Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
  • A61K31/525—Isoalloxazines, e.g. riboflavins, vitamin B2
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/14—Blood; Artificial blood
  • A61K35/19—Platelets; Megacaryocytes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/10—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person
  • A61K41/17—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person by ultraviolet [UV] or infrared [IR] light, X-rays or gamma rays
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
  • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
  • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
  • A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
  • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
  • A61L2/0082—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using chemical substances
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
  • A61M2202/00—Special media to be introduced, removed or treated
  • A61M2202/04—Liquids
  • A61M2202/0413—Blood

Definitions

• Red blood cells whole blood collected from volunteer donors for transfusion recipients is typically separated into its components, red blood cells, platelets, and plasma, by apheresis or other known methods. Each of these fractions are individually stored and used to treat a multiplicity of specific conditions and disease states.
• the red blood cell component is used to treat anemia
• the concentrated platelet component is used to control bleeding
• the plasma component is used frequently as a source of Clotting Factor VIII for the treatment of hemophilia.
• Blood screening procedures may miss contaminants, and sterilization procedures, which do not damage cellular blood components but effectively inactivate all or reduce infectious viruses and other microorganisms, are needed in the art.
• Bacteria can easily be introduced to blood components by at least two different means. First, if the donor is experiencing a mild bacteremia, a condition comprising bacteria in the blood, the blood will be contaminated, regardless of the collection or storage method. Adequate donor histories and physicals will decrease but not eliminate this problem. See B. J. Grossman et al., Transfusion 31 :500 (1991).
• a second, more pervasive source of contamination is the venepuncture employed when drawing blood. Even when "sterile" methods of skin preparation are employed, it is extremely difficult to sterilize the crypts around the sweat glands and hair follicles. During venepuncture, this contaminated skin is often cut out in a small “core” by a sharp needle. This core can serve to "seed" the blood bag with bacteria that may grow and become a risk to the recipient.
• platelets which have been treated with a photosensitizer and light to inactivate or reduce pathogens which may be present may show reactivation of pathogens during long-term storage after such a treatment.
• platelets may show high activation and low extended shape change response by day 5 of storage, both of which may be indications of cytoskeletal changes in the platelets. Such changes may be indications of platelet damage due to the storage conditions. It is therefore necessary to improve the quality of stored photoradiated platelets.
• glucose In cells, food is oxidized to produce high-energy electrons that are converted to stored energy. This energy is stored in high-energy phosphate bonds in ATP. Ingested sugars are broken down by enzymes that split them into a six-carbon molecule called glucose. Glucose may also be provided to cells in media or storage solutions. The breakdown of glucose to provide energy to cells is an important mechanism in cellular metabolism. This mechanism, known as glycolysis, produces ATP (adenosine triphosphate) in the presence or absence of oxygen. The production of ATP is essential for cellular energy metabolism. Glucose enters the cell by special molecules in the membrane called “glucose transporters.” Once inside the cell, glucose is broken down to make ATP in two pathways.
• the first pathway requires no oxygen and is called anaerobic metabolism.
• Anaerobic metabolism or glycolysis occurs in the cytoplasm outside the mitochondria.
• glucose is broken down into pyruvate, a three-carbon molecule.
• This conversion involves a sequence of nine enzymatic steps that create phosphate-containing intermediates.
• Each reaction is designed to produce hydrogen ions (electrons) that can be used to make energy in the form of ATP. Only two ATP molecules can be made by one molecule of glucose run through this pathway. This pathway is also used to produce two lactate molecules from every one glucose molecule.
• citric acid cycle is also known in the art as the Kreb's cycle or the tricarboxylic acid (TCA) cycle.
• TCA tricarboxylic acid
• the citric acid cycle occurs in the mitochondria and is the common pathway to completely oxidize fuel molecules, which are mostly acetyl CoA, the product from the oxidative decarboxylation of pyruvate. Acetyl CoA enters the cycle and passes through ten steps of reactions that yield energy (ATP) and C0 2 .
• glycolysis is a major source of the cell's ATP. This also occurs in an aerobic cell if the mitochondria of the cell are damaged in some way, thereby preventing the cell from entering the citric acid cycle.
• ATP is essential to continued cell function, when aerobic metabolism is slowed or prevented by lack of oxygen, anaerobic pathways for producing ATP are stimulated and become critical for maintaining cell viability.
• the pyruvate molecules instead of being degraded in the mitochondria, the pyruvate molecules stay in the cytosol and can be converted into ethanol and C0 2 (as in yeast) or into lactate (as in muscle).
• Lactate accumulation in cells causes an increased concentration of hydrogen ions and a decrease in pH.
• Blood cells in storage that experience a decrease in pH may be only undergoing glycolysis. Such a drop in pH indicates as well as contributes to a decrease in cell quality during cell storage.
• Factors which cause cells to enter glycolysis and thereby accumulate lactic acid or lactate include events which occur internally in a body such as strokes or infarctions, as well as external events such as treatment of the cells after removal from a body.
• An external treatment which might cause cells to accumulate lactate is a procedure to inactivate or reduce pathogens which might be contained in cells or fluids containing cells to be transfused into a recipient.
• pathogens which might be contained in cells or fluids containing cells to be transfused into a recipient.
• Currently used methods to sterilize pathogenic contaminants which may be present in blood or blood components can cause damage to the mitochondria of the cells being treated. If this occurs, the cells can only make ATP through the glycolysis pathway, causing a buildup of lactic acid in the cell and a subsequent drop in pH during storage.
• Mitochondria are critical subcellular organelles of blood components. They are involved in aerobic energy metabolism and the oxidative reactions therein. Mitochondria are sensitive to endogenous and exogenous influences and may be easily damaged or destroyed. Dysfunctional energy metabolism and, more severely, damaged mitochondria, lead to a decline in platelet quality and eventual cell death.
• a side effect of a pathogen reduction process is that when platelets are subjected to UV light, the mitochondria of the platelets have a greater chance of suffering at least some damage than when they have been subjected to visible light.
