WO2005097145A1 - Myocarditis treatment - Google Patents

Myocarditis treatment Download PDF

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Publication number
WO2005097145A1
WO2005097145A1 PCT/CA2005/000537 CA2005000537W WO2005097145A1 WO 2005097145 A1 WO2005097145 A1 WO 2005097145A1 CA 2005000537 W CA2005000537 W CA 2005000537W WO 2005097145 A1 WO2005097145 A1 WO 2005097145A1
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Prior art keywords
aliquot
blood
myocarditis
patient
symptoms
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PCT/CA2005/000537
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French (fr)
Inventor
Eldon R. Smith
Guillermo Torre-Amione
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Vasogen Ireland Limited
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Publication of WO2005097145A1 publication Critical patent/WO2005097145A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0023—Agression treatment or altering
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/10—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person
    • A61K41/17—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person by ultraviolet [UV] or infrared [IR] light, X-rays or gamma rays
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3681—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by irradiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3681—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by irradiation
    • A61M1/3683—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by irradiation using photoactive agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3687—Chemical treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/369—Temperature treatment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M2205/00—General characteristics of the apparatus
    • A61M2205/05—General characteristics of the apparatus combined with other kinds of therapy
    • A61M2205/051—General characteristics of the apparatus combined with other kinds of therapy with radiation therapy
    • A61M2205/053—General characteristics of the apparatus combined with other kinds of therapy with radiation therapy ultraviolet

Definitions

  • This invention relates to medical treatments , and to compositions of matter and procedures useful therein. More specifically, it relates to alleviation of symptoms of inflammatory disorders of the heart, notably myocarditis, and use of compositions therein.
  • the myocardium is the muscular layer of the heart, composed of myocytes.
  • Myocarditis is an inflammatory condition of the myocardium, commonly caused by viral infections, and less commonly by bacterial or parasitic infections. The condition may also be caused by exposure to chemicals or allergic reactions to certain medications, and it can be associated with autoimmune diseases.
  • C hagas disease has as its major cardiovascular manifestation an extensive myocarditis that typically becomes evident years after the initial infection. In myocarditis, the heart muscle becomes inflamed and weakened, and this commonly causes symptoms of heart failure.
  • Myocarditis is commonly a precondition leading to congestive heart failure. It is estimated that 25-30% of cases of chronic congestive heart failure might result from a myocarditis. For a significant proportion of these patients, treatment of the inflammation of the heart at an appropriate stage would likely
  • 8 ⁇ ffl TE SHEET prevent or at least decelerate progression to chronic (congestive) heart failure. Recognizing that congestive heart failure is now the commonest cause of hospital admissions in the age group over 65, this condition has major implications to the health system. The successful diagnosis and treatment of myocarditis would therefore be a valuable tool for the clinician, in order to avoid or delay the onset- of congestive heart failure. Acute viral myocarditis is, however, not easy to diagnose, since its symptoms are similar to those common to viral infections. In the case of other types of myocarditis, the symptoms of the bacterial infection, protozoal infection, heart damage due to substance abuse, poisons, hypersensitivity reactions, or immune system damage are also often vague.
  • the diagnosis of viral myocarditis may be supported by the identification of the virus in stool, throat washings, blood, myocardium, or pericardial fluid.
  • ECG abnormalities accompanying a viral or bacterial infection disorder, a hypersensitivity adverse reaction or an immune disorder are an indicator of probable myocarditis resulting from the original disorder.
  • United States Patent 4,968,483 Mueller describes an apparatus for treating mammalian blood simultaneously with UV radiation, elevated temperature and oxygen/ozone gaseous mixture, bubbled through the blood.
  • TITUTE SHEET simultaneous treatment with ozone/oxygen, UV radiation and elevated temperature e.g. in an apparatus as described in the Mueller patent above, and re-injecting the treated blood aliquot intramuscularly to the patient.
