - MMP-1 is implicated in human plaque instability This is supported by the finding that CRP localises in atherosclerotic arteries and, in coronary atherectomy specimens, histological features of plaque instability were associated with CRP immunoreactivity (Kobayashi et al, 2003) Many inflammatory molecules have been identified in plaque material characteristic of atherogenesis and it is now widely accepted that chronic inflammation contributes to the progression of plaque CRP has been shown to be an activator of complement (Siegel et al, 1974, Volanakis 1982, Wolbink et al, 1996) The expression of both complement proteins and CRP are up-regulated in plaque and, in the case of CRP, the level of expression of CRP mRNA is >10 times that in normal arteries (Yasojima et al, 2001 ) CRP also activates a number of transcription factors in vascular smooth muscle cells, including NF- kappa B, leading to the induction of a number of inflammatory factors including MCP-1 and IL-6, suggesting that CRP plays a further role in atherogenesis by activating vascular smooth muscle cells (Hatton et al, 2003) an important event in the development of atherosclerotic plaque

The function of CRP is a primitive host defence protein that acts by interacting with specific oxidised phospholipids on the surface of bacteria facilitating their internalisation

Thus, CRP has activities that suggest an intimate involvement and facilitation of the events associated with early plaque formation as well as in the inflammatory pathology and instability of plaque

Inflammatory responses are often accompanied by thrombotic events CRP has been shown to demonstrate a number of pro-thrombotic activities Thus, CRP decreases the expression of endothelial nitric oxide synthase (Venogupal et al, 2002) - the source of the endothelial vasodilator nitric oxide - as well as decreasing the release of prostacyclin from endothelial cells (Venogupal et al, 2003) The decrease in these platelet inhibitors is accompanied by an effect of CRP increasing the production of the pro-thrombotic factors endothelin 1 (Verma et al, 2002) and plasminogen activator ιnhιbιtor-1 expression (Devaraj et al, 2003a) from endothelial cells CRP also induces the syrthesis of tissue factor by peripheral blood monocytes (Cermak et al, 1993) Experimentally, human CRP transgenic mice show an enhanced rate of thrombotic occlusion following injury of the femoral artery (Danenberg et al, 2003), providing further evidence that CRP has. an overall pro-thrombotic activity The above demonstrates that elevated CRP levels in patients are predictive of being at greater risk of suffering atherosclerotic and/or thrombotic disorders

Atherosclerotic and/or thrombotic disorders which affect the cardiovascular system are considered to be cardiovascular disorders

Accordingly, treatments that lower serum CRP levels in the blood of a mammalian patient would be particularly beneficial in lowering of the risk of that patient being afflicted with one or more mammalian disorders associated with high serum levels of CRP, including atherogenesis and thrombosis

SUMMARY OF THE INVENTION

The present invention is based upon the discovery that blood treated with at least one stressor such as ozone, will, upon administration to a mammalian patient, cause a rapid decrease in the serum level of CRP in a mammalian patient Accordingly, the treated blood may be used to reduce elevated serum CRP levels in a mammalian patient

As per above, elevated levels of serum CRP in a patient are predictive of ^*uture adverse events in that patient including atherogenesis and thrombosis (as well as atherosclerotic and thrombotic disorders), and to a degree causative of them This invention is directed to methods for lowering serum CRP in those patients exhibiting elevated serum CRP levels It also relates, in one aspect, to use of diagnostic methods and data therefrom to select groups of patients likely to derive most benefit from the treatment

According to a first aspect, this invention provides use in preparation of a medicament for reducing the serum level of C-reactive protein (CRP) in a mammalian patient and consequently reducing the risk of that patient's subsequently developing a cardiovascular disorder, for administration to patient who has been diagnosed with elevated serum CRP level, of an aliquot of the patient's own blood which has been treated ex vivo with at least one stressor which is an oxidizing agent, an electromagnetic emission or elevated temperature Preferably, the elevated serum CRP level is at least 1 0 mg/L More preferably, the elevated serum CRP level is at least 3 0 mg/L

