[go: up one dir, main page]

WO2011162805A2 - Inactivation de prion à l'aide d'ozone - Google Patents

Inactivation de prion à l'aide d'ozone Download PDF

Info

Publication number
WO2011162805A2
WO2011162805A2 PCT/US2011/001103 US2011001103W WO2011162805A2 WO 2011162805 A2 WO2011162805 A2 WO 2011162805A2 US 2011001103 W US2011001103 W US 2011001103W WO 2011162805 A2 WO2011162805 A2 WO 2011162805A2
Authority
WO
WIPO (PCT)
Prior art keywords
ozone
fluid
infectious
biological
gas
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2011/001103
Other languages
English (en)
Other versions
WO2011162805A3 (fr
Inventor
Joseph S. Latino
Steven A. Keyser
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Acquisci Inc
Original Assignee
Acquisci Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Acquisci Inc filed Critical Acquisci Inc
Publication of WO2011162805A2 publication Critical patent/WO2011162805A2/fr
Publication of WO2011162805A3 publication Critical patent/WO2011162805A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

  • This invention relates to therapeutic methods for inactivating infectious prion proteins in a biological fluid, and specifically relates to methods of prion protein inactivation using ozone-related therapies.
  • ozone has been used as a disinfectant or sterilizing agent in a wide variety of applications. These include fluid-based technologies such as: purification of potable water, disinfection of wastewater and sewage, and inactivation of pathogens in biological fluids. Ozone has also been used in the past as a topical medicinal treatment, as a systemic therapeutic and as a treatment of various fluids that were subsequently used to treat a variety of diseases. Specifically, there have been numerous attempts utilizing a variety of technologies to inactivate viruses in biological fluids.
  • the methods of the invention involve subjecting an amount of a fluid containing infectious prion proteins to an amount of ozone delivered by an ozone delivery system that is entirely structured of ozone-inert materials or construction to assure accurate evaluation of the amount of ozone absorbed by the fluid containing infectious prion proteins.
  • the method may provide for maintaining the biological integrity of the biological fluid.
  • the method employs an ozone-delivery system for delivering and manufacturing a precise, measured amount of an
  • ozone/oxygen admixture which is able to measure, control and report and
  • the system may include improved gas-fluid contacting devices that maximize gas-fluid mass transfer. All surfaces of the system which come into contact with ozone, including all pathways leading to and away from one or more gas-fluid contacting devices, and the gas-fluid contacting devices themselves, are made from ozone-inert construction materials that do not absorb ozone or introduce contaminants or deleterious byproducts of oxidation into a fluid.
  • treatment systems of the present invention using an ozone delivery system are illustrated in the appended drawing, which schematically illustrates what is currently considered the best mode for carrying out the invention:
  • FIG. 1 illustrates, in a schematic diagram, alternate methods of carrying out treatment of a fluid comprising a discontinuous flow method
  • FIG. 2 illustrates, in a schematic diagram, a method of carrying out treatment of a fluid from a patient, comprising a continuous loop format method.
  • a gas-fluid contacting device includes a plurality of such gas-fluid contacting devices, and reference, for example, to a
  • protein is a reference to a plurality of similar proteins, and equivalents thereof.
  • an "ozone/oxygen admixture” refers to a concentration of ozone in an oxygen carrier gas.
  • concentration utilized by those skilled in the art include: micrograms of ozone per milliliter of oxygen, parts (ozone) per million (oxygen) by weight ('ppm') and parts per million by volume ('ppmv').
  • ppmv is defined as the molar ratio between ozone and oxygen.
  • One ppmv ozone is equal to 0.00214 micrograms of ozone per milliliter of oxygen.
  • one ppm ozone equals 0.00143 micrograms of ozone per milliliter of oxygen.
  • 1 % ozone equals 14.3 micrograms of ozone per milliliter of oxygen. All units of concentration and their equivalents are calculated at standard temperature and pressure (i.e. 25°C at 1 atmosphere).
  • live-ozone is the amount of ozone contained within a volume of an ozone/oxygen admixture that is delivered to a fluid, and is synonymous with the delivery of a measured amount of ozone.
  • absorbed-ozone is the amount of delivered ozone that is actually absorbed and utilized by an amount of fluid, and is synonymous with a quantifiable "absorbed-dose of ozone.”
  • residual-ozone is the amount of delivered-ozone that is not absorbed such that:
  • residual-ozone delivered-ozone - absorbed-dose of ozone.
  • An “interface” is defined as the contact between a fluid and an ozone/oxygen admixture.
  • Interface-time is defined as the time that a fluid resides within a gas-fluid contacting device and is interfaced with an ozone/oxygen admixture.
  • Interface surface area is defined as the dimensions of the surface within a gas-fluid contacting device over which a fluid flows and contacts an ozone/oxygen admixture.
  • Elapsed-time is the time that a fluid circulates throughout an ozone delivery system, including passage through one or more gas-fluid contacting devices, connecting tubing and an optional reservoir.
  • Ozone-inert materials are defined as construction materials that do not react with ozone in a manner that introduces contaminants or deleterious byproducts of oxidation of the construction materials into a fluid, and materials that do not absorb ozone.
  • Non-reactive is defined as not readily interacting with other elements or compounds to form new chemical compounds.
  • Measured-data is defined as information collected from various measuring components (such as an inlet ozone concentration monitor, exit ozone concentration monitor, gas flow meter, fluid pump, data acquisition device, humidity sensor, temperature sensor, pressure sensor, absorbed oxygen sensor) throughout the system.
  • Calculated-data is defined as the mathematical treatment of measured-data by a data acquisition device.
  • Absorption of ozone by a biological fluid is defined as the phenomenon wherein ozone reacts with the fluid being treated by a variety of mechanisms, including oxidation. Regardless of the mechanism involved, the reaction occurs instantaneously, and the products of this reaction include oxidative products, of which lipid peroxides are an example.
  • biological fluid is defined as a composition originating from a biological organism of any type.
  • biological fluids include blood, blood products and other fluids, such as saliva, urine, feces, semen, milk, tissue, tissue samples, homogenized tissue samples, gelatin and any other substance having its origin in a biological organism.
  • biological fluids may also include synthetic materials
  • a substance having its origin in a biological organism such as a vaccine preparation containing alum and a virus (the virus being the substance having its origin in a biological organism), cell culture media, cell cultures, viral cultures, and other cultures derived from a biological organism.
  • a "blood product” is defined as including blood fractionates and therapeutic protein compositions containing proteins derived from blood. Fluids containing biologically active proteins other than those derived from blood may also be treated by the method.
  • In vivo use of a material or compound is defined as the introduction of a material or compound into a living human, mammal or vertebrate.
  • In vitro use of a material or compound is defined as the use of the material or compound outside a living human, mammal or vertebrate, where neither the material nor compound is intended for reintroduction into a living human, mammal or vertebrate.
  • An example of an in vitro use would be the analysis of a component of a blood sample using laboratory equipment.
  • Ex vivo use of a process is defined as using a process for treatment of a biological material such as a blood product outside of a living human, mammal, or vertebrate. For example, removing blood from a human and subjecting that blood to a method to treat a cardiovascular disease is defined as an ex vivo use of that method if the blood is intended for reintroduction into that human or another human.
  • synthetic media is defined as an aqueous synthetic blood or blood product storage media consisting wholly of dilute, reproducible solution of chemically pure, known inorganic and/or organic compounds. Artificial media of exactly known, reproducible composition are called synthetic or chemically defined media.
  • a "pharmaceutically acceptable carrier” or “pharmaceutically acceptable vehicle” is defined as any liquid including: water, saline, a gel, salve, solvent, diluent, fluid ointment base, liposome, micelle, giant micelles or combinations thereof, which is suitable for use in contact with a living animal or human tissue without causing adverse physiological responses, and which does not interact with the other components of the composition in a deleterious manner.
  • prion or “prions” are defined as a pneumonic for protein infectious particles, having been abbreviated as PrP.
  • the terms “prion” or “prions” refer to all prion protein and do not distinguish between different isoforms.
  • PrPC represents the normal cellular prion protein that is present in healthy people. This form is rich in alpha-helical conformation and is soluble and protease-sensitive.
  • infectious prion proteins is defined as a group of prion isoforms which may include PrPsc, PrPres, PrP 27-30 as well as other prion derivatives.
  • PrPsc represents the disease-associated misfolded prion protein that is present in individuals affected by transmissible spongiform encephalopathies (TSE), is infectious, largely in the beta-sheet conformation, is insoluble and is mostly protease-resistant.
  • PrPres refers to a beta-sheet-rich, protease-resistant prion protein (wherein proteinase K is commonly the protease), which may or may not be identical to PrPsc.
  • PrPres is used to refer to protease- resistant protein that is produced in vitro and which has not been experimentally shown to be infectious.