  • a process for prophylaxis, or alleviation of the symptoms, of myocarditis in a mammalian patient which comprises extracting an aliquot of blood from the mammalian patient, treating the aliquot extracorporeally by subjection to oxidative stress and electromagnetic emission, and re-administering the treated aliquot to the patient, in a quantity and in a manner to result in prophylaxis of or alleviation of the symptoms of myocarditis in the patient.
  • a further aspect of the present invention is use in the manufacture of a medicament for prophylaxis, or alleviation of the symptoms, of inflammation of the myocardium consequent upon elevated levels of TNFV in the myocardium of a mammalian patient, of an aliquot of autologous blood which has been treated extracorporeally by subjection to oxidative stress and electromagnetic emission.
  • FIGURE of accompanying drawings is a graphical presentation of the results obtained from the specific Example below, namely a bar graph of TNF ⁇ expression in mouse myocardia of control animals and mouse myocardia of animals treated in accordance with the invention.
  • an aliquot of blood is extracted from a mammalian subject, preferably a human, and the aliquot of blood is treated ex vivo with certain stressors, described in more detail below.
  • the terms "aliquot”, “aliquot of blood” or similar terms used herein include
  • the effect of the stressors is to modify the blood, and/or the cellular or non-cellular fractions thereof, contained in the aliquot.
  • the modified aliquot is then re-introduced into the subject's body by any method suitable for vaccination, preferably selected from intra-arterial injection, intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, and oral, nasal or rectal administration. Intramuscular injection is most preferred.
  • the stressors to which the aliquot of blood is subjected ex vivo according to the method of the present invention are an oxidative environment and an electromagnetic emission, simultaneously or sequentially.
  • the aliquot of blood is in addition subjected to mechanical stress.
  • mechanical stress is suitably that applied to the aliquot of blood by extraction of the blood aliquot through a conventional blood extraction needle, or a substantially equivalent mechanical stress, applied shortly before the other chosen stressors are applied to the blood aliquot.
  • This mechanical stress may be supplemented by the mechanical stress exerted on the blood aliquot by bubbling gases through it, such as ozone/oxygen mixtures, as described below.
  • the aliquot has a volume sufficient that, when re- introduced into the subject's body, at least partial alleviation of a cardiac inflammatory disorder is achieved in the subject.
  • the volume of the aliquot is up to about 400 ml, preferably from about 0.1 to about 100 ml, more preferably from about 5 to about 15 ml, even more preferably from about 8 to about 12 ml, and most preferably about 10 ml.
  • the aliquot should contain the number of blood cells which would ordinarily be contained in whole blood of the aforementioned volumes, e.g. 10 3 to 10 12 .
  • a temperature stress blood temperature above or below body temperature
  • 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 cardiac inflammation disorder 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, alleviation of the disorder will be achieved.
  • 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 55°C.
  • 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 40°C to about 50°C, even more preferably from about 40°C to about 44°C, and most preferably about 42.5 ⁇ 1°C.
  • 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 10°C to about 30°C, and even more preferably from about 15°C to about 25°C.
  • the oxidative environment stressor can be the application to the aliquot of solid, liquid or gaseous oxidizing agents.
  • Chemical oxidants such as hydrogen peroxide can be used.
  • 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
  • SUBSTITUTE SHEET 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 are preferably 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.
  • the gas stream has an ozone content of up to about 300 ⁇ g/ml, preferably up to about 100 ⁇ g/ml, more preferably about 30 ⁇ g/ml, even more preferably up to about 20 ⁇ g/ml, particularly preferably from about 10 ⁇ g/ml to about 20 ⁇ g/ml, and most preferably about 14.5 ⁇ 1.0 ⁇ g/ml.
  • the gas stream is suitably supplied to the aliquot at a rate of up to about 2.0 litres/min, 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 O.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/min.
  • 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.
  • UV-A wavelengths from about 315 to about 400 nm
  • UV-B wavelengths from about 280 to about 315
  • an appropriate dosage of such UV light can be obtained from lamps with a combined power output of from about 45 - 65 mW/cm 2 .
  • ⁇ UTE ' SHEE the surface of the blood of from about 0.025 to about 10 joules/cm 2 , preferably from about 0.1 to about 3.0 joules/cm 2 .