In another aspect, the present invention provides a method of diagnosing the need for treatment of mammalian patients to lessen the risk of the patients' subsequently developing cardiovascular disorders and subsequently treating those patients determined to be in need thereof, which comprises determining the serum CRP levels of a group of mammalian patients, selecting from said group a sub-group of patients evidencing elevated serum CRP level, and treating members of said sub-group with an effective amount of auiologous blood which has been treated ex vivo with at least one stressor which is an oxidizing agent, an electromagnetic emission or elevated temperature to reduce the serum CRP level in that patient In a further aspect, the cardiovascular disorder is selected from the group comprising atherosclerotic and thrombotic disorders According to another aspect of the invention, there is provided the use in preparation of a medicament for treatment and prophylaxis of elevated serum CRP levels in a mammalian patient, of treated autologous blood

In yet another aspect of the invention, there is provided a method for decreasing the subsequent development of elevated serum CRP related disorders in mammalian patients, which comprises testing the group of patients for the presence of one or more risk factors associated with cardiovascular disorders selected from the group comprising high blood pressure, high cholesterol levels, diabetes, obesity, tobacco smoking, stress, body mass index exceeding 25, excessive alcohol consumption, hormone replacement therapy, and familial history of cardiovascular events of a group of mammalian patients, selecting from said group a sub-group of patients having one or more of Ihe risk factors associated with cardiovascular disorders, testing members of said subgroup for serum CRP levels,, selecting from said sub-group a sub-sub-group of patients with elevated serum CRP levels, ard treating members of said sub-sub-group with an effective amount of ireated autologous blood

In <a further aspect, the elevated serum CRP associated disorder is selected from the group comprising atherosclerotic and thrombotic disorders Currently, elevated serum CRP levels are considered to be those higher than 1 0 mg/L If elevated, the patient is then treated with a pharmaceutically effective amount treated autologous blood

The preparation of the treated autologous blood for use in the present invention preferably comprises extracting from the subject an aliquot of blood of volume about 0 01 ml to about 400 ml, and contacting the alicuot of blood, extracorporeal^, with an immune system-stimulating effective amount of ozone gas and an electromagnetic transmission

Following administration to the mammalian patient, the treated blood is believed to interact rapidly with the immune system which results in lowering of serum CRP levels

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graphical representation of the clinical predictive value of serum CRP levels in respect of relative risk of future cardiovascular events

Figure 2 is a graphical representation of the change in serum CRP levels in patients participating in the study described in Example 1 below, from baseline to weeks 26 and 50

DESCRIPTION OF PREFERRED EMBODIMENTS According to the present invention, blood from patients having elevated serum CRP levels (for example, above the accepted low risk level, currently accepted to be less than 1 mg/L), is treated extracorporeal^, with at least one stressor such as an electromagnetic emission or ozone (including combinations of stressors) The treated blood is then re-injected to the patient, with consequent lowering of serum CRP levels and concomitant lowering of risk to the patient developing cardiovascular disease and other conditions in which elevated serum CRP levels is a known risk factor

In humans, a serum CRP level of at least 1 0 mg/L indicates that the patient is at risk of developing an adverse future cardiovascular disease or event Cardiovascular events are a set of adverse clinical events that could be exemplified, but not limited, to coronary heart disease (defined by the presence of prolonged typical chest pain, ECG changes, enzymes, and percutaneous transluminal angioplasty or coronary artery bypass grafting), congestive heart failure, transient ischaemic attack or stroke, or peripheral atherosclerotic disease

In some instances, serum levels of CRP as low as 0 7 mg/L can be construed as elevated particularly if there is familial history (MDS, Inside Diagnostics, April 2003, Rifai, N , and Ridker, P M , Proposed Cardiovascular Risk Assessment Algorithm using High Sensitivity C- Reactive Protein and Lipid Screening, CIm Chem ,2001 , 47 28-30) Se'um CRP levels of greater than about 3 0 mg/L indicate that the patient is at high risk of developing an adverse future cardiovascular disease or event Minor elevations in the serum level of CRP have been shown to be associated with future major cardiovascular risk ((Pfutzner A, Forst T , Diabetes Technol Ther 2006 Feb,8(1 ) 28-36), For example, a minor serum CRP level elevation in respect of atherosclerotic events could be defined as an elevation of serum CRP levels between 3 and 10 mg/L (Kushner I, et al , What does minor elevation of C-reactive protein signify^Am J Med 2006 Feb, 119(2) 166 e17-28)), is associated with a great many diverse conditions, some of which are, or may prove to be, causal It is contemplated that some patients having low serum CRP levels (ι e less than 1 mg/L) and having defined risk factors could benefit from treatment according to the invention, as even small increases (although not high enough to fall within the high risk group) in serum CRP levels would indicate an increased risk for such patients For example, when such patients are regularly monitored for serum CRP levels and there is an indication of a slight but statistically significant increase in serum CRP levels, the patient can then be treated according to the invention to decrease the risk of future cardiovascular disease or events