• Mitochondria are present in all oxygen-utilizing organisms in which energy in the form of adenosine triphosphate (ATP) is generated and oxygen is reduced to water.
• ATP adenosine triphosphate
• ATP adenosine triphosphate
• a substantial byproduct of ATP generation is the formation of potentially toxic oxygen radicals. For example, it is estimated that 1-2% of all reduced oxygen yields superoxide (0 2 -) and hydrogen peroxide (H 2 0 2 ).
• ROS reactive oxygen species
• Singlet oxygen (0 2 )
• OH hydroxyl radicals
• This invention provides a method for treating a fluid comprising a cellular blood component to improve a vital quality of said cellular blood component, said method comprising adding an effective, substantially non-toxic amount of a mitochondrial enhancer to said fluid wherein said mitochondrial enhancer is selected from the group consisting of alloxazines, endogenous alloxazines, non-endogenous alloxazines, endogenously-based derivative alloxazines, endogenous photosensitizers, and non-endogenous photosensitizers.
• the concentration of mitochondrial enhancer in the fluid can be from any amount sufficient to provide measurable enhancement of a vital quality of a cell in the fluid up to a toxic amount.
• the mitochondrial enhancer is present at a final concentration from about one to about 200 micromolar.
• This invention also provides methods for increasing the storage life of cellular blood components, extending platelet storage life, treating a cell comprising a mitochondrion, treating a fluid comprising cells containing mitochondria to improve a quality of said fluid, said methods comprising adding an effective, substantially non- toxic amount of a mitochondrial enhancer to said fluid wherein said mitochondrial enhancer is selected from the group consisting of alloxazines, endogenous alloxazines, non-endogenous alloxazines, endogenously-based derivative alloxazines, endogenous photosensitizers, and non-endogenous photosensitizers.
• Fluids treatable by the methods of this invention include fluids containing living cells with mitochondria or fluids that come into contact with living cells such as peritoneal solutions, blood, and fluids comprising a blood product.
• Cells treatable by the methods of this invention include plant cells, animal cells, yeast cells, cellular blood components, platelets, and cells in a wound surface.
• the fluid is optionally exposed to photoradiation greater than ambient light.
• the photoradiation may be of sufficient energy to activate a photosensitizer in the fluid.
• the wavelength of the light can be in the visible or ultraviolet spectrum.
• the fluid can be performed at a time selected from the group consisting of before, after, and simultaneously with treating the fluid with mitochondrial enhancer.
• the fluid can be of energy between about 5 J/cm 2 and about 150 J/cm 2 .
• the photoradiation can also be of sufficient energy to substantially reduce pathogens which may be present in the fluid.
• Pathogens which can be reduced by the methods of this invention include extracellular and intracellular viruses, bacteria, bacteriophages, fungi, blood-transmitted parasites, and protozoa, and mixtures of any two or more of the foregoing.
• the photosensitizer can be the same as the mitochondrial enhancer that is added to the fluid.
• the concentration of photosensitizer can be any amount sufficient to provide a measurable reduction of pathogens in the fluid up to an amount which would be toxic to the cells.
• the photosensitizer is present at a concentration from about 1 to about 200 micromolar.
• the cellular blood component is not stored prior to said treating.
• the cellular blood component is stored prior to said treating. When the cellular blood component is stored prior to treating, it is stored for an amount of time between about 1 hour and about 7 days prior to said treating. Alternatively the cellular blood component is stored for more than about one hour prior to said treating.
• Mitochondrial enhancers useful in the practice of this invention include 7,8-dimethyl-lO-ribityl isoalloxazine, 7,8-dimethylalloxazine, 7,8,10-trimethylisoalloxazine, alloxazine mononucleotide, isoalloxazine-adenosine dinucleotide, vitamin Kl, vitamin Kl oxide, vitamin K2, vitamin K5, vitamin K6, vitamin K7, vitamin K-S(II), and vitamin L.
• the mitochondrial enhancer is 7,8-dimethyl-10-ribityl isoalloxazine it is in the fluid at a concentration of about one to about 200 micromolar.
• Additional mitochondrial enhancers useful in the practice of this invention include molecules of the formula:
• Rl, R2, R3, R4, R5 and R6 can be, independently from one another, selected from the group consisting of hydrogen, optionally substituted alcohol, straight chain or cyclic saccharide, amino acid, amine, polyamine, polyether, polyalcohol, sulfate, phosphate, carbonyl, glycol, halogen selected from the group consisting of chlorine, bromine and iodine, aldehyde, ketone, carboxylic acid and ascorbate. These compounds may also act as photosensitizers.
• a cellular blood component treatable by the methods of this invention is platelets.
• the cellular blood component is stored for more than about one hour after said mitochondrial enhancer is added.
• the methods of this invention can also include adding nitric oxide to the fluid, adding quencher to the fluid, adding process enhancer to the fluid, adding oxygen to the fluid, and/or adding glycolysis inhibitor to the fluid.
• Vital qualities that are improved by the methods of this invention include oxygen consumption, rate of oxygen consumption, lactate production, rate of lactate production, pH, rate of pH change, activation, hypotonic shock response, glucose consumption, rate of glucose consumption, platelet swirl, platelet aggregation, carbon dioxide production, rate of carbon dioxide production, cell count, and extent of shape change.
• the oxygen consumption is increased by at least about 5%.
• the rate of lactate production is decreased by at least about 25%.
• the pH is increased by at least about 0.1 units.
• the hypotonic shock response is increased by at least about 5%.
• the glucose consumption is decreased by at least about 10%.
• the platelet swirl is increased by at least about 5%.
• the platelet aggregation is decreased by at least about 5%.
• the carbon dioxide production is increased by at least about 5%.
• the cell count is increased by at least about 5%.
• the extent of shape change is increased by at least about 5%.
• the activation is decreased by at least about 5%.
• Figure 1 is a graph showing the effect of mitochondrial enhancer on platelet swirl (0-4 units) of cellular blood components as a function of storage time (days).
• Figure 2 is a graph showing the effect of mitochondrial enhancer on hypotonic shock response (HSR), % reversal, of platelets as a function of storage time (days).
