WO2001089601A1

WO2001089601A1 - Method and apparatus for ozone decontamination of biological liquids

Info

Publication number: WO2001089601A1
Application number: PCT/US2001/015706
Authority: WO
Inventors: Gerard V. Sunnen
Original assignee: Medizone International Inc
Current assignee: Medizone International Inc
Priority date: 2000-05-24
Filing date: 2001-05-16
Publication date: 2001-11-29
Anticipated expiration: 2002-11-24
Also published as: AU2001261627A1

Abstract

The present invention relates to a delivery system and a treatment protocol designed to accurately deliver ozone to biological liquids. To effect the accurate delivery of ozone to such liquids, ozone is delivered to the liquid via a gas permeable membrane while the liquid passes along the opposite side of the membrane via a narrow channel.

Description

METHOD AND APPARATUS FOR OZONE DECONTAMrNATION OF BIOLOGICAL LIQUIDS

BACKGROUND OF THE INVENTION

Ozone (O₃) has unique biological properties which have wide application in various medical fields. As early as the First World War, ozone's antipathogenic properties were used to treat infected wounds, mustard gas burns, and fistulas. Ozone has proven to be a promising approach to the treatment of additional pathogenic infections such as viral infections. Preliminary research on hepatitis C infection has shown that reduction of viral load by means of ozone therapy can significantly normalize hepatic enzymes (SGOT, SGPT, alkaline phosphatase) and improve patient health. In fact, volunteers administered ozone therapy achieved a viral load reduction on the order of 5 log, or 99.9%.

Traditional methods of administering ozone to patients have relied mainly on using sterile bottles to collect the patient's blood. Subsequently, usually by means of a syringe, a dose of ozone/oxygen gas is added to the blood. U.S. Patent No. 4,632,980 describes a typical method where ozone is bubbled through the blood. U.S. Patent No. 4,968,483 describes an alternative method where bottles are rotated on rollers so that a thin film of blood lining the inside of the rotating bottle may more evenly be exposed to ozone. U.S. Patent No. 5,882,591 describes dispersing blood into fine droplet and exposing the droplets to ozone. However, these methods present several drawbacks. Injecting a gaseous mixture into blood subjects the blood to foaming which creates physical characteristics making ozone exposure uneven. The rolling action of rotating bottles and the dispersion of blood into fine droplets causes cellular damage and hemolysis due to mechanical stress.

Thus, there is a need for a method of interfacing ozone with blood that would expose the blood to minimal mechanical trauma, afford a low hemolysis rate, and would allow for an even distribution and homogeneous transfer of the ozone into the blood.

SUMMARY OF THE INVENTION According to one aspect of the invention, there is provided a method of reducing the viral load in blood by contacting the blood with a sufficient amount of ozone wherein the blood is passed through a cartridge comprising a gas permeable membrane and the exterior of the membrane is exposed to ozone. The ozone passes through the membrane and into the blood at a controlled rate. The path for the blood is sufficiently narrow so that the blood is uniformly exposed to the ozone.

According to another aspect of the invention, there is provided an apparatus for disinfecting biological liquids with ozone. The apparatus comprises an ozone generator for producing a controlled concentration of ozone and a cartridge comprising a gas permeable membrane and a narrow passage along the membrane for the biological liquid. The invention also provides an apparatus comprising a cartridge which further comprises a gas permeable membrane and a narrow passage along the membrane for the biological liquid. The path for the biological liquid is sufficiently narrow so that the biological liquid is uniformly exposed to ozone passing through the membrane and into the biological liquid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a schematic circuit diagram of an ozone generator according to one embodiment of the present invention.

FIG. 2A illustrates a schematic circuit diagram of a cartridge having a gas permeable membrane according to one embodiment of the present invention.

FIG. 2B illustrates a side view of the cartridge of FIG. 2 A.

DETAILED DESCRIPTION OF THE INVENTION According to the invention, methods and delivery systems for contacting a biological liquid with ozone to decrease the amount of pathogenic agents in the biological liquid is provided. Biological liquids include, but are not limited to blood and blood products such as plasma, and serum. The system comprises a medical grade ozone generation portion and a delivery portion which is referred to as a cartridge. The cartridge comprises a gas permeable membrane which allows for a controlled amount of ozone to be delivered to the blood.

The concentration of ozone delivered to a patient must be accurately quantified. Reasons include (1) the fact that ozone in too high concentrations is toxic to whole blood cellular element and causes hemolysis; (2) that immune system stimulation of cytokines occurs within an optimal range; and (3) that different members of viral families have different susceptibilities to ozone challenge. Ozone, therefore, like many medications, may be said to have a therapeutic window. Below the lower limits of the window few biological or otherwise therapeutic effects occur, while beyond the window toxic effects are noted. Administration of increasing dosages of ozone to whole blood shows that beyond a certain threshold there is a rise in the rate of hemolysis. This threshold,, depending upon various parameters, is reached at about 40 to about 60 micrograms per milliliters (ug/ml), and becomes significant when higher levels are attained.