  • PrP 27-30 refers to the resistant protein core that remains after protease treatment of PrPsc or PrPres. It comprises the last two-thirds of the protein.
  • TSEs transmissible spongiform encephalopathies
  • PrP prion protein
  • BSE bovine spongiform encephalopathy
  • anti-prion activity is defined as the result of a process that renders an infectious prion protein-contaminated biological fluid non-infectious.
  • vCJD new variant Creutzfeldt-Jakob disease
  • biologically-active is defined as being capable of effecting a change in the living organism or component thereof.
  • biologically-active with respect to biologically-active protein does not refer to proteins that are part of the infectious prion protein being inactivated.
  • a non-infectious biological fluid is defined as a biological fluid being unable to cause one or more diseases.
  • biological integrity of a biological fluid refers to a fluid that subsequent to the application of the inactivation methods described herein, sufficiently maintains its replacement value for intended use in vivo or in vitro use or processes.
  • “Western blotting” is defined as a technique involving the separation of proteins on an electrophoretic gel for identification by immunological techniques.
  • Cell blotting is defined as an immunochemical-detection method developed to rapidly detect the earliest stages of de novo prion infection of cultured cells.
  • ScN2a cells chronic scrapie infected neuroblastoma cell line
  • N2a cells normal neuroblastoma cell line
  • cell blotting detects proteinase K-resistant PrP ScN2a/N2a ratios of 1 :100 as compared to 1 : 10 for Western blotting.
  • animal bioassays are defined as methodologies utilizing, for example, mice, Syrian hamsters and transgenic mice, which allow for the
  • biological fluids are treated with an ozone/oxygen admixture to produce a product free of infectious prions, the resulting product being useful in other biological processes or testing.
  • byproducts of ozonation are generated by the treatment of biological fluids with an ozone/oxygen admixture.
  • the byproducts may, for example, include lipid peroxides that are a result of the absorption of an absorbed-dose of ozone by the biological fluid from the delivered-ozone.
  • the presence of lipid peroxides is quantifiable by assay.
  • a direct relationship can be derived between the absorbed-dose of ozone absorbed by a biological fluid and these quantifiable lipid peroxides.
  • the lipid peroxides generated by inactivation of infections prion proteins in biological fluids may be pharmacologically active and possess intrinsic anti-prion activity.
  • a quantifiable pharmacologically-active anti- prion amount of lipid peroxides may be generated when a biological fluid has absorbed an absorbed-dose of ozone.
  • a fluid additive such as a diluent or fluid carrier
  • a fluid additive may be treated with an ozone/oxygen admixture in accordance with the described methods to produce a fluid additive that is effective in rendering the biological fluid to which the treated fluid is added non-infectious for prion activity.
  • a biological fluid may be rendered noninfectious for deleterious prion activity by an indirect method that does not expose the cellular components of the biological fluid to potentially harmful conditions.
  • INFECTIOUS PRION PROTEINS As reported by Prusiner, prion diseases or transmissible spongiform encephalopathies are neurodegenerative disorders that can be sporadic, inherited or acquired by infection. The underlying pathogenic event in all prion diseases is a conformational modification of the normal or cellular prion protein
  • PrPC is soluble in non-denaturing detergents, whereas PrPsc is insoluble; PrPC is readily digested by proteases (i.e. proteinase K), whereas PrPsc is mostly resistant, and digestion results in the formation of an amino-terminally truncated fragment known as PrP 27-30.
  • proteases i.e. proteinase K
  • PrPsc is mostly resistant, and digestion results in the formation of an amino-terminally truncated fragment known as PrP 27-30.
  • PrPsc has been shown to catalyze the conversion of PrPC into a protease-resistant PrPsc-like form in vitro.
  • the final, processed form of PrP from the hamster contains amino acids 23- 231 from the original translation product of 253 amino acids.
  • Peptide 1-22 is cleaved as signal peptide during trafficking, and peptide 232-253 is replaced by the
  • glycosylphosphatidylinositol- anchor ('GPI-anchor').
  • the cellular form is attached via this anchor to the outer membrane.
  • Asparagine residues 181 and 97 carry highly branched glycosyl groups with sialic acid substitutions.
  • PrP is always isolated as a mixture of three forms; unglycosylated, with one glycosyl-, and with two glycosyl- groups.
  • a disulphide bridge is formed between Cys179 and Cys214.
  • PrPC is constitutively expressed in neurons as a GPI-anchored protein and is localized into the cholesterol and sphingolipid-rich domains of the plasma membrane, termed lipid rafts. These lipid domains are defined biochemically by their insolubility in nonionic detergents at low temperature and thus are also termed detergent-resistant membranes (DRMs).
  • DRMs detergent-resistant membranes
  • pharmacological treatments that modify the lipid composition of the DRMs result in decreased efficiency of prion propagation in N2a cells infected with scrapie.
  • recombinant transmembrane PrPC lacking the GPI anchor is not located in DRMs and acts as a poor substrate for the formation of PrPres in neuroblastoma cells and in a transgenic mouse model.
  • In vitro studies using a cell- free system have shown that the insertion of PrPres into the DRMs is required for the conversion of PrPC into PrPres.
  • PrPsc According to the "protein only" hypothesis for prion misfolding, PrPsc interacts directly with the host-encoded PrPC, which is then converted to PrPsc. PrPsc accumulates in the nervous tissue, and causes disease when it reaches a critical threshold.
  • PrPC to PrPsc conversion is mediated by chaperone(s), and molecular chaperones may include Hsp104 and GroEL.
  • Hsp104 and GroEL Several lines of evidence indicate that the PrPC to PrPsc conversion is mediated by chaperone(s), and molecular chaperones may include Hsp104 and GroEL.
  • This protein temporarily named Protein X, remains to be identified and may be a cellular chaperone.
  • vCJD variant CJD
  • LRS lymphoreticular system
  • Biological Products The use of ozone for prion inactivation in biological fluids is of particular interest as the safety of the blood supply is an issue of universal concern.
  • materials to be treated to render them non-infectious include: whole blood, packed red cells, platelets and plasma (both fresh and fresh frozen plasma) and aqueous compositions containing biologically active proteins derived from blood or blood fractionates.
  • Factor VIII Von Willebrand Factor, Factor IX, Factor X, Factor XI, Hageman Factor, prothrombin, antithrombin III, fibronectin, plasminogen, plasma protein fraction, immune serum globulin, modified immune globulin, albumin, plasma growth hormones, somatomedin, plasminogen- streptokinase complex, ceruloplasmin, transferrin, haptoglobin, antitrypsin and prekallikrein may be treated by one embodiment of the inactivation method.
  • a method of inactivating infectious prion proteins in biological fluids while maintaining the biological integrity of the fluid is provided.
  • the biological fluid is a blood component.
  • the blood product is either plasma, red blood cell preparations or platelet concentrates.
  • a method of inactivation is performed in a blood bank or similar setting, wherein the biological fluid is passed through a gas-fluid contacting device and subjected to the methods described herein. The blood product is then suitable for in vitro or in vivo use.
  • Biological fluids are generally aqueous in nature and in many cases are in a fluid state compatible with treatment by the method described herein. However, in those instances where required (i.e. blood and blood fractionates), a compatible aqueous buffered carrier may be added to lower viscosity, promote anticoagulation, or to otherwise permit the method to occur more effectively.
  • lipid peroxides are reproducibly generated as an example of a reactive oxidative intermediate byproduct of the process, in a carrier fluid to be subsequently added to a biological fluid.
  • Platelet additive solutions may contain physiological saline solution, buffers, and other components including magnesium chloride and sodium gluconate. These solutions are useful carriers for platelet concentrates and allow for maintenance of cell quality and metabolism during storage, reduce plasma content and extend storage life.
  • the pH of such solutions is preferably between about 7.0 and 8.0.
  • Creutzfeldt-Jakob disease (CJD) and other prion-related diseases are known to resist conventional sterilization procedures.
  • Methods to inactivate infectious prion proteins include alkali, low pH, high hydrostatic pressure and detergent-proteolytic enzyme combinations-- all agents that are incompatible with maintaining the biological integrity of a biological fluid.
  • PrPsc An infectious prion, abbreviated PrPsc, represents the disease-associated misfolded prion protein that is present in individuals affected by transmissible spongiform encephalopathies.
  • TSEs are neurodegenerative diseases that include Creutzfeldt-Jakob disease, chronic wasting disease, scrapie, and bovine spongiform encephalopathy.
  • the detection method can be theoretically defined as the measurement of the level of infection, with disease as the result of exposure to the material.
  • the threshold for infection below which the inactivation method is complete is then taken to be the level of inactivation which is sufficient to prevent the disease from occurring due to contact with the biologically derived material. It is recognized that in this practical scenario, it is not essential that the methods result in “total inactivation”. That is to say, “substantial inactivation” will be adequate as long as the treated biologically derived material contains residual prions in an amount insufficient to cause disease, defined as "noninfectious".
  • the inactivation methods described render the infectious prion proteins substantially inactivated.
  • the inactivation method renders the infectious prion proteins in blood preparations inactivated and it is inferred to be substantially inactivated, or non-infectious, as defined above.