• HSR hypotonic shock response
• Figure 3 is a graph showing the effect of mitochondrial enhancer on pH of the stored fluid as a function of storage time (days).
• Figure 4 is a graph showing the effect of mitochondrial enhancer on % extent of shape change (ESC) of platelets as a function of storage time (days).
• Figure 5 is a graph showing the effect of mitochondrial enhancer on lactate production by platelets as a function of storage time (days).
• Figure 6 is a graph showing the effect of mitochondrial enhancer on the rate of lactate production by platelets as a function of mitochondrial enhancer concentration.
• Figure 7 is a graph showing the effect of mitochondrial enhancer on lactate production by platelets as a function of storage time (days).
• Figure 8 is a graph showing the effect of mitochondrial enhancer on glucose consumption by platelets as a function of storage time (days).
• Figure 9 is a graph showing the effect of mitochondrial enhancer on p-selectin expression (% activation), by platelets as a function of storage time (days).
• Figure 10 is a graph showing the effect of mitochondrial enhancer on oxygen consumption by platelets as a function of storage time (days).
• Figure 11 is a graph showing the effect of mitochondrial enhancer on reduction kinetics of vaccinia virus as a function of photoradiation exposure time (delivered energy).
• Figure 12 is a graph showing the effect of various concentrations of mitochondrial enhancer on reduction of Herpes Virus 2 (HSV-2) as a function of photoradiation exposure time (delivered energy).
• Figure 13 is a graph showing the effect of various energy doses on reduction of S. epidermidis as a function of concentration of mitochondrial enhancer (micromoles).
• Figure 14 is a graph showing the effect of various concentrations of mitochondrial enhancer on reduction of ⁇ X174 as a function of delivered photoradiation energy.
• mitochondria enhancer refers to a composition which enhances a vital quality of mitochondria or of cells containing mitochondria.
• Mitochondrial enhancers useful in the practice of this invention include, but are not limited to alloxazines, endogenous alloxazines, non-endogenous alloxazines, endogenously based derivative alloxazines, endogenous photosensitizers, and non- endogenous photosensitizers.
• ambient light refers to natural light such as sunlight, including sunlight through glass or plastic, or overhead room light, such as from incandescent, fluorescent, and/or halogen bulbs. Ambient light is generally not of enough energy and/or of the appropriate wavelengths to sufficiently activate a photosensitizer in a solution to substantially reduce pathogens therein.
• substantially inactivate pathogens refers to reducing the ability of pathogens to reproduce, preferably by killing them.
• the treated fluid comprises a cellular blood component
• the level of pathogens in the fluid can be decreased such that the cellular blood component may be safely administered to a patient.
• substantially reduce pathogens refers to reducing the ability of pathogens to reproduce, preferably by killing them.
• the treated fluid comprises a cellular blood component
• the level of pathogens in the fluid can be decreased such that the cellular blood component may be safely administered to a patient.
• storage refers to the amount of time after formulation or collection before a fluid is utilized for its intended purpose.
• storage of blood or blood product refers to time between the collection of the blood or blood product and the utilization of the blood or blood product for its intended purpose, such as the administration of that blood product to a patient.
• an amount of mitochondrial enhancer sufficient to improve storage life refers to an amount that measurably increases a vital cell quality.
• glycolysis inhibitor refers to compositions that interfere with the biochemical pathway of glycolysis. 2-deoxy-D-glucose is an example of a glycolysis inhibitor.
• nitric oxide refers to increasing the amount of nitric oxide within a fluid.
• nitric oxide may be added to a fluid by any method known in the art. Methods for adding nitric oxide to a fluid include, but are not limited to, adding liquids, solids, or gases containing nitric oxide and adding nitric oxide generators.
• Nitric oxide generators are chemicals that are able to react, directly or indirectly, to produce nitric oxide. Nitric oxide generators may react with components already in a fluid to produce nitric oxide, or they may require the addition of one or more different nitric oxide generators to the fluid, with which they may react to produce nitric oxide.
• Nitric oxide generators that do not require the addition of one or more different nitric oxide generators are nitric oxide donors.
• Nitric oxide donors are well known in the art (Bauer et al., (1995) Advances in Pharmacology 34:361 and U.S. Patent No. 6,232,434) and are available for purchase from companies such as Cayman Chemical, Ann Arbor, MI.
• Nitric oxide donors include, but are not limited to L-arginine, N-acetyl-L-cysteine, DEA-NO, DETA-NO, DETA-NONOate, PAPA-NO, sodium nitroprusside, and nitroglycerine.
• Liquids containing nitric oxide include, but are not limited to liquids comprising two nitric oxide generators combined in a fluid to produce nitric oxide, saline in which nitric oxide gas has been bubbled, and nitric oxide-saturated water.
• adding oxygen refers to adding oxygen to a fluid to increase the dissolved oxygen in the fluid to an amount greater than would be present in the fluid when the fluid is under an air atmosphere at ambient conditions without mixing.
• process enhancer refers to a composition that enhances a pathogen reduction process.
• Process enhancers can be included in photoradiation processes of this invention.
• Such enhancers include antioxidants or other agents to prevent damage to desired fluid components or to improve the rate of reduction of microorganisms and are exemplified by adenine, histidine, cysteine, tyrosine, tryptophan, ascorbate, N-acetyl-L-cysteine, propyl gallate, glutathione, mercaptopropionylglycine, dithiothreotol, nicotinamide, BHT, BHA, lysine, serine, methionine, glucose, mannitol, trolox, glycerol, vitamin E, alpha tocopherol acetate, and mixtures thereof.
• agitator refers to an apparatus which can agitate, e.g. shake or rotate, the container containing the product to be irradiated, such as the Helmer platelet incubator/agitator (Helmer Company, Noblesville, IN).
• 100% plasma carryover and “100% PCO” refer to plasma to which about 20% by volume of anticoagulant has been added. 100% PCO is therefore about 80% plasma. By a similar calculation, 90% PCO is about 72% plasma.