  • four such lamps are used.
  • the time for which the aliquot is subjected to the stressors is normally within the time range of up to about 60 minutes. The time depends to some extent upon the chosen intensity of the electromagnetic emission, the temperature, the concentration of the oxidizing agent and the rate at which it 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 about 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.
  • the aliquot of blood it is preferred to subject the aliquot of blood to a mechanical stressor, as well as the chosen stressor(s) discussed above. Extraction of the blood aliquot from the patient through an injection needle constitutes the most convenient way of obtaining the aliquot for further extracorporeal treatment, and this extraction procedure imparts a suitable mechanical stress to the blood aliquot.
  • the mechanical stressor may be supplemented by subsequent processing, for example the additional mechanical shear stress caused by bubbling as the oxidative stressor is applied.
  • the blood aliquot may be treated with the stressors using an apparatus of the type described in aforementioned U.S. Patent No. 4,968,483 to Mueller, incorporated herein by reference.
  • the aliquot is placed in a suitable, sterile, UV 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
  • E SHEET 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.
  • the oxygen/ozone gas mixture of known composition and controlled flow rate
  • compositions according to the present invention may be identified and selected according to diagnostic methods referred to earlier. Many cases of myocarditis are sub-clinical. Viral infection followed by cardiac involvement about two weeks after the onset of the viral illness (chest pain, myalgia, dyspnea, and/or left ventricular dysfunction) is an indicator of myocardial inflammation.
  • the enterovirus group with coxsackievirus B, adenovirus type C and serotypes 2 and 5 are reported to be viruses from the infection with which myocardial inflammation may follow, so that patients diagnosed with such an infection, followed by cardiac involvement, are a basis of selection of patients for treatment according to the invention. Chagas disease, caused by the protozoa Trypanosoma cruzi, is the most common cause of myocarditis in Central and South America.
  • 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.
  • 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.
  • 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
  • SUBSTITUTE SHEET 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.
  • 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 11 days being provided between the administration of the second and third aliquots.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • mice Male A/J mice were used, and were injected subcutaneously with porcine myosin to induce cardiac inflammation and simulate myocarditis (Neu, N., Rose, N.R., Beisel, K.W., Herskowitz, A., Gurri- Glass, G., and Craig, S.W. 1987. Cardiac Myosin Induces Myocarditis in Genetically Predisposed Mice, J. Immunol. 139:3630-3636).
  • mice were sacrificed and syngeneic blood collected via cardiac puncture.
  • the blood was treated with oxygen/ozone and UV at elevated temperature in a VC7000A treatment apparatus from Vasogen Inc., Mississauga, Canada.
  • This apparatus is similar in principle and operation to that described in the aforementioned Mueller patent. Briefly, 10 ml_ of blood was added to 2 ml_ of 4% sodium citrate and immediately transferred to a sterile single-use disposable blood container, which was placed in the VC7001A blood treatment apparatus for ex vivo treatment over a period of approximately 20 minutes.
  • the apparatus exposed the blood to oxidative stresses in the form of medical grade oxygen containing 14.5 ⁇ 1.0 ⁇ g/mL of ozone (STP), UV light at a nominal wavelength of 253.8 nm, with a total energy of 2.0 J/cm 2 , and at an elevated temperature of 42.5 ⁇ 1.0°C.
  • treated blood was immediately transferred to a sterile syringe ready for injection intramuscularly into the animals' leg muscle, in 50 ⁇ amounts.
  • mice A group of 4 control mice (Group A) were each given 3 weekly (day 14, day 28 and day 35) sc injections of porcine myosin (50 g/mouse), thereby inducing acute cardiac inflammation.