  • the inactivation method provides an ex vivo method of inactivating infectious prion proteins in biologically derived materials, most commonly fluids, prior to use in vivo or in vitro.
  • Methods to assess the inactivation of infectious prions may include cell-based assays including chronically scrapie-infected neuroblastoma cells (ScN2a) as a model for studying TSEs.
  • the cells produce PrPsc, permitting cellular processes associated with PrPsc production to be examined. This assay uses mouse
  • neuroblastoma N2a cell sublines that are highly susceptible to mouse prions, as shown by the accumulation of PrPres and infectivity.
  • susceptible N2a cells are exposed to prion-containing samples after being subjected to the inactivation method described, and are grown for several generations.
  • the proportion of cells that contain PrPres may be determined through an immunoblotting methodology.
  • Immunoblotting assays such as Western blotting or Cell blot assays rely on the proteinase K resistance of PrPsc as a distinguishing feature from native PrP and utilize monoclonal antibodies which recognize specific conserved epitopes in both prion isoforms.
  • Another possibility for biochemical differentiation is based on the insolubility of PrPsc in a nonionic detergent.
  • the lysate is suspended in the detergent, pelleted by ultracentrifugation and the subsequent pellet and supernatant fractions are analyzed immunochemically.
  • PrPC remains soluble in the supernatant whereas PrPsc is found insoluble in the pellet.
  • animal bioassays often utilizing susceptible, and in most cases transgenic, PrP-overexpressing mice, where the incubation time to clinical prion disease is measured, may be employed to evaluate the effectiveness of the inactivation method in preventing the transmission of PrPsc.
  • the inactivation method provides an ex vivo method of inactivating infectious prion proteins in biological fluids prior to use in vivo or in vitro.
  • the described methods inactivate infectious prion proteins present in biological fluids through a single procedural step without the need of additional inactivation processes.
  • the methods described herein for inactivation of infectious prion protein viruses may be complimentary or synergistic with other inactivation processes when used in conjunction with such processes, either prior to, subsequent to or contemporaneously with other inactivation methods.
  • These other inactivation methods may include targeted chemotherapeutics (e.g. frangible anchor-linked-effectors), photochemotherapeutics (e.g. psoralen derivatives), gamma-irradiation and
  • photodynamic antimicrobial chemotherapy e.g. thiazine dyes such as thionin, azure A, azure B, azure C, methylene blue and Toluidine blue, and riboflavin.
  • the inactivation method provides a means to inactivate infectious prion proteins while maintaining the biological integrity of the biologically-derived material for its intended use. Furthermore, the method allows inactivation of infectious prions at temperatures compatible with maintaining the biological integrity of the material, most commonly biological fluids.
  • the biological integrity of plasma may be measured by the functionality of its protein components either in whole plasma or after separation into plasma fractions.
  • the biological integrity of red blood cell and platelet preparations may be determined by the methods and criteria known by those skilled in the art and are similar to those used in establishing the suitability of storage and handling protocols.
  • the biological integrity of a biological fluid is a fluid that subsequent to the inactivation method described herein, has sufficiently maintained its replacement value for intended use in vivo or in vitro.
  • maintaining the biological integrity of a growth media or serum supplement can be measured as the sufficient retention of the functionality of the media to support growth and its other indicated uses subsequent to the inactivation process described herein.
  • Potential mechanisms to inactivate PrPsc-contaminated biological fluids sufficient to render it non-infectious may include processes that promote dis-aggregation of the PrPsc isoform, reduction in its beta-sheet content and enhancing sensitivity to proteinase K digestion.
  • Glycosylation of specific asparagine residues can modify the conformation of PrPC and affect its affinity for a particular conformer of PrPsc, which results in specific patterns of PrPsc deposition. These interactions between PrPsc and PrPC are likely to determine the rate of nascent PrPsc formation.
  • glycosylation modifies the stability of PrPsc and, hence, the rate of PrPsc clearance. Oxidation of these sugars leading to their perturbation or disruption is an additional potential site for inactivation of an infectious prion protein.
  • these sugar residues have been associated with binding of PrPC to Protein X which is considered the rate-limiting step in PrPsc formation under most circumstances. Possible alteration of these sugars may impede binding of chaperone proteins including Protein X affecting PrPsc conversion of native PrP.
  • another site of action may be the specific single disulfide bond between Cys 179 and Cys 214 which is responsible for the joining COOH-terminal helices required for PrPsc formation; a site potentially susceptible to oxidation.
  • a method is provided of inactivating infectious prion proteins while maintaining the biological integrity of the biological fluid producing a non-infectious biological fluid.
  • the method involves subjecting a quantity of a fluid containing infectious prions, to an amount of ozone delivered by an ozone delivery system.
  • Inactivation of infectious prions in biological fluids generates byproducts of ozonation, which may include lipid peroxides that are a result of the absorption of an absorbed-dose of ozone from the delivered-ozone by the fluid.
  • the lipid peroxides are quantifiable by a variety of known assays.
  • Oxidation of polyunsaturated fatty acids involves an allylic hydrogen abstraction followed by insertion of molecular oxygen; the resulting peroxyl radicals abstract hydrogens to form lipid peroxides.
  • ozone reacts directly with carbon-carbon double bonds in unsaturated fatty acids and that free radical lipid peroxides can be detected by electron spin resonance.
  • These lipid peroxides are relatively unstable compounds and decompose to form a series of reactive carbonyl compounds including malondialdehyde ('MDA') and 4-hydroxyalkenals ( ⁇ '). Measurement of either MDA or HAE may be utilized as an indicator of lipid peroxidation.
  • the assay for the quantification of lipid peroxides can be performed according to the thiobarbituric acid (TBA) procedure where the formation of the malondialdehyde-TBA adduct results in a fluorometrically assayable species at 532 nm under acidic conditions.
  • TSA thiobarbituric acid
  • the test measures the amount of malondialdehyde, a decomposition product of lipid peroxides.
  • the total amount of malondialdehyde MDA measured reflects the amount formed from lipid peroxides during the testing
  • An alternative method to the fluorometric determination in the thiobarbituiric acid assay for lipid peroxide quantification is through the use of High Pressure Liquid Chromatography. The procedure involves forming the thiobarbituric acid-malondialdehyde adduct, separating it on suitable column (i.e. uBondapak C-i 8 ), and measuring the absorbance at 546 nm.
  • An alternative method for quantification of lipid peroxides is based upon the formation of a stable chromophore between the MDA degradation product of lipid peroxides and N-methyl-2-phenylindole under acidic conditions, and assayable by spectroscopy at 586 nm. Both fluorometric and HPLC thiobarbituric detection
  • HAE Assay Analysis of the formation of 4-hydroxyalkenals ('4-HAE') can also be utilized as a measure of lipid peroxidation. Although 4-HAE represents relatively minor degradation products of lipid peroxidation as compared to MDA, they can also be used as an indicator of lipid peroxidation. The most abundant 4-hydroxyalkenal formed in lipid peroxidation is 4-hydroxy-2-nonenal (' ⁇ ').
  • the assay for HAE is based upon the formation of a stable chromophore with N-methyl-2-phenylindole under acidic conditions. In contrast to the assay for MDA, the acidic conditions for HAE assay involve the use of methanesulfonic acid. HAE may either be assayed individually or in conjunction with MDA by spectroscopy at 586 nm.
  • lipid peroxides lipid peroxides
  • 'LPs' lipid peroxides
  • These LPs are a product of ozonation at various sites of unsaturation found in the carbon chain lengths found in the aliphatic portion of the fatty acids that compose lipids.
  • these LPs can be quantified by conventional methods such as the formation of a malondialdehyde or hydroxyalkenal adduct by the assays described above.
  • Those skilled in the art will appreciate that other assays are available for the quantification of lipid peroxides.
  • adducts are subsequently quantified by fluorescence or absorption spectroscopy against an established standard curve.
  • the high reactivity and concomitant short half-life of those LPs derived from five (5) carbon chain lengths or less in an aqueous fluid, and which form more stable aldehydes, ketones, alcohols or other relatively stable oxygen containing compounds that do not contain a peroxide component are precluded from such measurement.
  • a large number of LPs that are derived from fatty carbon chain lengths in excess of five (5) carbons are sufficiently stable and therefore measurable.
  • the wide variety of LPs that are measured by these assays are represented as a total aggregate number of LPs.
  • a direct relationship can be derived between the absorbed-dose of ozone absorbed by a biological fluid and the aggregate number of LPs produced.
  • Quantifiable lipid peroxides may be pharmacologically-active and possess intrinsic anti-prion activity.
  • a quantifiable pharmacologically anti-prion amount of lipid peroxides may be generated when a biological fluid has absorbed an absorbed-dose of ozone.
  • Products of ozonation, including lipid peroxides can be quenched by the addition of a biocompatible agent.
  • biocompatible agents may include all known antioxidants including Vitamin A, Vitamin E, other tocopherol-containing compounds, glutathione, ascorbic acid, curcumin and activated charcoal.