• the balance of the platelet composition which is neither plasma nor anticoagulant can contain additional ingredients such as mitochondrial enhancer and/or photosensitizer.
• Anticoagulants known to the art are useful in the practice of this invention, including ACD-A (anticoagulant citrate dextrose formula A) and CPD (citrate phosphate dextrose).
• vitamin quality refers to an indicator of cellular blood component quality, i.e., a parameter of a fluid containing cells or a cellular blood component that can be measured to assess its quality.
• Indicators of cellular blood component quality are described below and include but are not limited to activation, hypotonic shock response, amount and rate of lactate production, amount and rate of glucose consumption, pH and rate of pH change, platelet swirl, platelet aggregation, amount and rate of oxygen consumption, amount and rate of carbon dioxide production, cell count (cell survival), and extent of shape change (ESC).
• to "improve a vital quality of a cellular blood component” refers to improving a parameter of a cellular blood component that can be measured to assess quality, including the previously mentioned parameters.
• An improved cellular blood component provides better results when utilized, for example, to treat patients. Measurement of these vital qualities provides information on the status of mitochondria health. Additional qualities which provide information on the status of mitochondria health include activation, hypotonic shock response, glucose consumption, platelet swirl, platelet aggregation, carbon dioxide production, cell count, and ESC.
• amount of mitochondrial enhancer effective to improve a vital quality refers to enough mitochondrial enhancer to cause a measurable improvement in a vital cell quality, but not so much as to be toxic to cells containing mitochondria.
• the mitochondrial enhancer of this invention is added in an amount sufficient to cause a measurable improvement in a vital quality.
• the mitochondrial enhancer riboflavin is added to blood cells, between about 0.1 micromolar and about 1 millimolar, between about 1 micromolar and about 200 micromolar, and between about 5 micromolar and about 75 micromolar final concentrations are effective.
• biologically active means capable of effecting a change in a living organism or component thereof.
• blood product and "blood component” as used herein include blood, plasma, blood constituents, and therapeutic protein compositions containing proteins derived from blood.
• cellular blood component refers to blood components that contain a substantial amount of or are cellular components of blood.
• Cellular blood components include platelets, erythrocytes (red blood cells), eosinophils, neutrophils, leukocytes (white blood cells), monocytes, lymphocytes, basophils, and blood stem cells. If a sample of plasma contains a substantial amount a cellular blood component, i.e. enough so that the cells therein are useful, such as platelets or white blood cells, it is a cellular blood component.
• a “blood component comprising platelets” includes platelets in plasma and platelets in media.
• cells in a wound surface refers to cells at or near the surface of a wound.
• Wounds towards the surface of a mammalian body can include white blood cells, red blood cells, fibroblasts, epidermal cells, and endothelial cells.
• Cells in a wound surface can include cells of ectodermal, mesodermal, and endodermal origin.
• Substantially non-toxic amounts of elements of this invention are those which do not destroy the biological activity of such fluid components other than microorganisms.
• pathogen refers to an individual pathogenic organism of one species, a plurality of such organisms of one species, or a plurality of pathogenic organisms of two or more species.
• inactivation with respect to the effects of a procedure described herein refers to reduction of a greater quantity of pathogens after using the procedure than in the absence of the procedure.
• increase pathogen reduction with respect to the effects of a procedure described herein refers to reduction of a greater quantity of pathogens after using the procedure than in the absence of the procedure.
• the pathogens which may be present in fluid and decontaminated by the processes of this invention typically include those selected from the group consisting of extracellular and intracellular viruses, bacteria, bacteriophages, fungi, blood- transmitted parasites, and protozoa, and mixtures of any two or more of the foregoing.
• one of the pathogens is a virus, it may be selected from the group consisting of human immunodeficiency virus (HIV), hepatitis A, B and C viruses, Sindbis virus, cytomegalovirus, vesicular stomatitis virus, herpes simplex viruses, e.g.
• pathogens are a bacteriophage, it may be selected from the group consisting of ⁇ X174, ⁇ 6, ⁇ , R17, T4, and T2, and mixtures of any two or more of the foregoing. If one of the pathogens is a bacterium, it may be selected from the group consisting of P. aeruginosa, S. aureus, S. epidermidis, E.
• coli K. pneumoniae, E. faecalis, B. subtilis, S. pneumoniae, S. pyrogenes, S. viridans, B. cereus, E. aerogenes, propionabacter, C. perfringes, E. cloacae, P. mirabilis, S. cholerasuis, S. liquifaciens, S. mitis, Y. enter colitica, P. fluorescens, S. enteritidis, C. freundii, and S. marcescens, and mixtures of any two or more of the foregoing.
• one of the pathogens is a protozoon, it may be P. falciparum.
• photosensitizer refers to any compound which absorbs radiation of one or more defined wavelengths and subsequently utilizes the absorbed energy to carry out a chemical process.
• the photosensitizers useful in this invention include any photosensitizers known to the art to be useful for reducing microorganisms. Examples of such photosensitizers include porphyrins, psoralens, dyes such as neutral red, methylene blue, acridine, toluidines, flavine (acriflavine hydrochloride) and phenothiazine derivatives, coumarins, quinolones, quinones, and anthroquinones.
• Photosensitizers of this invention may include compounds which preferentially adsorb to nucleic acids, thus focusing their photodynamic effect upon microorganisms and viruses with little or no effect upon accompanying cells or proteins. Other photosensitizers of this invention are also useful, such as those using singlet oxygen-dependent mechanisms. Photosensitizers useful in the practice of this invention include endogenous photosensitizers.
• activate a photosensitizer refers to altering a photosensitizer to make it capable of substantially reducing pathogens.
• An activated photosensitizer is capable of reducing microorganisms in a fluid, such as by interfering to prevent their replication. Specificity of action of the photosensitizer can be conferred by the close proximity of the photosensitizer to nucleic acid of the microorganism and this may result from binding of the photosensitizer to the nucleic acid.
• Nucleic acid includes ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Photosensitizers can also act by binding to cell membranes or by other mechanisms.