Leucocytes show good resistance to ozone because they have enzymes which protect them from oxidative stress. These enzymes include superoxide dismutase, glutathione, and catalase. Research has shown that platelets also maintain their integrity after ozone administration. In ozone viral load reduction therapy, the doses applied to blood do not disrupt its cellular elements. On the contrary, they tend to stimulate leucocyte function. Ozone increases the oxygen saturation (pO₂) in erythrocytes and enhances their pliability so that capillary circulation is facilitated.

In contrast to the relative stability of whole blood to ozone, virions are adversely affected by ozone in a variety of ways. Ozone disrupts envelope proteins, lipoproteins, lipids, and glycoproteins, and the presence of numerous double bonds in these unsaturated molecules makes virions vulnerable to the oxidizing effects of ozone. Double bonds are thus reconfigured, molecular architecture is disrupted and widespread breakage of the envelope ensues. Deprived of an envelope, virions cannot sustain nor replicate themselves.

In addition, the introduction of ozone into the serum portion of whole blood induces the formation of lipid and protein peroxides. While these peroxides are not toxic to the host in quantities produced by ozone therapy, they nevertheless possess oxidizing properties of their own which persist in the bloodstream for several hours. Lipid peroxides created by ozone administration show long-term antiviral effects which serve to further reduce viral load. This factor may explain in part the reason for the fact that ozonated blood in the amount processed in the treatment protocol (50 to 300 ml) is able to reduce the viral load value in the total blood volume (approximately 7 liters).

Ozone treatment provides additional benefits such as inducing the release of cytokines. Cytokines are proteins manufactured by several different types of cells which regulate the functions of other cells. Mostly released by leucocytes, they are important in mobilizing the immune response. Thus, the ozone-induced release of cytokines is a significant means for the reduction of circulating virions. Ozone action on viral particles in infected blood provides additional indirect benefits. One benefit is the modification of virions so that they are sufficiently dysfunctional so as to be nonpathogenic yet remain grossly structurally intact. This attenuation of viral particle functionality through slight modifications of the viral envelope by ozone eliminates pathogenicity and at the same time provides an immunogen to function as an auto vaccine. In view of the fact that so many mutational variants exist in any one afflicted individual, the creation of an antigenic spectrum of crippled virions provides for a unique host-specific stimulation of the immune system, thus designing what may be called a host-specific autovaccine. The viral load inhibition in this scenario offers unique therapeutic specificity in that the attenuated virions are species-specific to the host. The resulting antibodies formed in response to the host-specific virions provides for a therapeutic tactic which conventional vaccines have been unable to attain.

Alternatively, viral integrity may be completely destroyed by the ozone challenge resulting in fragmented circulating virions. These cleaved fragments in turn stimulate the immune system to elaborate antibodies. A great variety of viral fragments are created by this mechanism and the nature of the host immune response is likely to be idiosyncratic.

The ozone generation portion of the system comprises a source of oxygen connected to an ozone generator. Preferably, the generator provides an adjustable and consistent oxygen flow rate which will permit accurate control over the final ozone concentration. The oxygen channeled to the generator is preferably of medical grade purity.

To generate ozone, oxygen is imparted energy in order to split some if its molecules so that single oxygen atoms may then react with diatomic oxygen molecules to form ozone (O₃). Energy may come from different sources such as coronal discharge, ultraviolet radiation and microwave energy. A preferred method of generating ozone is by microwave energy.

Referring now in detail to the drawings, a biological liquid treatment system in accordance with the invention is illustrated generally in Figs. 1 and 2. The system comprises an ozone generator (Fig. 1) and a disinfecting gas and biological liquid interface apparatus, or cartridge (Fig.. 2). Ozone generator generates and administers a controlled amount of ozone to the biological liquid interface apparatus in order to disinfect the biological liquid. As shown in Fig. 1, the ozone generator 1 receives medical grade oxygen from an oxygen supply tank 2 regulated by control flow 3. The ozone generator 1 comprises a flow rate gauge 4 for measuring the exact oxygen flow rate entering the ozone generator 1, and a flow rate regulator 5 fine tuning the oxygen flow rate.

The conversion of oxygen into ozone is by way of an energy module 6. The energy module 6 utilizes an appropriate electrical discharge, such as, for example, coronal discharge, plasma discharge, UV radiation, or microwaves. An amperage digital gauge 9 measures, in amperes, the energy channeled into the energy module 6, while an amperage regulator 8 modulates energy output such that higher energy levels result in greater concentrations of ozone.