  • activated charcoal subsequent to quenching the products of ozonation, the activated charcoal may be removed. Furthermore, the use of activated charcoal may be used to remove the products of ozonation, including lipid peroxides. Subsequent to the contact of the activated charcoal with the ozone absorbed biological fluid, the charcoal may be removed.
  • Ozone Delivery System
  • An ozone delivery system utilized in the methods of the present invention to inactivate infectious prion proteins in biological fluids delivers a measured amount of an ozone/oxygen admixture and is able to measure, control, report and differentiate between the delivered-ozone and absorbed-dose of ozone.
  • the system provides a controllable, measurable, accurate and reproducible amount of ozone that is delivered to a controllable, measurable, accurate and reproducible amount of a biological fluid, and controls the rate of ozone absorption by the fluid resulting in a quantifiable absorbed-dose of ozone used in the treatment of cardiovascular diseases.
  • the system may accomplish this by using a manufacturing component, control components, measuring components, a reporting component and calculating component (such as an ozone generator, gas flow meter, fluid pump, variable pitch platform, data acquisition device, inlet ozone concentration monitor, and exit ozone concentration monitor) that cooperate to manufacture and deliver a measured, controlled, accurate and reproducible amount of ozone, i.e., the delivered-ozone, to a fluid through the use of one or more gas-fluid contacting devices that provides for the interface between the ozone/oxygen admixture and fluid.
  • a manufacturing component control components, measuring components, a reporting component and calculating component (such as an ozone generator, gas flow meter, fluid pump, variable pitch platform, data acquisition device, inlet ozone concentration monitor, and exit ozone concentration monitor) that cooperate to manufacture and deliver a measured, controlled, accurate and reproducible amount of ozone, i.e., the delivered-ozone, to a fluid through the use of one or more gas-fluid contacting devices
  • control components such as a gas flow meter, fluid pump, variable pitch platform, data acquisition device, inlet ozone concentration monitor and exit ozone
  • the system may instantly differentiate the delivered-ozone from the absorbed-dose of ozone.
  • the system utilizes (a gas flow meter, fluid pump, variable pitch platform, data acquisition device, inlet ozone concentration monitor, and exit ozone concentration monitor) control components, measuring components, a reporting component and calculating component that cooperate and instantly report data that may include the delivered-ozone, residual-ozone, absorbed-dose of ozone, interface-time, elapsed- time and the amount and flow rate of the fluid delivered to the gas-contacting device.
  • a particularly suitable ozone delivery system that may be used in carrying out the methods of the present invention is disclosed in U.S. Patent No. 7,736,494 and co-pending application Serial No. 12/813,371 , the contents of which are incorporated herein in their entirety.
  • the disclosed ozone delivery system is particularly and uniquely constructed such that all ozone-contacting surfaces of the device are made of ozone-inert material so that the amount of ozone that is actually absorbed by the biological fluid being treated is accurately determinable.
  • Ozone-inert materials include stainless steel, borosilicate, quartz, ceramic composites, PFA (copolymer of tetrafluoroethylene and perfluorinated vinyl ether from the perfluoroalkoxy group) and PTFE (polytetrafluoroethylene, TEFLON), and are further defined as being non-reactive within the concentration range of ozone manufactured and delivered by an ozone delivery system.
  • a delivery system including one or more gas-fluid contact devices may be constructed without the inclusion of any fluoropolymers, polyfluoroethylene, PFA or PTFE materials, in the event PTFE becomes a health concern.
  • FIG. 1 illustrates through a schematic flow chart illustration the structural and functional aspects of an ozone delivery system that may be utilized in carrying out the methods of the present invention.
  • oxygen flows from a pressurized cylinder (1-1), through a regulator (1-2), through a particle filter (1-3), and through a flow meter (1-4) where the oxygen and subsequent ozone/oxygen admixture flow rate is controlled and measured, through a pressure release valve (1-5), and through an ozone generator (1-6) where the concentration of the ozone/oxygen admixture is manufactured and controlled and where the admixture volume contains the delivered- ozone.
  • medical grade oxygen is the source gas utilized, as lesser grades of oxygen may include nitrogenous contaminants resulting in the formation of toxic nitrous oxides.
  • the ozone/oxygen admixture flows through a particle filter (1-3) to remove particulates, and through an optional moisture trap (1-7), to reduce moisture.
  • the admixture proceeds through an ozone inlet concentration monitor (1-8) that measures and reports the inlet ozone concentration of the ozone/oxygen admixture that contains the delivered-ozone. This real-time measurement may be based on ozone's UV absorption characteristics as a detection methodology.
  • the ozone/oxygen admixture then passes through a set of valves (1-9) used to isolate a gas-fluid contacting device for purging of gases.
  • the ozone/oxygen admixture may pass an optional humidity sensor (1-20) where humidity may be measured and recorded, through a gas-fluid contacting device (1-10) where it interfaces with a fluid.
  • the interface-time between an ozone/oxygen admixture and a fluid may be controlled through adjustment of the variable pitch platform as illustrated in Figure 3, the fluid pump (1-15), and the time controlling capacity of the data acquisition device (1-17).
  • the interface-time can be measured by the data acquisition device (1-17).
  • Temperature (1-21) and pressure (1-22) may be measured by the use of temperature and pressure sensors, respectively, inserted into their respective ports.
  • the resultant ozone/oxygen admixture containing the residual-ozone then exits the gas-fluid contacting device and flows through the exit purge valves (1-1 1 ), through a moisture trap (1-7), through an exit ozone concentration monitor (1-12), which may utilize a similar detection methodology as ozone concentration monitor (1-8), and that measures and reports the exit ozone/oxygen admixture concentration.
  • the exiting ozone/oxygen admixture then proceeds through a gas drier (1-13), through an ozone destructor (1-14) and a flow meter (1-19).
  • FIG. 1 further illustrates that a fluid flows through tubing, from the fluid pump (1 - 15), into the gas-fluid contacting device (1-10) where it interfaces with an
  • Insertion ports for temperature and pressure sensors may be located in the gas-fluid contacting device for the measurement of temperature and pressure, respectively.
  • the fluid After interfacing with the ozone/oxygen admixture, the fluid exits into tubing that may contain a port for an optional absorbed oxygen sensor (1-23), followed by a fluid access port allowing for fluid removal (1-24), and into an optional reservoir (1-16), where if configured in a closed loop, the fluid is circulated in a repetitive manner.
  • tubing may contain a port for an optional absorbed oxygen sensor (1-23), followed by a fluid access port allowing for fluid removal (1-24), and into an optional reservoir (1-16), where if configured in a closed loop, the fluid is circulated in a repetitive manner.
  • configurations may be utilized, including but not limited to, configurations similar to those used in dialysis for mammalian applications, for example as depicted in Figure 2.
  • a data acquisition device (1-17) such as DAQSTATION (Yokogawa), for example, has time measurement capabilities, reports, stores and monitors data instantly and in real-time, and performs various calculations and statistical operations on data acquired. All data is transmitted to the data acquisition device through data cables (1-18), including: data from ozone concentration monitors (1-8) and (1-12), flow meters (1-4) and (1-19), humidity sensor (1-20), temperature sensor (1-21 ), pressure sensor (1-22), fluid pump (1-15), and absorbed oxygen sensor (1- 23).
  • the elapsed time, a composite of both the interface time and the period of time that the fluid circulates through the other elements of the apparatus can be measured and controlled through the data acquisition device (1-17).
  • the ozone delivery system utilizes measuring components, reporting
  • components and calculating components such as an inlet ozone concentration monitor, exit ozone concentration monitor, gas flow meter, fluid pump, data acquisition device) that cooperate together to determine certain calculated-data including the delivered-ozone, the residual-ozone and the absorbed-dose of ozone.
  • Delivered-ozone is an amount of ozone calculated by multiplying the measured volume of ozone/oxygen admixtures, as reported by gas flow meters, by the measured concentration of ozone within the ozone/oxygen admixture as it enters the gas-fluid contacting device, as reported by the inlet ozone concentration monitor.
  • the measured volume of ozone/oxygen admixtures is calculated by multiplying the measured gas flow reported by gas flow meters, by the elapsed-time.
  • Residual-ozone is an amount of ozone calculated by multiplying the measured volume of ozone/oxygen admixtures, as reported by gas flow meters, by the measured concentration of ozone within the ozone/oxygen admixture exiting the gas- fluid contacting device, as reported by the exit ozone concentration monitor.
  • the measured volume of ozone/oxygen admixtures is calculated by multiplying the measured gas flow reported by gas flow meters, by the elapsed-time.
  • the quantifiable absorbed-dose of ozone is an amount of ozone calculated by subtracting the amount of residual-ozone from the amount of delivered-ozone.
  • the quantifiable absorbed-dose of ozone in the methods of the invention may range from 1 to 10,000,000 micrograms per milliliter of fluid, and may be between 1 and 10,000 ⁇ g per milliliter of fluid.