• endogenous means naturally found in a human or mammalian body, either as a result of synthesis by the body or because of ingestion as an essential foodstuff (e.g. vitamins) or formation of metabolites and/or byproducts in vivo.
• non-endogenous means not naturally found in a human or mammalian body, either as a result of synthesis by the body or because of ingestion of an essential foodstuff or formation of metabolites and/or byproducts in vivo.
• Examples of endogenous photosensitizers include alloxazines.
• Alloxazines are molecules comprising an alloxazine backbone.
• the term "alloxazine” includes isoalloxazines.
• Examples of endogenous photosensitizers include 7,8-dimethyl-10- ribityl isoalloxazine (riboflavin), 7,8,10-trimethylisoalloxazine (lumiflavin), 7,8- dimethylalloxazine (lumichrome), isoalloxazine-adenine dinucleotide (flavine adenine dinucleotide [FAD]), alloxazine mononucleotide (also known as flavine mononucleotide [FMN] and riboflavine-5-phosphate), vitamin Ks, vitamin L, their metabolites and precursors, and napththoquinones, naphthalenes, naphthols and their derivatives having planar mo
• Endogenously based derivative photosensitizers include synthetically derived analogs and homologs of endogenous photosensitizers which may have or lack lower (1-5) alkyl or halogen substituents of the photosensitizers from which they are derived, and which preserve the function and substantial non-toxicity thereof.
• endogenous photosensitizers or endogenously based derivative photosensitizers are used in the practice of this invention, particularly when such photosensitizers are not inherently toxic or do not yield toxic photoproducts after photoradiation, no removal or purification step is required after decontamination, and treated product can be directly returned to a patient's body or administered to a patient in need of its therapeutic effect.
• Non-endogenous photosensitizers that are mitochondrial enhancers and are based on endogenous structures, such as those described in U.S. Patent Application 09/420,652 are useful in the practice of this invention.
• These non-endogenous mitochondrial enhancers and endogenously based derivative mitochondrial enhancers are referred to herein as endogenously based derivative mitochondrial enhancers.
• The include molecules of the formula:
• R4 and R5 when Rl, R4 and R5 are CH 3 and R3 and R6 are H; R5 is not chloro when R4 is methoxy and Rl is ethyl-2'N-pyrrolidino and R2, R3, and R6 are hydrogen; Rl is not N,N-dimethylaminopropyl or N,N-diethylaminoethyl when R5 is chloro or methyl and R2, R3, R4 and R6 are hydrogen; R3 is not -NH(CH 2 CH 2 )C1 when R6 is -NH 2 and Rl, R2, R4 and R5 are H; Rl, R4, R5 are not all methyl groups when all of R2, R3 and R6 are hydrogens; Rl , R4, R5 and R2 are not all methyl groups when R3 and R6 are hydrogens; R2 is not carboxymethyl when Rl, R4 and R5 are methyl and R3 and R6 are hydrogen; R4 is not -NH 2 when Rl and
• R1-R6 are substituted with various substituents, as described elsewhere, except those previously known to the art.
• the substituents included in the compounds and used in the methods of the invention may be any substituent not having structures or reactivity which would substantially interfere with the desired microorganism neutralization of the microorganism neutralizer, as may readily be determined without undue experimentation by those skilled in the art.
• the foregoing isoalloxazine-related photosensitizers also function as enhancers of mitochondrial function.
• This invention provides methods for treating cell-containing fluids to improve a vital quality of the fluid by enhancing the vital quality and/or preventing damage to mitochondria within the cells.
• Compositions useful for treating fluids to enhance mitochondrial function and/or prevent damage to mitochondria comprise mitochondrial enhancers.
• Mitochondrial enhancers provided by this invention include alloxazines such as endogenous alloxazines, non-endogenous alloxazines, and endogenously based derivative alloxazines, and photosensitizers such as endogenous photosensitizers, non-endogenous photosensitizers, and endogenously based derivative photosensitizers.
• Molecules with alloxazine backbones are alloxazines.
• the methods provided by this invention comprise adding a substantially non-toxic amount of mitochondrial enhancer to a fluid, whereby the function of a mitochondrion within a cell in the fluid is enhanced. Mitochondria are enhanced when they are prevented from being damaged, when they are rejuvenated, and/or when a vital cell quality of a cell containing mitochondria is improved.
• a method step is performed on the fluid which damages mitochondria within cells in the fluid, such as storing or photoradiating to reduce pathogens which may be present within the fluid
• the mitochondrial enhancer prevents damage to or rejuvenates mitochondria within the cells.
• Mitochondrial enhancer may be added before, during, or after the damaging method step.
• the methods of this invention include the use of mitochondrial enhancer to prevent damage to and/or rejuvenate non-lymphocytic blood components before, during, and after a lymphocytic population reduction process.
• Any fluid comprising a cell containing a mitochondrion is treatable by the methods of this invention.
• Mitochondrial enhancer is used in an amount which is effective at preventing damage to and/or rejuvenating mitochondria and which is also non-toxic to the mitochondria, to the cells containing the mitochondria, and to the recipient of the treated fluid.
• Fluids treatable by the methods of this invention include blood, fluids comprising a cellular blood product or a cellular blood component, and bodily fluids.
• Cellular blood components treatable by the methods of this invention include platelets, blood, and plasma containing platelets or other cellular blood components.
• the mitochondrial enhancer When the fluid to be treated by the methods of this invention is to be consumed by a human or an animal, the mitochondrial enhancer must also be non- toxic to the human or animal, or be removed from the fluid before consumption.
• This invention also provides methods for using mitochondrial enhancer to treat cells having mitochondria.
• Cells treatable by the methods of this invention include cells known in the art to contain mitochondria, including plant cells, animal cells, and yeast cells.
• This invention also provides methods for using mitochondrial enhancer to treat a wound surface.
• Cells at or near a wound surface can include white blood cells, red blood cells, fibroblasts, epidermal cells, and endothelial cells.
• Cells in a wound surface can include cells of ectodermal, mesodermal, and endodermal origin. Treating mitochondria-containing cells in a wound surface with mitochondrial enhancer improves the health of the cells, prevents infection, and speeds healing.