  • results presented herein indicate a substantial reduction in myocardial TNF ⁇ expression following induction of inflammation with the experimental toxin myosin (porcine) as a result of treatment of the animals with syngeneic treated blood as described, either before or during the administration of the experimental toxin myosin (porcine) as a result of treatment of the animals with syngeneic treated blood as described, either before or during the administration of the experimental toxin myosin (porcine) as a result of treatment of the animals with syngeneic treated blood as described, either before or during the administration of the experimental toxin myosin (porcine) as a result of treatment of the animals with syngeneic treated blood as described, either before or during the administration of the experimental toxin myosin (porcine) as a result of treatment of the animals with syngeneic treated blood as described, either before or during the administration of the experimental toxin myosin (porcine) as a result of treatment of the animals with syngeneic treated blood
  • SUBSTITUTE SHEET toxin indicative of a significant reduction of cardiac inflammation including inflammation based acute myocarditis, resulting from toxin exposure and, by analogy, other forms of cardiac inflammation-inducing infections such as viral and bacterial infection and other causes.
  • the invention accordingly provides a potential inhibition of the progression of and treatment for myocarditis in mammalian patients.

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Abstract

Cardiac inflammation as exemplified by myocarditis in a mammalian patient is alleviated by a process which comprises extracting an aliquot of blood from the mammalian patient, treating the aliquot extracorporeally by subjection to oxidative stress and electromagnetic emission, and readministering the treated aliquot to the patient, in a quantity and in a manner to result in prophylaxis of or alleviation of the symptoms of myocarditis in the patient.

Description

MYOCARDITIS TREATMENT Field of the Invention
This invention relates to medical treatments , and to compositions of matter and procedures useful therein. More specifically, it relates to alleviation of symptoms of inflammatory disorders of the heart, notably myocarditis, and use of compositions therein.
Background of the invention and prior art
The myocardium is the muscular layer of the heart, composed of myocytes. Myocarditis is an inflammatory condition of the myocardium, commonly caused by viral infections, and less commonly by bacterial or parasitic infections. The condition may also be caused by exposure to chemicals or allergic reactions to certain medications, and it can be associated with autoimmune diseases. In South America, C hagas disease has as its major cardiovascular manifestation an extensive myocarditis that typically becomes evident years after the initial infection. In myocarditis, the heart muscle becomes inflamed and weakened, and this commonly causes symptoms of heart failure.
Myocarditis is commonly a precondition leading to congestive heart failure. It is estimated that 25-30% of cases of chronic congestive heart failure might result from a myocarditis. For a significant proportion of these patients, treatment of the inflammation of the heart at an appropriate stage would likely
8Ϊ ffl TE SHEET prevent or at least decelerate progression to chronic (congestive) heart failure. Recognizing that congestive heart failure is now the commonest cause of hospital admissions in the age group over 65, this condition has major implications to the health system. The successful diagnosis and treatment of myocarditis would therefore be a valuable tool for the clinician, in order to avoid or delay the onset- of congestive heart failure. Acute viral myocarditis is, however, not easy to diagnose, since its symptoms are similar to those common to viral infections. In the case of other types of myocarditis, the symptoms of the bacterial infection, protozoal infection, heart damage due to substance abuse, poisons, hypersensitivity reactions, or immune system damage are also often vague. The diagnosis of viral myocarditis may be supported by the identification of the virus in stool, throat washings, blood, myocardium, or pericardial fluid. ECG abnormalities accompanying a viral or bacterial infection disorder, a hypersensitivity adverse reaction or an immune disorder, are an indicator of probable myocarditis resulting from the original disorder.
United States Patent 4,968,483 Mueller describes an apparatus for treating mammalian blood simultaneously with UV radiation, elevated temperature and oxygen/ozone gaseous mixture, bubbled through the blood.
United States Patent 5,834,030 Bolton describes a process of increasing the nitric oxide concentration in human blood, with consequent improvement of various medical conditions including peripheral vascular disease, by extracting an aliquot of a patient's blood, subjecting, it extracorporeally to
TITUTE SHEET simultaneous treatment with ozone/oxygen, UV radiation and elevated temperature, e.g. in an apparatus as described in the Mueller patent above, and re-injecting the treated blood aliquot intramuscularly to the patient.
United States Patent 6,572,895 Torre-Amione et. al. describes the use of autologous blood which has been treated extracorporeally with oxygen/ozone, UV radiation and heat simultaneously, in the alleviation of symptoms of congestive heart failure.