Exactly how the treatment operates following this re-injection is not currently fully understood The following tentative explanation is offered for a better and more complete description of the invention, but is not to be considered as binding or limiting

T-cells, which are one kind of lymphocyte and which play a significant role in the control of the immune system, include CD-8 cells, and CD-4 cells otherwise known as T-helper cells, further sub-dividable into Th1 and Th2 cells The Th1 cells secrete pro-inflammatory cytokines such as interferon gamma (IFN-γ) The Th2 cells are considered to be regulatory cells and secrete regulatory cytokines, such as ιnterleukιn-4 (IL-4) A number of components of the treated blood of the present invention, possibly including HLA-DR and/or other MHC antigens released from the leucocyte cell surfaces have numerous effects including upregulating the Th2 cells in the patient's blood and/or locally at the site of the inflammation, thereby increasing the secretion of regulatory cytokines

A patient or a subject is identified as having a need for prophylaxis or treatment of disorders in which elevated serum CRP is associated with inci eased risk of adverse outcomes and the patient is determined as having elevated serum CRP levels (for example, above the accepted low risk level, currently considered to be less 1 0 mg/L) An anti- coagulated blood aliquot from the patient is prepared by exposing the aliquot to at least one stressor, in controlled amounts, the stressor being selected from among oxidizing agents such as ozone, ultraviolet radiation and elevated temperature Combinations of two or all three of such stressors are also preferred The resulting blood aliquot, after such treatment, can be re-injected into the patient

Preferably, the stressors to which the cells in the extracted blood aliquot are subjected are a temperature stress (blood temperature above body temperature), an oxidative environment such as a mixture of ozone and oxygen bubbled through the blood aliquot, and an electromagnetic emission, individually, in any combination, simultaneously or successively, but preferably simultaneously

In general, from about 0 01 ml to about 400 ml of blood may be treated according to the invention Preferred amounts are in the range of about 0 1 to about 200 ml More suitably, the aliquot for treatment has a volume of from about 0 1 to about 100 mis, preferably 1 to about 50 ml and most preferably 5 to 15 ml The method most preferably involves treating an aliquot of about 10 mis blood with ozone gas and an electromagnetic emission, then re-administering the treated blood to the patient by intramuscular injection

It is preferred, according to the invention, to apply a temperature stress (blood temperature above or below body temperature), in addition to the electromagnetic emission stress and the oxidative stress Preferably, all three of the aforementioned stressors are applied simultaneously to the aliquot under treatment, in order to ensure the appropriate modification to the blood Care must be taken to utilize an appropriate level of the stressors to thereby effectively modify the blood to alleviate the acute inflammatory condition in the subject

The temperature stressor warms the aliquot being treated to a temperature above normal body temperature or cools the aliquot below normal body temperature The temperature is selected so that the temperature stressor does not cause excessive hemolysis in the blood contained in the aliquot and so that, when the treated aliquot is injected into a subject, reduction of the serum CRP levels will be achieved Preferably, the temperature stressor is applied so that the temperature of all or a part of the aliquot is up to about 55°C, and more preferably in the range of from about -5°C to about 55⁰C

In some preferred embodiments of the invention, the temperature of the aliquot is raised above normal body temperature, such that the mean temperature of the aliquot does not exceed a temperature of about 55°C, more preferably from about 40⁰C to about 50°C, even more preferably from about 40°C to about 44°C, and most preferably about 42 5±1 °C

In other preferred embodiments, the aliquot is cooled below normal body temperature such that the mean temperature of the aliquot is within the range of from about -5°C to about 36 5°C, even more preferably from about 1 O⁰C to about 30⁰C, and even more preferably from about 15°C to about 25°C