• peritoneal fluid is the fluid within the peritoneal space that houses the gastrointestinal organs of the mammalian body.
• Peritoneal fluid can contain cells and can be treated by the methods of this invention.
• peritoneal fluid can be removed from a body, mitochondrial enhancer added, and the treated fluid administered back to a body.
• mitochondrial enhancer can be directly administered to peritoneal fluid inside a body, without removing peritoneal fluid.
• the mitochondrial enhancer in the peritoneal fluid inside the body enhances the mitochondrial function of cells within the fluid and/or lining the peritoneal space. Adding mitochondrial enhancer to saliva enhances the mitochondrial function of cells lining the digestive tract.
• the processes of collection and transfusion, the passage of time between collection and transfusion, and processes performed on blood and blood components between collection and transfusion may decrease the quality of the blood or the blood components.
• Change in the quality of blood or blood component are detectable as a change in a vital quality of the blood or blood component and can be assayed by any method known to the art.
• Indicators of cellular blood component quality include but are not limited to activation, hypotonic shock response, amount and rate of lactate production, amount and rate of glucose consumption, pH and rate of pH change, platelet swirl, platelet aggregation, amount and rate of oxygen consumption, amount and rate of carbon dioxide production, cell count, and extent of shape change (ESC).
• the processes of collection and transfusion, the passage of time between collection and transfusion, and processes performed on blood and blood components between collection and transfusion may decrease the quality of blood or cellular blood components by damaging mitochondria or mitochondrial processes that occur within the blood or cellular blood components.
• mitochondria or mitochondrial processes are damaged, the energy-producing metabolism of a cell switches to favor anaerobic glycolysis over oxidative phosphorylation in combination with the citric acid cycle and electron transport chain process, which occurs within the mitochondria, optionally in combination with glycolysis in the cytoplasm.
• lactate production is increased (lactate is a product of glycolysis), and the resulting increase in lactic acid causes a decrease in the pH of the fluid.
• indicators of cell quality that are also indicators of mitochondrial health include amount and rate of oxygen consumption, amount and rate of lactate production, pH and rate of pH change.
• Other indicators of cell quality including, but not limited to, activation, hypotonic shock response, amount, and rate of glucose consumption, platelet swirl, platelet aggregation, amount and rate of carbon dioxide production, cell count, and ESC, also provide information on mitochondrial health.
• Processes that can decrease the quality of blood or cellular blood components include, but are not limited to, the passage of time between collection and transfusion and photoradiation to reduce pathogens.
• the passage of time between collection and transfusion, or other use for collected blood or blood product may be as short as a few minutes to a few hours, to days, or as long as several weeks.
• whole blood components collected in an "open" (i.e., non-sterile) system must, under governmental rules, be transfused within twenty-four hours and in most cases within six to eight hours.
• red blood cells When whole blood components are collected in a "closed" (i.e., sterile) system, the red blood cells can be stored up to forty-two days (depending upon the type of anticoagulant and storage medium used), and plasma may be frozen and stored for even longer periods.
• platelet concentrate may be stored at room temperature for up to no more than five days.
• This invention provides a method for treating a fluid comprising a cellular blood component to improve a vital quality of said blood component, said method comprising adding a substantially non-toxic amount of a mitochondrial enhancer to said fluid wherein said mitochondrial enhancer is selected from the group consisting of endogenous alloxazines, non-endogenous alloxazines, endogenously-based derivative alloxazines, photosensitizers, endogenous photosensitizers, non- endogenous photosensitizers, and endogenously-based derivative photosensitizers.
• the fluid is not exposed, before, after, or during addition of a mitochondrial enhancer, to photoradiation greater than ambient light.
• Adding mitochondrial enhancer to a fluid comprising blood or a cellular blood component improves the quality of the blood or cellular blood component thereby increasing the allowed storage life of the blood or cellular blood component, allowing the blood or cellular blood component to be stored for a longer amount of time before it is administered to a patient.
• the FDA provides Guidance for Industry: An Acceptable Circular of Information for the Use of Human Blood and Blood Components (http://www.fda.gov/cber/gdlns/circbld.pdf) and allowed storage times of blood and blood components are known in the art.
• the vital cell qualities of platelets treated with mitochondrial enhancer are improved such that the treated platelets can be administered to a patient after seven days of storage.
• Mitochondrial enhancer may be added to a fluid before, after, and/or during storage. In the practice of this invention, if a fluid is utilized for its intended purpose immediately after creation or acquisition, it is considered to have not been stored. If time passes between creation or acquisition, including processing time, this is storage time. In one embodiment of this invention, mitochondrial enhancer is added at the beginning of storage. The beginning of storage is about the first 10% of total storage time. In another embodiment mitochondrial enhancer is added during the middle of storage. The middle of storage is about the central 80% of total storage time. In another embodiment, mitochondrial enhancer is added towards the end of storage. The end of storage is about the last 10% of total storage time.
• a fluid comprising blood or a cellular blood component has been stored for an amount of time between about one minute and about forty-five days before adding mitochondrial enhancer. In an embodiment of this invention, a fluid comprising blood or a cellular blood component has been stored for an amount of time between about one hour and about seven days before adding mitochondrial enhancer. In an embodiment of this invention, the fluid comprises platelets that have been stored for six days before mitochondrial enhancer is added to the fluid. In an embodiment of this invention, a fluid comprising blood or a cellular blood component is stored for an amount of time between about one hour and about five days after adding mitochondrial enhancer.
• a fluid comprising blood or a cellular blood component is stored for an amount of time between about one minute and about forty-five days before adding mitochondrial enhancer, and the fluid to which mitochondrial enhancer had been added is subsequently stored for an amount of time between about one minute and about forty-five days.
• a fluid comprising platelets is stored for an amount of time up to about three years after adding mitochondrial enhancer.
• a fluid comprising a cellular blood component is stored for an amount of time up to about three years before adding mitochondrial enhancer.
• the method also comprises exposing the fluid to photoradiation of energy greater than ambient light.