The generator 1 also comprises a cooling element 10. Cooling element 10 includes an intake 11 and an outlet 12. The heat produced by the energy module 6 is dissipated by water entering the intake 11 and exiting the outlet 12 of the cooling element 10.

The generator 1 further comprises a bar pressure gauge 13 for measuring the internal gas pressure of the generator.

The amount of generated ozone is monitored by an ozone analyzer 14 and displayed by an ozone digital gauge 15.

The generator 1 also includes a computer interface port 16 connected to the components of the generator 1. The computer interface port 16 provides an external computer system with data that enables a clinician to adjust the treatment parameters of the generator 1, such as, for example, the oxygen flow rate, the internal system pressure, the ozone concentration, the amperage output to the energy module 6, the time functions, and memory functions.

An ozone exit flow gauge 17 measures the ozone/oxygen flow rate as they exit the generator through ozone exit port 18. An ozone re-entry port 19 accepts ozone that is returned to the generator for purposes of reversion to oxygen by, for example, an ozone destructor unit 20.

Further information about the operation and construction of ozone generators is provided in United States Patent No. 5,052,382, entitled "Apparatus for the Controlled Generation and Administration of Ozone" to Wainwright.

As illustrated in Fig. 2, the disinfecting gas and biological liquid interface apparatus comprises a collection receptacle 21 and a cartridge 27. The collection receptacle 21 includes a container having an entry port 22 and an exit port 25. The container contains an anticoagulant, such as, for example, citrate or heparin, and features graduated horizontal markings indicating the volume of blood contained within the receptacle 21. The exit port 25 permits blood to pass through a conduit to a control valve 26. Control valve 26 regulates the blood entering the cartridge 27.

Cartridge 27 includes an interior wall, a liquid flow inlet 29, a liquid flow outlet 31, an ozone inlet 32 and an ozone outlet 33. The ozone inlet 32 and ozone outlet 33 of the cartridge 27 are connected by conduits (not shown) to the ozone exit port 18 and the ozone reentry port 19 of the generator 1.

Cartridge 27 contains a membrane 30. Membrane 30 is gas permeable to permit the diffusion of gas mixture quickly, efficiently and consistently. The latter requirement is important because several materials used as membranes cannot sustain their molecular integrity in the face of prolonged ozone exposure. Membranes whose gas diffusion capacity depends upon the presence of micropores are often found to lose efficiency. Electron microscopy shows that micropores exposed to ozone are apt to show loss of patency, probably through oxidation of the polymer molecules lining their lumens. It is preferred that membranes according to this invention be made from materials which do not have micropores but nevertheless permit the diffusion of ozone/oxygen through the mesh of their molecular makeup. Non-limiting examples of membranes for use according to the invention include silicon, polytetrafluoroethylene (PTFE), expanded PTFE, cellulose, polycarbonate, polysulfone, metal, and ceramic membranes. Such membranes are known in the art and described, for example, in Resting and Fritzsche Polymeric Gas Separation Membranes 1993, John Wiley & Sons, New York and in the Encyclopedia of Chemical Technology, Fourth Ed., Volume 18, 1995, John Wiley & Sons, New York pp. 135- 193. The membranes for use according to this invention are preferably flat-sheet membranes.

Membrane 30 includes a first side and a second side. The first side of membrane 30 and the interior wall of the cartridge 27 define an area for circulating ozone from the ozone inlet 32 to the first side of the membrane. A portion of the second side of the membrane defines a narrow passageway in communication with the liquid flow inlet 29 and the liquid flow outlet 31. In operation, the ozone passes through the membrane 30 to treat the liquid flowing through the passageway created by a portion of the second side of the membrane 30. In one embodiment, the cartridge 27 is constructed so that blood flows through a narrow passageway defined by a bilayered membrane 37. The distance separating the bilayers needs to be narrow enough so as to permit uniform exposure of blood to ozone. At the same time this membrane bilayer must be wide enough to allow for adequate blood flow. This distance is about 5 cm to about 0.1 mm in width. A more preferred distance is about 5 mm to about 0.5 mm and a most preferred distance is about 1.5 mm to about 0.5 mm in width. The bilayer is held in its configuration by internal trabeculae 38 and/or by external buttresses 39. The O₃/O₂ (or any other ozone gas mixture) maintained outside the membrane 34 diffuses through the membrane and into the blood at a constant rate. The blood inside the membrane flows at a constant rate. Integrating the ozone concentration, the ozone diffusion rate of the membrane, and the blood flow rate through the cartridge, the dosage of ozone administered to the blood can easily be determined and regulated.