Alternatively, the blood sample is heated while being subjected to an electromagnetic emission until the blood reaches a predetermined temperature (preferably about 42 5+1 Celsius) at which point bubbling of ozone gas through the blood is commenced The concurrent electromagnetic emission/ozone treatment is then maintained for a predetermined period of time, preferably about 3 minutes

Another alternative method involves subjecting the blood to electromagnetic emission/ozone while heating to a predetermined temperature (preferably about 42 5+1 ° Celsius), then either ending the treatment once the predetermined temperature is reached, or continuing electromagnetic emission/ozone treatment for a further period of time, most preferably about 3 minutes

The oxidative environment stressor can be the application to the aliquot of solid, liquid or gaseous oxidizing agents Chemical oxidants such as hydrogen peroxide or sodium butyrate can be used Preferably, it involves exposing the aliquot to ozone gas More preferably, it involves exposing the aliquot to a mixture of medical grade oxygen and ozone gas, most preferably by bubbling through the aliquot, at the aforementioned temperature range, a stream of medical grade oxygen gas having ozone as a minor component therein The ozone content of the gas stream and the flow rate of the gas stream should be selected such that the amount of ozone introduced to the blood aliquot, either on its own or in combination with other stressors, does not give rise to excessive levels of cell damage such that the therapy is rendered ineffective Suitably, the gas stream has an ozone content of up to about 300 μg/ml, preferably up to about 100 μg/ml, more preferably about 30 μg/ml, even more preferably up to about 20 μg/ml, particularly preferably from about 10 μg/ml to about 20 μg/ml, and most preferably about 14 5±1 0 μg/ml The gas stream is suitably supplied to the aliquot at a rate of up to about 2 0 litres/mm, preferably up to about 0 5 litres/min, more preferably up to about 0 4 litres/min, even more preferably up to about 0 33 litres/min, and most preferably about 0 24±0 024 litres/min The lower limit of the flow rate of the gas stream is preferably not lower than 0 01 litres/min, more preferably not lower than 0 1 litres/min, and even more preferably not lower than 0 2 litres/mm

The electromagnetic emission stressor is suitably applied by irradiating the aliquot under treatment from a source of an electromagnetic emission while the aliquot is maintained at the aforementioned temperature and while the oxygen/ozone gaseous mixture is being bubbled through the aliquot Preferred electromagnetic emissions are selected from photonic radiation, more preferably UV, visible and infrared light, and even more preferably UV light The most preferred UV sources are UV lamps emitting primarily UV-C band wavelengths, i e at wavelengths shorter than about 280 nm Such lamps may also emit amounts of visible and infrared light Ultraviolet light corresponding to standard UV-A (wavelengths from about 315 to about 400 nm) and UV-B (wavelengths from about 280 to about 315) sources can also be used For example, an appropriate dosage of such UV light, applied simultaneously with the aforementioned temperature and oxidative environment stressors, can be obtained from lamps with a combined power output of from about 45

- 65 mW/cm ² Up to eight such lamps surrounding the sample container holding the aliquot, with a combined output at 253 7 nm of 15

- 25 watts, operated at an intensity to deliver a total UV light energy at the surface of the blood of from about 0 025 to about 10 joules/cm², preferably from about 0 1 to about 3 0 joules/cm² Preferably, four such lamps are used The time for which the aliquot is subjected to the stressors can be from a few seconds to about 60 minutes The time depends to some extent upon the chosen intensity of the electromagnetic emission, the temperature and the concentration of the rate at which the oxidizing agent is supplied to the aliquot Some experimentation to establish optimum times may be necessary on the part of the operator, once the other stressor levels have been set Under most stressor conditions, preferred times will be in the approximate range of from about 2 to about 5 minutes, more preferably around 3 minutes The starting blood temperature and the rate at which it can be warmed or cooled to a predetermined temperature, tends to vary from subject to subject Warming is suitably by use of one or more infrared lamps placed adjacent to the aliquot container Other methods of warming can also be adopted