  • All measured-data including measured data from the gas flow meters, inlet and exit ozone concentration monitors, the fluid pump, temperature sensors, pressure sensors, absorbed oxygen sensor and humidity sensors are reported to a data acquisition device.
  • the data acquisition device has instant, real-time reporting, calculating and data storing capabilities to process all measured data.
  • the data acquisition device may use any measured data or any combination of measured data as variables to produce calculated-data. Examples of calculated-data may include delivered-ozone, residual-ozone, absorbed-dose of ozone, absorbed-dose of ozone per unit volume of fluid, and the quantifiable absorbed-dose of ozone per unit volume of fluid per unit time.
  • An ozone delivery system particularly suitable to the present invention includes an ozone generator for the manufacture and control of a measured amount of an ozone/oxygen admixture and where the admixture volume contains the delivered- ozone.
  • An ozone generator capable of producing ozone in a concentration range between 10 and 3,000,000 ppmv of ozone in an ozone/oxygen admixture may be employed.
  • Ozone/oxygen admixture concentrations entering the gas-fluid contacting device are instantly and constantly measured in real time, through an inlet ozone concentration monitor that may utilize UV absorption as a detection methodology.
  • a flow meter controls and measures the delivery of the delivered-ozone in an ozone/oxygen admixture to the gas-fluid contacting device at a specified admixture flow rate.
  • Ozone/oxygen admixture flow rates are typically in the range between 0.1 and 5.0 liters per minute.
  • Measurement of the humidity of the ozone/oxygen admixture delivered to the gas-fluid contacting device may be included through the use of a humidity sensor.
  • a humidity sensor port may be provided in the ozone/oxygen admixture connecting tubing; however, it can be placed in a variety of locations.
  • the humidity sensor may be located in the connecting tubing prior to the admixture's entrance into gas-fluid contacting device.
  • Measurement of the temperature within the gas-fluid contacting device during the interface-time may be provided by inclusion of a temperature sensor port in the gas fluid contacting device through which a temperature sensor may be inserted.
  • the temperature at which ozone/oxygen admixtures interface fluids ranges from 4°C to 100°C, and may be performed at ambient temperature, 25°C, for example.
  • the temperature at which the interface occurs can be controlled by placing the gas-fluid contacting device, optional reservoir, and both gas and fluid connecting tubing in a temperature controlled environment and/or by the addition of heating or cooling elements to the gas-fluid contact device.
  • Measurement of the pressure within the gas-fluid contacting device during the interface-time is provided by inclusion of a pressure sensor port in the gas-fluid contacting device through which a pressure sensor may be inserted.
  • the pressure at which an ozone/oxygen admixtures interfaces with a fluid ranges from ambient pressure to 50 psi and may be performed between ambient pressure and 3 psi, for example.
  • a pressure sensor port may be provided in each gas-fluid contacting device to measure and report the pressure at which the interface occurs.
  • the concentration of the ozone/oxygen admixtures exiting the gas-fluid contacting device, and where the admixture volume contains the residual-ozone, are instantly and constantly measured in real time through an exit ozone concentration monitor that may utilize UV absorption as a detection methodology.
  • a fluid pump controls and measures the flow rate of the fluid delivered to the gas-fluid-contacting device at a specified fluid flow rate.
  • Fluid flow rates through the gas-fluid contacting device typically will range from 1 ml to 100 liters per minute, and for example, may be between 1 ml to 10 liters per minute.
  • the fluid is generally contained within a closed-loop design and may be circulated through the gas-fluid contacting device once or multiple times.
  • Measurement of the amount of oxygen absorbed into a fluid while it interfaces with the ozone/oxygen admixture within the gas-fluid contacting device may be provided through the use of an absorbed oxygen sensor.
  • the sensor is inserted within the absorbed oxygen sensor port located in the tubing as it exits the gas-fluid contacting device.
  • Measurement of absorbed oxygen may be recorded in various units, including ppm, milligrams/liter or percent saturation.
  • the ozone delivery system may also include a fluid access port for fluid removal.
  • the port is generally located in the tubing member after the fluid exits through the fluid exit port of the gas-fluid contacting device and prior to an optional reservoir.
  • Calculated-data in carrying out the methods of the present invention include delivered-ozone, residual-ozone, and the quantifiable absorbed-dose of ozone.
  • Measurement of the volume of the ozone/oxygen admixture delivered can be calculated though data provided from the flow meter and the time measurement capability of the data acquisition device. Measurement of the volume of fluid delivered to the gas-fluid contacting device can be calculated by the data acquisition device utilizing fluid flow rate data transmitted from the fluid pump.
  • the elapsed-time can be measured and controlled through the data acquisition device.
  • the elapsed-time that the fluid circulates through the apparatus, including the gas-fluid contacting device, and is interfaced with an ozone/oxygen admixture can vary, generally for a duration of up to 120 hours.
  • the interface-time may also be measured by the time measuring capacity of the data acquisition device.
  • the interface-time between a fluid and an ozone/oxygen admixture may be controlled through a composite of controls. These controls include the angle of the gas fluid contacting device, the fluid flow rate via the fluid pump, and the time controlling capacity of the data acquisition device.
  • the interface-time may vary in duration of up to 720 minutes, and generally within duration of up to 120 minutes.
  • Controllable variables for an ozone delivery system may include delivered amounts and concentrations of ozone in the entering ozone/oxygen admixtures, fluid flow rates, admixture flow rates, temperature in the gas-fluid contacting device, interface-time between fluid and admixture, and the elapsed-time that the fluid may circulate through the apparatus and interface with an ozone/oxygen admixture.
  • Measurable variables may include ozone/oxygen admixture flow rates, amounts and concentrations of ozone in the entrance and exit ozone/oxygen admixtures, fluid flow rates, temperature and pressure in the gas-contacting device, humidity of the entrance admixture to the gas-fluid contacting device, absorbed oxygen by the fluid, interface-time and elapsed-time.
  • Data representing controllable variables and measurable variables acquired by the apparatus allows for a variety of calculations including delivered-ozone, residual- ozone, quantifiable absorbed-dose of ozone, quantifiable absorbed-dose of ozone per unit volume of fluid and the quantifiable absorbed-dose of ozone per unit volume of fluid per unit time.
  • FIG. 2 illustrates, in another aspect of the invention, that blood, as the infectious biological fluid, can be removed from a patient and extracorporeal ⁇ interfaced with an ozone/oxygen admixture.
  • Blood may be circulated in a continuous loop format in a venovenous extracorporeal exchange format as will be appreciated by one of skill in the art.
  • this continuous loop can be established through venous access of the antecubital veins of both right and left arms. Prior to establishing an
  • a patient may optionally be anticoagulated with heparin or any other suitable anticoagulant known to those skilled in the art.
  • the oxygen flows from a pressurized cylinder (2-1), through a regulator (2-2), through a particle filter (2-3) to remove particulates, through a flow meter (2-4) where the oxygen and subsequent ozone/oxygen admixture flow rate is controlled and measured.
  • the oxygen proceeds through a pressure release valve (2- 5), through an ozone generator (2-6) where the concentration of the ozone/oxygen admixture is manufactured and controlled and where the admixture volume includes the delivered-ozone.
  • the ozone/oxygen admixture flows through an optional moisture trap (2-7), to reduce moisture.
  • the admixture proceeds through an inlet ozone concentration monitor (2-8) that measures and reports the inlet ozone concentration of the ozone/oxygen admixture that contains the delivered-ozone. This real-time measurement may be based on ozone's UV absorption characteristics as a detection methodology.
  • the ozone/oxygen admixture then passes through a set of valves (2-9) used to isolate a gas-fluid contacting device for purging of gasses.
  • the ozone/oxygen admixture may pass an optional humidity sensor (2-20) where humidity may be measured and recorded, and into a gas-fluid contacting device (2-10) where it interfaces with fluid.
  • the interface-time between fluid and ozone/oxygen admixture may be controlled through adjustment of a variable pitch platform, a fluid pump and the time controlling capacity of the data acquisition device.
  • the interface-time may then be measured by the data acquisition device (2-17).
  • Temperature (2-21) and pressure (2-22) may be measured by the use of optional temperature and pressure sensors, respectively, inserted into their respective ports.
  • the resultant ozone/oxygen admixture containing the residual-ozone exits the gas-fluid contacting device and flows through the exit purge valves (2-11), through a moisture trap (2-7), through an exit ozone concentration monitor (2-12), which may utilize a similar detection methodology as the inlet ozone concentration monitor (2-8), that measures and reports the exit ozone/oxygen admixture concentration.
  • the exiting ozone/oxygen admixture then proceeds through a gas drier (2-13), through an ozone destructor (2- 14) and a flow meter (2-19).
  • intravenous blood flows from the patient through tubing through a pressure gauge (2-27) which monitors the pressure of the blood flow exiting the patient.
  • the pressure of the blood exiting the patient ranges from a negative pressure of 100 - 200 mm Hg, and may be between a negative pressure of 150 and 200 mm Hg, with a maximum cutoff pressure of minus 250 mm Hg.