The above references are incorporated herein in their entirety.
Summary of the Invention
It has now been discovered that treatment of an aliquot of blood from a patient, extracorporeally, with an oxidative stressor and electromagnetic emission (e.g. UV radiation), and re-injection of the treated blood aliquot to the patient, acts to reduce the amount of pro-inflammatory cytokine TNFα expression in the patient's heart in response to cardiac inflammation-causing factors. This discovery provides the basis for prophylaxis and treatment of cardiac inflammatory conditions in mammalian patients, including human patients, such as myocarditis. The ability to treat cardiac inflammatory conditions such as myocarditis offers the potential for treating patients for one of the contributing causes of more serious cardiac problems such as chronic (congestive) heart failure and thereby helping to avoid the progression of inflammatory conditions into chronic heart failure.
SUBSTITUTE SHEET Thus, according to one aspect of the present invention, there is provided a process for prophylaxis, or alleviation of the symptoms, of myocarditis in a mammalian patient, which comprises extracting an aliquot of blood from the mammalian patient, treating the aliquot extracorporeally by subjection to oxidative stress and electromagnetic emission, and re-administering the treated aliquot to the patient, in a quantity and in a manner to result in prophylaxis of or alleviation of the symptoms of myocarditis in the patient.
A further aspect of the present invention is use in the manufacture of a medicament for prophylaxis, or alleviation of the symptoms, of inflammation of the myocardium consequent upon elevated levels of TNFV in the myocardium of a mammalian patient, of an aliquot of autologous blood which has been treated extracorporeally by subjection to oxidative stress and electromagnetic emission.
Brief Reference to the Drawings
The single FIGURE of accompanying drawings is a graphical presentation of the results obtained from the specific Example below, namely a bar graph of TNFα expression in mouse myocardia of control animals and mouse myocardia of animals treated in accordance with the invention.
Description of the Preferred Embodiments.
According to a preferred process of the present invention, an aliquot of blood is extracted from a mammalian subject, preferably a human, and the aliquot of blood is treated ex vivo with certain stressors, described in more detail below. The terms "aliquot", "aliquot of blood" or similar terms used herein include
' whole blood, separated cellular fractions of the blood including platelets, separated non-cellular fractions of the blood including plasma, and combinations thereof. The effect of the stressors is to modify the blood, and/or the cellular or non-cellular fractions thereof, contained in the aliquot. The modified aliquot is then re-introduced into the subject's body by any method suitable for vaccination, preferably selected from intra-arterial injection, intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, and oral, nasal or rectal administration. Intramuscular injection is most preferred.
The stressors to which the aliquot of blood is subjected ex vivo according to the method of the present invention are an oxidative environment and an electromagnetic emission, simultaneously or sequentially.
Preferably also, the aliquot of blood is in addition subjected to mechanical stress. Such mechanical stress is suitably that applied to the aliquot of blood by extraction of the blood aliquot through a conventional blood extraction needle, or a substantially equivalent mechanical stress, applied shortly before the other chosen stressors are applied to the blood aliquot. This mechanical stress may be supplemented by the mechanical stress exerted on the blood aliquot by bubbling gases through it, such as ozone/oxygen mixtures, as described below.
Suitably, in human subjects, the aliquot has a volume sufficient that, when re- introduced into the subject's body, at least partial alleviation of a cardiac inflammatory disorder is achieved in the subject. Preferably, the volume of the aliquot is up to about 400 ml, preferably from about 0.1 to about 100 ml, more preferably from about 5 to about 15 ml, even more preferably from about 8 to about 12 ml, and most preferably about 10 ml. When a cellular fraction is used instead of whole blood, the aliquot should contain the number of blood cells which would ordinarily be contained in whole blood of the aforementioned volumes, e.g. 103 to 1012. 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 cardiac inflammation disorder 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, alleviation of the disorder 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 55°C.
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 40°C 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 10°C to about 30°C, and even more preferably from about 15°C to about 25°C.