• the method also comprises performing a pathogen reduction process on the fluid.
• the pathogen reduction process comprises exposing the fluid to photoradiation of sufficient energy to substantially reduce pathogens which may be present in the fluid.
• a pathogen reduction process is performed before adding mitochondrial enhancer to the fluid.
• mitochondrial enhancer is added during a pathogen reduction process.
• a pathogen reduction process is performed after adding mitochondrial enhancer.
• mitochondrial enhancer is added during a pathogen reduction process, resulting in a pathogen reduction process being performed before, during, and after adding mitochondrial enhancer.
• pathogen reduction processes utilizing photoradiation also typically utilize a photosensitizer.
• the method also comprises adding a photosensitizer to the fluid.
• a mitochondrial enhancer may also be a photosensitizer and vice versa.
• the photosensitizer is the same as the mitochondrial enhancer utilized.
• the step of adding photosensitizer to the fluid is performed by adding mitochondrial enhancer to the fluid.
• Pathogens that are substantially reduced by the methods of this invention include extracellular and intracellular viruses, bacteria, bacteriophages, fungi, blood- transmitted parasites, protozoa, and mixtures of any two or more of the foregoing.
• an amount of photoradiation is chosen that substantially reduces pathogens without destroying desired biological activities within the fluid.
• an amount of photoradiation is chosen that minimally damages or decreases desired biological activities within the fluid.
• visible photoradiation is of about 419nM.
• ultraviolet radiation is of about 320nM.
• mitochondrial enhancer protects or rejuvenates fluid components from damage caused by photoradiation steps of a pathogen reduction process performed on the fluid, enabling the use of more photoradiation energy which in turn enables better pathogen reduction.
• mitochondrial enhancer is added to a fluid comprising platelets before a pathogen
• the pathogen reduction process utilizes more than about 5 J/cm , more than about 30
• mitochondrial enhancer is added more than once.
• the mitochondrial enhancer is riboflavin, also known as 7,8-dimethyl-lO-ribityl isoalloxazine.
• riboflavin is added to a final concentration in the fluid of about 1 micromolar to about 200 micromolar. In one embodiment about ten micromolar is added. In another embodiment about 50 micromolar is added.
• Riboflavin can be a photosensitizer and a mitochondrial enhancer. In one embodiment, riboflavin is added in an amount between about five micromolar and about 100 micromolar.
• At least one vital quality of the fluid being treated e.g. a fluid comprising blood or cellular blood component
• a fluid comprising blood or cellular blood component is improved after adding mitochondrial enhancer to the fluid.
• Any indicator of cell quality known in the art may be measured.
• cells consume glucose and make two lactate molecules, which lowers the pH.
• the specified lower limit for pH of the surrounding fluid is about 6.2 as measured at 22°C. Limits can be different in different countries. A fixed amount of glucose is provided to cells in storage. If the cells use up the glucose too quickly, they will die. A slower consumption of glucose is better, resulting in less lactose production and maintenance of a pH above 6.2.
• Glucose consumption, lactose production, and pH indicators of cell quality are measured in rate as well as absolute change.
• Three activity parameters that can be measured to determine whether platelets and other cellular blood components have retained their functional ability after storage are: cell count, hypotonic stress response, and aggregation, as induced by collagen in combination with adenosine diphosphate (ADP).
• indicators of cell quality measured in the practice of this invention include activation, hypotonic shock response, amount and rate of lactate production, amount and rate of glucose consumption, pH, rate of pH change, platelet swirl, platelet aggregation, amount and rate of oxygen consumption, amount and rate of carbon dioxide production, cell count, and/or ESC.
• Indicators of cell quality are typically measured periodically, e.g., cell quality for platelets is typically measured each hour or on Days 1, 3, 5, and/or 7 after adding mitochondrial enhancer. All methods known in the art for measuring indicators of cell quality are useful in the practice of this invention. Oxygen concentration, carbon dioxide concentration, and pH may be measured using a blood gas analyzer.
• P-selectin also known as GMP-140, measures activation. When cells are activated, p-selectin is expressed and appears on the surface of the cells. Platelet cells must retain the ability to activate when they are taken out of long-term storage to function normally for transfusion purposes. Cells need to be activated in vivo, so premature activation in vitro needs to be prevented. There is improved recovery and survival of platelets in vivo when p-selectin is kept low (Transfusion 2002;42:847-854 and Transfusion 2002;42:1333-1339).
• FDA Food and Drug Administration
• Expression of p-selectin is optionally measured before adding mitochondrial enhancer and then at one or more time points after adding mitochondrial enhancer. At a selected time point, the percentage of cells expressing p-selectin is measured for both treated and untreated samples. The percentage of cells expressing p-selectin is the percent of cells activated (percent activation).
• cellular activation is decreased as compared to cellular activation in a fluid to which mitochondrial enhancer has not been added.
• activation is measured by detecting alpha granule release, such as by the presence of p-selectin expression, but any method known in the art may be used.
• Activation can be decreased by at least about 3%, by at least about 10%, and up to at least about 15%. In an embodiment of this invention, activation can be decreased by an amount between about 5% and about 50%.
• Hypotonic stress response is an assay used to determine if platelets have retained metabolic viability. It measures the ability of the cells to respond to osmotic shock after about a ten-minute recovery period. Percent HSR measures the percentage of cells that are able to recover in about ten minutes. The percentage of cells that are able to recover is the percent of reversal. The specified lower limit for HSR is about 36%.
• This assay is a photometric measurement of the platelets' ability to overcome the addition of a hypotonic solution. This activity reflects cell function (i.e., ability to maintain a functional membrane water pump) and is indicative of platelet recovery following storage. Hypotonic stress response has been demonstrated to be an important indicator of platelets' ability to survive in circulation following transfusion.
• hypotonic stress response represents an important parameter for evaluating platelet biochemistry following storage.
• hypotonic shock response is increased, as compared to hypotonic shock response in a fluid to which mitochondrial enhancer has not been added.