  • the blood flows through a fluid pump (2-15) and is optionally admixed with heparin or other suitable anticoagulant as provided by an optional heparin pump (2-16).
  • the blood then passes through the gas-fluid contacting device (2-10) where it interfaces with the ozone/oxygen admixture containing the delivered- ozone.
  • Ports for the insertion of sensors may be located in the gas-fluid contacting device for the measurement of temperature and pressure, respectively.
  • the fluid After interfacing with the ozone/oxygen admixture, the fluid exits into tubing that may contain a port for an optional absorbed oxygen sensor (2-23) followed by a fluid access port (2-24).
  • the blood continues through an air/emboli trap (2-25) that removes any gaseous bubbles or emboli.
  • the blood then continues through a fluid pump (2-26) and then into a pressure gauge (2-28) which monitors the pressure of the blood flow before returning to the patient.
  • the pressure of the blood entering the patient ranges from a pressure of 100 - 200 mm Hg, and may be between 150 and 200 mm Hg, with a maximum cutoff pressure of 250 mm Hg.
  • the blood continues through a priming fluid access port (2-29) that allows for the removal of the priming fluid from the extracorporeal loop. The blood is then re-infused directly into the patient.
  • a data acquisition device (2-17) such as a DAQSTATION (Yokogawa), for example, has time measurement capabilities, reports, stores and monitors data instantly and in real-time, and performs various calculations and statistical operations on data acquired. All data is transmitted to the data acquisition device through data cables (2-18), including: data from ozone concentration monitors (2-8) and (2-12), flow meters (2-4) and (2-19), humidity sensor (2-20), temperature sensor (2-21 ), pressure sensor (2-22), fluid pumps (2-15) and (2-26), pressure gauges (2-27) and (2-28), and absorbed oxygen sensor (2-23).
  • the elapsed time, a composite of both the interface time and the period of time that the fluid circulates through the other elements of the apparatus can be measured and controlled through the data acquisition device (2-17).
  • Other configurations for an extracorporeal blood circuit are possible and the circuit illustrated in FIG. 4 is but one example.
  • Gas-fluid contacting devices may be included in an ozone delivery system to increase the surface area of a fluid to be treated allowing for an increase in the mass transfer efficiency of the ozone/oxygen admixture.
  • Gas-fluid contacting devices may encompass the following properties: closed and isolated from the ambient atmosphere, gas inlet and outlet ports for the entry and exit of ozone/oxygen admixtures, fluid inlet and outlet ports for the entry and exit of a fluid, components (temperature sensor, pressure sensor and data acquisition device) for the
  • Gas-fluid contacting devices include designs that encompass surfaces that may be horizontal or approaching a horizontal orientation. These surfaces may include ridges, indentations, undulations, etched surfaces or any other design that results in a contour change and furthermore, may include any pattern, regular or irregular, that may disrupt the flow, disperse the flow or cause turbulence. These surfaces may or may not contain holes through which a fluid passes through. The surface of the structural elements may have the same or different pitches. Designs of gas-fluid contacting devices may include those that involve one or more of the same shaped surfaces or any combination of different surfaces, assembled in any combination of ways to be encompassed within the device which may include cones, rods, tubes, flat and semi-flat surfaces, discs and spheres.
  • the interface between an ozone/oxygen admixture and a fluid may be accomplished by the use of a gas-fluid contact device that generates a thin film of the fluid that interfaces with the ozone-oxygen admixture as it flows through the device.
  • a gas-fluid contact device that generates a thin film of the fluid that interfaces with the ozone-oxygen admixture as it flows through the device.
  • generation of any interface that increases the surface area of the fluid and thereby maximizes the contact between a fluid and an admixture, may be used. Additional examples include the generation of an aerosol through atomization or nebulization.
  • the interface-time within a gas-fluid contacting device is measurable, controllable, calculable and reportable. Furthermore, the interface-time may be for duration of up to 720 minutes, generally however, for duration of up to 120 minutes. Following the interface-time, the fluid exits the gas-fluid contacting device containing the quantifiable absorbed-dose of ozone.
  • the elapsed-time a composite of both the interface-time and the time for circulation of a fluid through other elements of an ozone delivery system is also measurable, controllable, calculable and reportable. This elapsed-time is for duration of up to 120 hours.
  • the pressure at the interface between fluid and ozone/oxygen admixture within a gas-fluid contacting device may be measured. Measurement of pressure within the device may be accomplished through the use of a pressure sensor inserted at the pressure port of the gas-fluid contacting device. The pressure at which an
  • ozone/oxygen admixture interfaces with a fluid ranges from ambient pressure to 50 psi and may be performed between ambient pressure and 3 psi.
  • the temperature within a gas-fluid contacting device may be controlled by housing the device such that the connecting tubing containing both gas and fluid and an optional reservoir are maintained in a controlled temperature environment.
  • a flow hood that provides for temperature regulation is an example of a controlled
  • thermoelectric device may provide for the control of temperature. Measurement of temperature within the device may be accomplished through the use of a
  • the temperature at which ozone/oxygen admixtures interface fluids ranges from 4°C to 100°C, and may be performed at ambient temperature, 25°C, for example.
  • Gas-fluid contacting devices as disclosed in U.S. Patent No. 7,736,494 may be utilized in the ozone-delivery systems to carry out the methods of the present invention. Such gas-fluid contacting devices may be utilized individually or in conjunction with other such devices, whether they are similar or dissimilar in construction, design or orientation. In the event that multiple devices are utilized, either of the same design, or a combination of different gas-fluid contacting devices of different designs, these devices may be arranged one after the other in succession (in series), making a single device out of multiple individual contact devices.
  • a fluid flowing through the different contact devices flows in series, from the fluid exit port of one contact device to the fluid entrance port of the next, until passing through all the devices.
  • the ozone/oxygen admixture may flow in a number of arrangements.
  • the ozone/oxygen admixture flows through different contact devices in series, from the admixture exit port of one contact device to the admixture entrance port of the next.
  • the ozone/oxygen admixture may flow directly from the admixture source to the entrance port of each different contact device.
  • Another alternative is a combination of the foregoing examples where the ozone/oxygen admixture flows from the exit port of some devices to the entrance port of other devices and in addition, to the entrance of some devices directly from the admixture source.
  • the resultant fluid from the terminal device can either be collected or returned to the original device and recirculated.
  • interface time between the fluid and ozone/oxygen admixture is controllable, and can be adjusted based on the individual pitch chosen for each device in series, or by adding additional devices to the series.
  • the overall interface surface area will range from 0.01 m 2 for an individual device, and upwards based on the number of devices serially utilized.
  • Table 1 An example of data measured and calculated by the described ozone delivery system that utilizes a fluid target described herein is included in Table 1.
  • Newborn Calf Serum commercially obtained was utilized as the target fluid.
  • a variable pitch device with variable pitch platform as disclosed in U.S. Patent No. 7,736,494, was employed as the gas-fluid contacting device. The following initial conditions were utilized; 300 ppmv ozone inlet concentration, 145 ml initial fluid volume, 1000 ml per minute gaseous flow rate, 189 ml per minute fluid flow rate counter current to the ozone/oxygen admixture flow. Incremental reductions in fluid volume are due to sampling of fluid through the fluid access port. Table 1
  • Incremental reductions in fluid volume are due to sampling of fluid through the fluid access port.
  • Fluid-contacting surfaces including gas-fluid contacting devices constructed from ozone-inert material, may be treated with a human serum albumin (HSA) solution to prevent platelet adhesion, aggregation and other related platelet phenomena in the instances when a biological fluid to be treated contains platelets (i.e. whole blood, platelet concentrates).
  • HSA solutions ranging between 1 and 10% may be employed.
  • An HSA solution prepared in a biocompatible bacteriostatic buffer solution will be passaged throughout the gas-fluid contacting device. Subsequent to passage, the HSA solution will be drained from the device.
  • the gas-fluid contacting device and all surfaces that are in contact with the biological fluid during the method described are consequently primed for use with platelet-containing biological fluids.
  • the biological fluid is whole blood or a derivative thereof.
  • Derivatives include plasma, red blood cell preparations and platelet concentrates.
  • the method would be particularly suitable for treatment of red blood cell and platelet preparations on a single donor basis. That is, an individual blood donation, once fractionated into its respective blood derivative(s), would subsequently be treated to inactivate infectious prion proteins contained therein. The treated blood derivative would then be suitable for in vivo use.
  • the biological fluid is a unit of whole blood containing infectious prion proteins.