The oxidative environment stressor can be the application to the aliquot of solid, liquid or gaseous oxidizing agents. Chemical oxidants such as hydrogen peroxide can be used. 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
SUBSTITUTE SHEET 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 are preferably 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/min, 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 O.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/min.
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
7
ΓUTE'SHEEΓ 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 is normally within the time range of up to about 60 minutes. The time depends to some extent upon the chosen intensity of the electromagnetic emission, the temperature, the concentration of the oxidizing agent and the rate at which it 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 about 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.
As noted above, it is preferred to subject the aliquot of blood to a mechanical stressor, as well as the chosen stressor(s) discussed above. Extraction of the blood aliquot from the patient through an injection needle constitutes the most convenient way of obtaining the aliquot for further extracorporeal treatment, and this extraction procedure imparts a suitable mechanical stress to the blood aliquot. The mechanical stressor may be supplemented by subsequent processing, for example the additional mechanical shear stress caused by bubbling as the oxidative stressor is applied.
In the practice of the preferred process of the present invention, the blood aliquot may be treated with the stressors using an apparatus of the type described in aforementioned U.S. Patent No. 4,968,483 to Mueller, incorporated herein by reference. The aliquot is placed in a suitable, sterile, UV 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
E SHEET 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.
Subjects potentially treatable with compositions according to the present invention may be identified and selected according to diagnostic methods referred to earlier. Many cases of myocarditis are sub-clinical. Viral infection followed by cardiac involvement about two weeks after the onset of the viral illness (chest pain, myalgia, dyspnea, and/or left ventricular dysfunction) is an indicator of myocardial inflammation. The enterovirus group with coxsackievirus B, adenovirus type C and serotypes 2 and 5 are reported to be viruses from the infection with which myocardial inflammation may follow, so that patients diagnosed with such an infection, followed by cardiac involvement, are a basis of selection of patients for treatment according to the invention. Chagas disease, caused by the protozoa Trypanosoma cruzi, is the most common cause of myocarditis in Central and South America.
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
SUBSTITUTE SHEET 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 11 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 invention is further illustrated and described with reference to the following specific example.
10
SUBSTITUTE SHEET Specific Example
In this experimental Example, male A/J mice were used, and were injected subcutaneously with porcine myosin to induce cardiac inflammation and simulate myocarditis (Neu, N., Rose, N.R., Beisel, K.W., Herskowitz, A., Gurri- Glass, G., and Craig, S.W. 1987. Cardiac Myosin Induces Myocarditis in Genetically Predisposed Mice, J. Immunol. 139:3630-3636).
Approximately 12-15 mice were sacrificed and syngeneic blood collected via cardiac puncture. The blood was treated with oxygen/ozone and UV at elevated temperature in a VC7000A treatment apparatus from Vasogen Inc., Mississauga, Canada. This apparatus is similar in principle and operation to that described in the aforementioned Mueller patent. Briefly, 10 ml_ of blood was added to 2 ml_ of 4% sodium citrate and immediately transferred to a sterile single-use disposable blood container, which was placed in the VC7001A blood treatment apparatus for ex vivo treatment over a period of approximately 20 minutes. During this time the apparatus exposed the blood to oxidative stresses in the form of medical grade oxygen containing 14.5 ± 1.0 μg/mL of ozone (STP), UV light at a nominal wavelength of 253.8 nm, with a total energy of 2.0 J/cm2, and at an elevated temperature of 42.5 ± 1.0°C. After treatment, treated blood was immediately transferred to a sterile syringe ready for injection intramuscularly into the animals' leg muscle, in 50 μ\ amounts.
A group of 4 control mice (Group A) were each given 3 weekly (day 14, day 28 and day 35) sc injections of porcine myosin (50 g/mouse), thereby inducing acute cardiac inflammation. A second group of 4 mice, group B, were each given pre-treatment injections of treated blood prepared as described
11
BSTITUTE SHEET above, 50 μl/mouse at each injection, on day 0, day 1 and day 14. Subcutaneous injections of porcine myosin as above were administered to each mouse of Group B on days 14, 28 and 35. A third group of 4 mice, Group C, were given injections of treated blood on days 14, 28 and 35, with 50 μg myosin injections on the same days. All the animals were sacrificed on day 42. The experiment was repeated a second time, but using 4 control animals instead of 3.