In the practice of the preferred process of the present invention, the blood aliquot (or the separated cellular fractions of the blood, or mixtures of the separated cells, including platelets, these various leucocyte-combinations, along with whole blood, being referred to collectively throughout as the "aliquot") may be treated with the stressors using an apparatus of the type described in U S Pat No 4,968,483 Mueller The aliquot is placed in a suitable, sterile, L)V light- transmissive container, which is fitted into the machine The UV lamps are switched on for a fixed period before the gas flow is applied to the aliquot providing the oxidative stress, to allow the output of the UV lamps to stabilize The UV lamps are typically on while the temperature of the aliquot is adjusted to the predetermined value, e g 42 5±1 °C Then the oxygen/ozone gas mixture, of known composition and controlled flow rate, is applied to the aliquot, for the predetermined duration of up to about 60 minutes, preferably 2 to 5 minutes and most preferably about 3 minutes as discussed above, so that the aliquot experiences all three stressors simultaneously In this way, blood is appropriately modified according to the present invention to achieve the desired effects

Thus, the invention also provides a method of stimulating or activating the immune system in a human by contacting about 0 01 ml to about 400 ml of blood from a human with an immune system-stimulating amount of ozone gas and ultraviolet radiation, followed by administering the treated blood to a human It is believed that this stimulation or activation of the immune system may have the effect of lowering serum CRP levels in patients already having elevated serum CRP levels and suffering from or risk of suffering from disorders in which elevated serum CRP levels are associated with increased risk of adverse outcomes Similarly, the invention provides a method of lowering or preventing the increase of elevated serum CRP levels (for example, above the accepted low risk level, currently accepted to be less 1 mg/L) in a human by contacting about 0 01 ml to about 400 ml of blood from a human with an immune system-stimulating amount of ozone gas and ultraviolet radiation, followed by administering the treated blood to a human

A subject preferably undergoes a course of treatments, each individual treatment comprising removal of a blood aliquot, treatment thereof as described above and re-administration of the treated aliquot to the subject A course of such treatments may comprise daily administration of treated blood aliquots for a number of consecutive days, or may comprise a first course of daily treatments for a designated period of time, followed by an interval and then one or more additional courses of daily treatments In one preferred embodiment, the subject is given an initial course of treatments comprising the administration of 1 to 6, more preferably 4 to 6 aliquots of treated blood In another preferred embodiment, the subject is given an initial course of therapy comprising administration of from 2 to 4 aliquots of treated blood, with the administration of any pair of consecutive aliquots being either on consecutive days, or being separated by a rest period of from 1 to 21 days on which no aliquots are administered to the patient, the rest period separating one selected pair of consecutive aliquots being from about 3 to 15 days In a more specific, preferred embodiment, the dosage regimen of the initial course of treatments comprises a total of three aliquots, with the first and second aliquots being administered on consecutive days and a rest period of 1 1 days being provided between the administration of the second and third aliquots For optimum effectiveness of the treatment, it is preferred that no more than one aliquot of modified blood be administered to the subject per day, in one or more injection sites, and that the maximum rest period between any two consecutive aliquots during the course of treatment be no greater than about 21 days It may be preferred to subsequently administer additional courses of treatments following the initial course of treatments Preferably, subsequent courses of treatments are administered following a rest period of several weeks or months, preferably at least about three weeks, after the end of the initial course of treatments In one particularly preferred embodiment, the subject receives a second course of treatments comprising the administration of one aliquot of treated blood every 30 days following the end of the initial course of treatments, for a period of 6 months It may also be preferred in some circumstances to follow one or more of the above-described courses of treatment by periodic "booster" treatments, if necessary, to maintain the desired effects of the present invention For example, it may be preferred to administer booster treatments at intervals of 3 to 4 months following the initial course of treatment

It will be appreciated that the spacing between successive courses of treatments should be such that the positive effects of the treatment of the invention are maintained, and may be determined on the basis of the observed response of individual subjects

The present invention also includes a process for the selection of patients who have a familial background of disorders in which elevated serum CRP levels are associated with increased risk of adverse outcomes and who have elevated serum CRP levels (for example, above the accepted low risk level, currently considered to be less than 1 0 mg/L), whereby such patients will have blood withdrawn and treated with at least one stressor such as an electromagnetic transmission or ozone (including a combination of stressors) The treated blood is then re-injected to the patient with consequent lowering of serum CRP levels and concomitant lowering of risk to the patients developing or having progression of adverse outcomes related to disorders in which elevated serum CRP levels are associated with inci eased risk of adverse outcomes, especially atherogenesis and atherosclerosis, but most particularly thrombosis