  • the whole blood may be fractionated, after being inactivated using the methods of the present invention, into both cellular (red blood cells and platelets) and non-cellular components (plasma) for intended in vivo use.
  • plasma derivatives may be the biological fluid to be inactivated as an aggregate product, to be later separated for individual use in vivo.
  • large pooled volumes of plasma or plasma-derived Factor concentrates would be subjected to inactivation by the method described by incorporating a gas-fluid contacting device compatible with these volumes.
  • These applications may be performed by the plasma fractionating industry wherein inactivation of infectious prion proteins may become an integral step in the production of plasma fractionates for in vivo use.
  • this method of infectious prion protein inactivation may be required as a mandatory process in the production of animal-derived sera.
  • fetal calf serum is added to another infectious prion protein-free biological fluid and the mixture is subjected to an ozone/oxygen admixture through an ozone delivery device having all ozone- contacting pathways constructed of ozone-inert material.
  • Mammalian serum derivatives are a common constituent in culture media and other biological fluids used in the culture of cell-lines, viruses, bacteria and other microorganisms. A potential occurrence during the culture of these microorganisms is the simultaneous
  • an infectious prion protein contaminant in the culture system that renders the culture system infectious for future use. It is recognized that the source of this contamination is derived from the addition of the biological fluid component into the culture system. Treatment allows the inactivation of infectious prion proteins within the biological fluid constituent while maintaining the biological integrity of the biological fluid constituent. The resulting addition of the infectious prion protein-inactivated biological constituent will, therefore, not cause contamination in the culture system.
  • This example is also applicable to the addition of a biological fluid to a synthetic media wherein the only biological fluid in the system is being treated by the methods of the present invention prior to addition to the synthetic media.
  • Another example of this application is the addition of a biological fluid to a synthetic media in the
  • ozone delivering ozone to a 10% bovine serum fluid in phosphate-buffered saline (PBS) at a flow rate of 0.25 liters per minute, consisting of a 1000 ppmv inlet ozone/oxygen admixture concentration at a 2.0 liter per minute admixture flow rate, and for a duration of 30 minutes, results in an absorbed-dose of ozone of approximately 500 pg/ml of fluid. This yields approximately 12 ⁇ MDA/ml of serum.
  • PBS phosphate-buffered saline
  • the lipid peroxides generated by this method form a dose-response relationship with the absorbed-dose of ozone absorbed by the biological fluid. More specifically, there is a direct correlation between absorbed- ozone and the lipid peroxides generated. Table 4 below depicts the direct relationship between ozone absorbed by a 10% bovine serum in PBS and the degree of lipid peroxide generation as represented by MDA concentration.
  • the resulting quantifiable lipid peroxides may be considered pharmacologically- active and possess intrinsic anti-prion activity.
  • a fluid additive such as an aqueous buffered carrier media
  • an ozone/oxygen admixture in accordance with the processes described herein to absorb ozone directly. Absorption of an absorbed-dose of ozone may cause the generation of byproducts of ozonation in the aqueous buffered carrier media.
  • These byproducts which may include lipid peroxides, may be used to inactivate infectious prion proteins in biological fluids by first generating the byproducts in the carrier media and subsequently adding this byproduct-containing medium to the biological fluid containing an infectious prion protein. Concurrently, the biological integrity of the biological fluid will be maintained.
  • a fluid additive in the form of 10% human serum albumin (HAS) in phosphate-buffered saline was contacted with an ozone/oxygen admixture in an ozone delivery system as described herein where the ozone concentration was 1000 ppmv, the gas flow rate was 2.0 liters/min, and the biofluid flow rate was 250 ml/hr.
  • the ozone contacting process was continued for 30 minutes, and was sufficient to result in an absorbed-dose of ozone of approximately 500 pg/ml of fluid, and yielded approximately 12 pmole MDA/ml of serum.
  • the treated HAS solution was then added to a cellular platelet or red blood cell concentrate [1/10 (v/v)].
  • the quantifiable lipid peroxides in the treated additive were measured as an MDA adduct with thiobarbituric acid and was shown to exhibit anti-prion activity while in solution with the cellular concentrate.
  • the treated HSA-cellular concentrate was stored under standard conditions for up to 72 hours. Samples of the cellular mixture were then assayed for infectious prions by standard techniques to confirm lack of prion infectivity.
  • the MDA- detected lipid peroxides were subsequently quenched though the addition of a biocompatible agent, namely Vitamin E in an amount sufficient to obtain a final concentration of 12 to 37 pmol/L.
  • the biological integrity of the biological fluid may be of no consequence.
  • Byproducts of ozonation in biological fluids e.g., reactive oxygen intermediates which may include lipid peroxides, may be generated by allowing these byproducts to continue to be generated for a period of time up to 72 hours after the completion of the absorption of ozone and prior to use of the biological fluid.
  • byproducts of ozonation in aqueous carrier media may be generated by allowing these byproducts to be generated for a period of up to 72 hours after the completion of the absorption of an absorbed-dose of ozone and prior to the
  • the products of ozonation reactive oxygen intermediates which may include lipid peroxides
  • a biocompatible agent may include all known antioxidants including Vitamin A, Vitamin E, other tocopherol-containing compounds, glutathione, ascorbic acid, curcumin and activated charcoal.
  • activated charcoal may be removed.
  • the use of activated charcoal may remove the products of ozonation, reactive oxygen intermediates including lipid peroxides. Subsequent to the contact of the activated charcoal with the ozone-absorbed biological fluid, the charcoal may be removed.
  • bovine serum which is a classical additive to promote growth and proliferation of cells in culture, is treated with an ozone/oxygen admixture to produce a serum free of prion contamination.
  • This process is typically applied to an acellular fluid like sera and is subjected to much more aggressive processing conditions (i.e. duration of up to 2 hrs) that are necessary to inactivate the infectious prions, as described in the spiked sera experiment of Example 6, below.
  • the data derived from this example is counter-intuitive to the skilled expert because theoretically there should not be any differentiation between a prion protein and the balance of other proteins in sera in terms of their propensity for oxidation.
  • test data demonstrate inactivation of the infectious prion form (PrPsc) by apparently changing its glycosylation pattern (removing the diglycosylated form that is considered the infectious isoform) while maintaining the sera's ability to maintain 85% of its cell growth and proliferation capabilities as a cell culture supplement.
  • Bovine sera (20% v/v) spiked with PrPSc inocula derived from scrapie-infected mouse brain homogenates (strain 139A) were contacted with an ozone/oxygen admixture using the ozone delivery device described herein where the ozone concentration was 1200 ppmv, the gas flow rate was 1 .0 liters/min and the biofluid flow rate was 200 ml/hr.
  • Contact of the bovine sera with the ozone/oxygen admixture was continued for 120 minutes.
  • Prion infectivity in the treated bovine sera was determined by using a N2a cell culture protocol to determine whether prions were acquired from brain homogenate and propagated by the exposed cells.
  • the methods include subjecting an amount of a fluid containing infectious prion proteins to an amount of ozone delivered by an ozone delivery system, especially of the type being constructed of ozone inert materials along all ozone-contacting pathways.
  • the methods utilize an ozone-delivery system for delivering and manufacturing a measured amount of an ozone/oxygen admixture that is able to measure, control and report and differentiate between delivered-ozone and the absorbed-dose of ozone. All gas contact surfaces of the system, including one or more gas-fluid contact devices are made from ozone-inert construction materials that do not absorb ozone or introduce contaminants or deleterious
  • the methods applied to biological fluids generates byproducts of ozonation, which may include lipid peroxides that are a result of the absorption of an absorbed-dose of ozone from the delivered-ozone by the fluid.
  • the resulting lipid peroxides are quantifiable by assay for lipid peroxides.
  • a direct relationship between the absorbed-dose of ozone absorbed by a biological fluid and the quantifiable lipid peroxides exists.
  • the lipid peroxides generated may be pharmacologically active and possess intrinsic anti-prion activity. Furthermore, these lipid peroxides can be quenched with a variety of antioxidants.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Endocrinology (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Apparatus For Disinfection Or Sterilisation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des procédés d'inactivation de protéines prions infectieuses dans un liquide biologique afin de produire un liquide biologique non infectieux, les procédés comprenant le traitement d'une quantité d'un liquide contenant des protéines prions infectieuses par un mélange ozone/oxygène distribué par un système de distribution d'ozone, grâce à quoi les liquides biologiques sont rendus exempts de prions infectieux. Les procédés décrivent également le traitement d'un additif liquide par de l'ozone afin de rendre l'additif liquide pharmaceutiquement actif pour une activité anti-prion.
PCT/US2011/001103 2010-06-21 2011-06-21 Inactivation de prion à l'aide d'ozone Ceased WO2011162805A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US39811810P 2010-06-21 2010-06-21
US61/398,118 2010-06-21