Cardiac tissue was examined for myocardial TNFα expression. Myocardial TNFα expression measurement was accomplished by immunohistochemical analysis, using an appropriate stain for TNFα, and examining areas of the sectioned myocardium visually to determine the percentage of total myocardium area staining positive for TNFα expression. The results are presented graphically on the accompanying Figure. The vertical axis represents the percentage of the total area staining positive for TNFα expression. These results are the averages of all of the animals from the groups, in both experiments. Thus, the results from control Group A are the average for 7 animals, and the results from groups B and C are both averages from 8 animals. Numerically, the average total area staining positive for TNFα expression in respect of the control animals was 2.25, compared with 0.375 for the pretreated animals of group B and 0.6 for the treated animals of group C.
The results presented herein indicate a substantial reduction in myocardial TNFα expression following induction of inflammation with the experimental toxin myosin (porcine) as a result of treatment of the animals with syngeneic treated blood as described, either before or during the administration of the
12
SUBSTITUTE SHEET toxin, indicative of a significant reduction of cardiac inflammation including inflammation based acute myocarditis, resulting from toxin exposure and, by analogy, other forms of cardiac inflammation-inducing infections such as viral and bacterial infection and other causes. The invention accordingly provides a potential inhibition of the progression of and treatment for myocarditis in mammalian patients.
13
SUBSTITUTE SHEET

Claims

WHAT IS CLAIMED IS:
1. Use in the preparation of a medicament for prophylaxis, or alleviation of the symptoms of, inflammation of the myocardium consequent upon elevated TNFV in the myocardium, in a mammalian patient, of an aliquot of autologous blood which has been treated extracorporeally by subjection to oxidative stress and electromagnetic emission.
2. Use as in claim 1 wherein the mammalian patient is a human.
3. Use as in claim 1 or claim 2 wherein the oxidative stress and electromagnetic emission are applied to the blood aliquot simultaneously.
4. Use as in any of claims 1 - 3 wherein the blood aliquot has a volume of from 0.1 - 100ml_.
5. Use according to any preceding claim wherein the electromagnetic emission is uv radiation
6. Use according to any preceding claim wherein the oxidative stress is oxygen/ozone gaseous mixture.
7. Use according to any preceding claim wherein the electromagnetic emission is uv radiation of UV-C wavelengths.
8. Use according to any preceding claim wherein the blood aliquot is maintained at a temperature of from 40° - 50° C during subjection to the oxidative stress and electromagnetic emission.
9. Use according to any preceding claim in preparation of a medicament for treating myocarditis.
14
ITUT SHEET
10. A process for alleviation of the symptoms of myocarditis in a mammalian patient, which comprises extracting an aliquot of blood from the mammalian patient, treating the aliquot extracorporeally by subjection to oxidative stress and electromagnetic emission, and re- administering the treated aliquot to the patient, in a quantity and in a manner to result in alleviation of the symptoms of myocarditis in the patient.
11. A process for alleviation of the symptoms of myocarditis in a human patient, which comprises selecting a human patient exhibiting symptoms of myocarditis, extracting an aliquot of blood from the patient, treating the aliquot extracorporeally by subjection to oxidative stress and electromagnetic emission, and re-administering the treated aliquot to the patient, in a quantity and in a manner to result in alleviation of the symptoms of myocarditis in the patient.
12. Use in the manufacture of a medicament for alleviation of the symptoms of cardiac inflammation as exemplified by myocarditis in a mammalian patient, of an aliquot of autologous blood which has been treated extracorporeally by subjection to oxidative stress and electromagnetic emission.
15
SUBSTITUTE SHEET
PCT/CA2005/000537 2004-04-12 2005-04-08 Myocarditis treatment WO2005097145A1 (en)

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