The invention may also be useful in pre-conditioning individuals having elevated serum CRP levels but not exhibiting symptoms of disorders in which elevated serum CRP levels are associated with increased risk of adverse outcomes, to prevent or retard the progression of the disorders in which elevated serum CRP levels are associated with increased risk of adverse outcomes The; prophylaxis or treatment methods described herein may be administered in combination with one or more other modalities Examples of other preferred modalities include, but are not limited to, statin therapy Administration in combination includes, for example, administration of the treated blood described herein, prior to, during or after administration of the other one or more modalities One of skill in the art will be able to determine the administration schedule and dosage

EXAMPLE 1

This example describes some results from a phase III, double blind, randomized clinical trial in about 550 patients suffering from peripheral arterial disease (PAD) Male and female patients of age 40 years or older, were randomized to the study through about 50 centers in the United States and Canada Inclusion in the study was based on a history of significant intermittent claudication (Fontaine II), an ankle brachial index (ABI) of < 0 85, and an absolute claudication distance (ACD) consistently and reproducibly demonstrated to be > 50 meters on a treadmill operated at a constant speed with an incremental grade (Gardner protocol) For diabetic patients with calcified blood vessels, toe brachial index (TBI) of < 0 7 was used for inclusion instead of ABI Patients were randomly allocated between the treatment group and the control group and were to be tracked in the study for 50 weeks A number of primary, secondary and tertiary endpoints were assessed One of the tertiary endpoints was the measurement of serum CRP levels using a high sensitivity assay at weeks 26 and 50

Protocol Patients received multiple treatments in two phases, an induction phase and a maintenance treatment phase The induction phase was spread over two weeks during which three treatments were administered (either treated blood or placebo) at days 1 , 2, and 14 The maintenance phase, which began four weeks after the last treatment of the induction phase, consisted of single treatments given at four-week intervals out to 46 weeks

Each individual treatment comprised the following steps

1 Collection of 10 ml of a patient's venous blood into 2 ml of sodium citrate USP as an anti-coagulant to prevent the blood from clotting during the treatment

2 Transfer of the citrated blood sample to a sterile, disposable low density polyethylene container

3 Ex vivo treatment of the blood sample performed over a period of approximately 20 minutes by simultaneous exposure to elevated temperature of 42 5+1 O⁰C, a gas mixture of medical grade oxygen containing 14 5+1 0 μg/inl of ozone, at a flow rate of 240+24 ml/minute (at STP) for 3 minutes, and ultraviolet light at a wavelength of 253 7+10 nm

4 Transfer of treated blood from the sterile disposable container to a sterile syringe

5 Intramuscular injection of 8-10 ml of treated blood into the gluteal muscle of the same patient, following injection with a local anaesthetic (1 ml of 2% Novocaine or equivalent)

Approximately 35 minutes after blood collection, 10 ml of saline was administered to patients in the placebo group applying the same injection procedures used for the treated blood The; ex vivo treatment of the blood sample in step (3) above was performed with an apparatus as generally described in U S Patent No 4,968,483 to Mueller et al

Patients had blood withdrawn at the start of their involvement in the tria , week 26, and week 50 to test for serum CRP levels, using known test methods in a commercial test laboratory, to determine the change in serum CRP levels over time for both treatment and placebo groups

As seen in Figure 1 (from Ridker, P M and Cook, N , Circulation, 2004, 109 1955-59), serum CRP levels are a predictive indicator of relative risk of future cardiovascular events For example, a person having a CRP serum concentration of more than 20 mg/L, has about 8 times more risk of developing future cardiovascular events in comparison to a person having a serum CRP concentration of less than 0 5 mg/L