Publications (2)

Publication Number Publication Date
WO2011162805A2 true WO2011162805A2 (fr) 2011-12-29
WO2011162805A3 WO2011162805A3 (fr) 2012-04-12

Family

ID=45372000

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/001103 Ceased WO2011162805A2 (fr) 2010-06-21 2011-06-21 Inactivation de prion à l'aide d'ozone

Country Status (1)

Country Link
WO (1) WO2011162805A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11426505B2 (en) 2014-09-15 2022-08-30 Sangair Ab Apparatus and method for contacting blood with ozone

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0339924B1 (fr) * 1988-04-29 1995-03-29 Medizone International, Inc. Appareil pour la génération et l'administration contrôlée d'ozone
EP0653945A1 (fr) * 1992-07-31 1995-05-24 WAINWRIGHT, Basil, E. Appareil et procede d'inactivation du virus de l'immunodeficience humaine
WO2003066112A1 (fr) * 2002-02-06 2003-08-14 Curozone Ireland, Ltd. Procede et appareil de sterilisation par l'ozone destines a denaturer les prions

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11426505B2 (en) 2014-09-15 2022-08-30 Sangair Ab Apparatus and method for contacting blood with ozone

Also Published As

Publication number Publication date
WO2011162805A3 (fr) 2012-04-12

Similar Documents

Publication Publication Date Title
Schallmoser et al. Production and quality requirements of human platelet lysate: a position statement from the working party on cellular therapies of the international society of blood transfusion
Leiter et al. Platelets in neurodegenerative conditions—friend or foe?
Foster Prions and blood products
US20020131958A1 (en) Method for purifying a biological composition
Lacroux et al. Impact of leucocyte depletion and prion reduction filters on TSE blood borne transmission
Burnouf et al. Current strategies to prevent transmission of prions by human plasma derivatives
Reichl et al. Studies on the removal of a bovine spongiform encephalopathy‐derived agent by processes used in the manufacture of human immunoglobulin
Brown Creutzfeldt‐Jakob disease: reflections on the risk from blood product therapy
JP4523222B2 (ja) プリオンおよびウイルスの除去プロセス
US20050051497A1 (en) Viral inactivation using ozone
Neisser‐Svae et al. Prion removal effect of a specific affinity ligand introduced into the manufacturing process of the pharmaceutical quality solvent/detergent (S/D)‐treated plasma OctaplasLG®
WO2011162805A2 (fr) Inactivation de prion à l'aide d'ozone
Boczkowska-Radziwon et al. Ozonation of whole blood results in an increased release of microparticles from blood cells
Nomura et al. Performance evaluation of bactericidal effect and endotoxin inactivation by low‐temperature ozone/hydrogen peroxide mixed gas exposure
JP4524045B2 (ja) 蛋白質含有液体の処理
Morales et al. Reduction of prion infectivity in packed red blood cells
EP1385871B1 (fr) Processus destine a inactiver des prions dans des lipoproteines
Sy et al. Human prion diseases
Svae et al. Prion safety of transfusion plasma and plasma-derivatives typically used for prophylactic treatment
US7252720B2 (en) Removal of prion infectivity
Leunda et al. Laboratory activities involving transmissible spongiform encephalopathy causing agents: risk assessment and biosafety recommendations in Belgium
Galbraith Transmissible spongiform encephalopathies and tissue cell culture
Mohsen et al. Study the effect of chlorine gas in some blood criteria among the workers in the water purification plants in Babylon governorate
KR20090109546A (ko) 생물학적 유체로부터 전염성 해면상 뇌병증 병원체의 직교적 제거 방법
WO2010147677A2 (fr) Traitement de troubles inflammatoires, de maladies cardiovasculaires et d'attaque cérébrale ischémique aiguë par de l'ozone

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11798502

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11798502

Country of ref document: EP

Kind code of ref document: A2