The cartridge seen from a frontal perspective 28 (FIG. 2 A), and a side view perspective 35 (FIG. 2B), shows a Cartridge Blood Inflow Port 29. The Ozone Entry Port 32 accepts the O₃/O₂ mixture from the generator which circulates within the cartridge outside its membrane in the Cartridge O₃/O₂ Space 34. The Ozone Exit Port 33 returns ozone/oxygen to the generator's Destructor Unit 20. The Cartridge Blood Exit Port 31 channels ozonated/oxygenated blood to the patient.

All publications, patents and articles referred to herein are expressly incorporated herein in toto by reference thereto. The following examples are presented to illustrate the present invention but are in no way to be construed as limitations on the scope of the invention. It will be recognized by those skilled in the art that numerous changes and substitutions may be made without departing from the spirit and purview of the invention.

EXAMPLES

In this protocol, viral load determines the frequency of treatments and the duration of therapy. Since HBV, HCV, and HIV all show cycles of viremia, this therapy aims at repression of viral expression over long periods of time in order to decrease morbidity and mortality and to increase longevity and quality of life.

The patient is prepared for venipuncture. A volume of blood is withdrawn and is channeled into the sterile collection receptacle containing anticoagulant (citrate or heparin). Depending upon the clinical situation at hand, this volume of blood may range from 50 to 300 milliliters. In the event that the patient's veins are easily accessible, blood may be made to flow directly from the venipuncture intravenous line to the collection receptacle. In situations where veins have poor accessibility a syringe is used. The collection receptacle is constructed of soft transparent plastic. This serves to allow viewing of the blood being treated and, importantly, to minimize cell injury which occurs in hard containers. The collection bag is connected to the cartridge so that blood moves from the collection bag to the cartridge by gravity feed. Ozone is produced by the generator at a predetermined concentration and flow rate commensurate with the patient's clinical and laboratory status. Concentrations used in viral load reduction therapy range from 30 to 100 ug/ml. Flow rates approximate 1/2 to one liter per minute. A conduit from the generator feeds ozone to the cartridge. Another conduit returns unspent ozone to the ozone destructor of the generator so that ozone does not diffuse into the treatment area.

Blood is allowed to flow through the cartridge. In so doing it becomes ozonated and oxygenated. Flow rate through the cartridge is calculated so that depending upon ozone, concentration in the cartridge and ozone permeability through the membrane, ozone dosing to the blood is determined. Since the blood outflow from the cartridge may be directly connected to the same intravenous conduit used to withdraw blood from the patient, ozonated/oxygenated blood is accordingly returned to the patient via this same route.

Treatment is continued every 2 to 4 days for an average number of sessions approximating ten. It is important to note, however, that treatment course is predicated upon sequential clinical and laboratory determinations. Laboratory measures include a comprehensive blood screen including hepatic enzyme quantification, and/or viral load determinations by polymerase chain reaction (PCR) or other nucleic acid assay known in the art. In the event that viral load reduction is judged to be satisfactory, treatment may be halted after fewer than ten sessions and the patient monitored thereafter at regular intervals.

Claims

I claim:

1. A method of reducing a viral load in a biological liquid by treating the liquid with ozone, wherein the method comprises the steps of: passing the biological liquid through a cartridge comprising a gas permeable, flat- sheet membrane, and contacting the gas permeable membrane with the ozone for a time and under conditions sufficient to inactivate the virus in the biological liquid.

2. The method according to claim 1 , where in the biological liquid is blood or a blood product.

3. The method according to claim 1, wherein the membrane is bilayer membrane.

4. The method according to claim 3, wherein the membrane is a silicone membrane.

5. The method according to claim 4, wherein the distance between the bilayers is between about 5 cm and about 0.1 mm.

6. The method according to claim 4, wherein the distance between the bilayers is between about 5 mm and about 0.5 mm.

7. The method according to claim 4, wherein the distance between the bilayers is between about 1.5 mm and about 0.5 mm.

8. An apparatus for disinfecting a biological liquid with ozone, the apparatus comprising: a hollow cartridge including an interior wall, a liquid flow inlet, a liquid flow outlet, an ozone inlet and an ozone outlet; a gas permeable, flat-sheet membrane having a first side and a second side and contained within the cartridge, the first side of the membrane and the interior wall of the cartridge defining an area for circulating ozone from the ozone inlet to the first side of the membrane, a portion of the second side of the membrane defining a narrow passageway in communication with the biological liquid flow inlet and the liquid flow outlet, wherein the ozone passes through the membrane to treat the liquid flowing through the passageway.

9. The apparatus of claim 8, wherein the membrane is a bilayer membrane.

10. The apparatus of claim 9, wherein the membrane is a silicone membrane.

11. The apparatus of claim 10, wherein the distance between the bilayers is between 0.1 mm and 5 cm.

12. The apparatus of claim 10, wherein the distance between the bilayers is between 0.5 mm and 5 mm.

13. The apparatus of claim 10, wherein the distance between the bilayers is between 0.5 mm and 1.5 mm.