Serum CRP levels were measured in patients participating in the clinical trial described above As seen in Figure 2, there was a significant reduction in serum CRP levels of patients (modified intent to treat group which was the group which received at least one treatment injection and had at least one post-treatment treadmill test, "MITT"), who were administered the treated blood in comparison to placebo (MI TT group), when comparing the serum CRP levels at week 26 (p=0 09) and week 50 (p=0 008) to the baseline values (based on a ran< analysis of covanance using a Cochran Mantel-Haenszel Test adjusting for pooled site, on modified ridit scores (non-parametric Rank ANCOVA approach) At week 26, the MITT treatment group serum CRP levels decreased about 1 mg/L from baseline levels, while the MITT placebo group serum CRP levels increased slightly from baseline levels At week 50, the MITT treatment group serum CRP levels decreased about 1 mg/L from baseline levels, while the MITT placebo group serum CRP levels increased about 0 5 mg/L from baseline levels

The serum CRP levels were also determined for a sub-group of MITT patients having high serum CRP levels (greater than 3 mg/L and less than 50 mg/L) There were significant reductions in the serum CRP levels in this sub-group at both weeks 26 and 50 (p=0 004 and p=0 009 respectively) in comparison to the MITT placebo groups, relative to baseline values

Adverse cardiovascular events or disease associated with C-reactive protein levels in a mammalian patient refer to those cardiovascular events wherein damage to the cardiovasculature of the patient occurs Such damage can arise from hypertension, myocardial infarction, atherosclerosis, restenosis, and the like Adverse cardiovascular events also include, but are not limited, to coronary heart disease (defined by the presence of prolonged typical chest pain, ECG changes, enzymes, and percutaneous transluminal angioplasty or coronary artery bypass grafting), congestive heart failure, transient ischaemic attack or stroke, or peripheral atherosclerotic disease

Claims

WHAT IS CLAIMED IS

Use in preparation of a medicament for reducing the serum level of C-reactive protein (CRP) in a mammalian patient and consequently reducing the risk of that patient's subsequently developing a cardiovascular disorder, for administration to patient who has been diagnosed with elevated serum CRP level, of an aliquot of the patient's own blood which has been treated ex vivo with at least one stressor which is an oxidizing agent, an electromagnetic emission or elevated temperature

Use according to claim 1 wherein the patient has been diagnosed with elevated serum CRP level of at least 1 0 mg/L

Use according to claim 2 wherein the patient has been diagnosed with elevated serum CRP level of at least 3 0 mg/L

Use according to claim 1 , claim 2 or claim 3 wherein the blood aliquot has been treated with at least two of said stressors

Use according to any preceding claim wherein the blood aliquot has been treated with all three said stressors

Use according to claim 5 wherein the electromagnetic radiation stressor is UV radiation

Use according to claim 6 wherein the oxidizing agent stressor is gaseous ozone/oxygen mixture

Use according to any preceding claim wherein the patient has additionally been diagnosed with the presence of one or more risk factors associated with cardiovascular disorders selected from the group comprising high blood pressure, high cholesterol levels, diabetes, obesity, tobacco smoking, stress, body mass index exceeding 25, excessive alcohol consumption, hormone replacement therapy, and familial history of cardiovascular events A method of diagnosing the need for treatment of mammalian patients to lessen the risk of the patients' subsequently developing cardiovascular disorders and subsequently treating those patients determined to be in need thereof, which comprises determining the serum CRP levels of a group of mammalian patients, selecting from said group a sub-group of patients evidencing elevated serum CRP level, and treating members of said sub-group with an effective amount of autologous blood which has been treated ex vivo with at least one stressor which is an oxidizing agent, an electromagnetic emission or elevated temperature to reduce the serum CRP level in that patient The method of claim 9 wherein the cardiovascular disorders is an atherosclerotic or thrombotic disorders The method of claim 9 or claim 10 wherein the elevated serum CRP level is at least 3 0 mg/L The method of claim 8 wherein said oxidizing agent comprises ozone gas The method of claim 12 wherein the oxidizing agent comprises a mixture of medical grade oxygen and ozone gas The method of any of claims 9 -13 wherein the electromagnetic emission is ultraviolet radiation The method of claim 14 wherein the ozone gas and ultraviolet radiation are applied to the blood aliquot simultaneously, whilst the blood aliquot is at a temperature of from 40 -50 Celsius. Use in preparation of a medicament for treatment and prophylaxis of elevated serum CRP levels in a mammalian patient, of treated autologous blood which has been treated ex vivo with at least one stressor which is an oxidizing agent, an electromagnetic emission or elevated temperature.