• HSR can be increased by at least about 5%, by at least about 20%, and up to at least about 50%. In an embodiment of this invention, HSR can be increased by an amount between about 5% and about 25% as measured on Day 5 after adding mitochondrial enhancer.
• Platelet swirl is a subjective, qualitative indicator of cell quality. When a blood bag is squeezed, healthy cells will swirl, creating a pattern which can be observed by the light reflecting off and through the cells. Platelet swirl is scored on a scale of from zero to three or four, with three or four being the healthiest. The quantity of cells swirling and the strength of the swirl are two characteristics that are considered. Platelet swirl is optionally measured before adding mitochondrial enhancer and then at one or more time points after adding mitochondrial enhancer. At a selected time point, platelet swirl is measured for both treated and untreated samples.
• platelet swirl is increased, as compared to platelet swirl in a fluid to which mitochondrial enhancer has not been added. Platelet swirl can be increased by at least about 5% and by at least about 20%. In an embodiment of this invention, platelet swirl can be increased by an amount between about 5% and about 1000% as measured on Day 5 after adding mitochondrial enhancer.
• the pH of the fluid is increased and the rate of pH decrease can be decreased, as compared to pH and rate of pH decrease in a fluid to which mitochondrial enhancer has not been added.
• the increased rate of pH increase can also be measured, if pH increases.
• pH is optionally measured before adding mitochondrial enhancer and then at one or more time points after adding mitochondrial enhancer.
• pH is measured for both treated and untreated samples.
• the amount of change in pH in pH units is the pH of treated samples - pH of untreated samples.
• pH is measured at two or more selected time points, wherein at least one of the time points occurs after adding mitochondrial enhancer. Time point 2 is measured after time point 1.
• the rate of pH decrease is the absolute value of the percent change in rate of pH change.
• the rate of pH decrease can be decreased by at least about 2%, by at least about 20%, and up to at least about 40%. In an embodiment of this invention, the rate of pH decrease can be decreased by an amount between about 2% and about 50%.
• the pH can be increased by at least about 0.1 units, by at least about 0.2, by at least about 0.35, and up to at least about 0.5. In an embodiment of this invention, the pH can be increased by an amount between about 0.1 and about 0.75. In an embodiment of this invention, the rate of pH decrease can be decreased by an amount between about 15% and about 50% on Day 5. In an embodiment of this invention, the pH is increased by an amount between about 0.1 and about 0.5 pH units, about twenty-four hours after mitochondrial enhancer is added to platelets that were stored for six days after apheresis.
• the rate of lactate production by cells in the fluid being treated and the amount of lactate produced are decreased, as compared to rate of lactate production and amount of lactate produced in a fluid to which mitochondrial enhancer has not been added.
• Amount of lactate in a fluid can be measured by any method known in the art. Amount of lactate is optionally measured before adding mitochondrial enhancer and then at one or more time points after adding mitochondrial enhancer. Amount of lactate can be measured as the concentration of lactate in the fluid. At a selected time point, amount of lactate is measured for both treated and untreated samples.
• the percent change of lactate production is the ((amount of lactate in treated samples - amount of lactate in untreated samples) / amount of lactate in untreated samples) * 100. If the percent change of lactate production is negative, the percent decrease in lactate production due to adding mitochondrial enhancer is the absolute value of the percent change of lactate production.
• amount of lactate is measured at two or more selected time points, wherein at least one of the time points occurs after adding mitochondrial enhancer. Time point 2 is measured after time point 1.
• the rate of change of lactate production is calculated for both treated and untreated samples.
• the percent change in the rate of lactate production is ((rate of change of lactate production of treated - rate of change of lactate production of untreated) / rate of change of lactate production of untreated) * 100. If the rate of lactate production of treated and the rate of lactate production of untreated are positive, and the percent change in rate of lactate production is negative, the rate of lactate production is decreased by adding mitochondrial enhancer. The decrease in the rate of lactate production is the absolute value of the percent change in rate of lactate production.
• the rate of lactate production can be decreased by at least about 5%, by at least about 50%, by at least about 80% and up to at least about 125%. In an embodiment of this invention, the rate of lactate production can be decreased by an amount between about 25% and about 100%. In an embodiment of this invention, the amount of lactate produced can be decreased by an amount between about 15% and about 100%. In an embodiment of this invention, the amount of lactate produced can be decreased by about 75%. In an embodiment of this invention, the rate of lactate production can be decreased by an amount between about 75% and about 100% about twenty- four hours after mitochondrial enhancer is added to platelets.
• the rate of glucose consumption by cells in the fluid, such as cellular blood components, and the amount of glucose consumed are decreased, as compared to rate of glucose consumption and amount of glucose consumed in a fluid to which mitochondrial enhancer has not been added.
• Glucose consumption is decreased by adding mitochondrial enhancer because adding mitochondrial enhancer enables a cell to better utilize mitochondrial biochemical pathways (citric acid cycle and electron transport chain) to generate energy, which generate more energy per glucose molecule compared to cytoplasmic biochemical pathways (glycolysis). Additionally, in platelets, mitochondrial biochemical pathways for generating energy don't require glucose.
• Glucose consumption can be measured by measuring the amount of glucose remaining in the fluid at a selected time point.
• the decrease in glucose consumption can be measured as the increase in the amount of glucose remaining in the fluid.
• the amount of glucose remaining in a fluid can be measured by any method known in the art.
• the amount of glucose remaining is optionally measured before adding mitochondrial enhancer and then at one or more time points after adding mitochondrial enhancer.
• the amount of glucose can be measured as the concentration of glucose (e.g. molecules/volume or molecules/cells) in the fluid.
• amount of glucose is measured for both treated and untreated samples.
• the percent change of glucose consumption is the ((amount of glucose in treated samples - amount of glucose in untreated samples) / amount of glucose in untreated samples) * 100. By this calculation, the percent change of glucose remaining in the fluid is the percent change of glucose consumption due to adding mitochondrial enhancer.
• the amount of glucose remaining in the fluid is measured at two or more selected time points, wherein at least one of the time points occurs after adding mitochondrial enhancer.
• Time point 2 is measured after time point 1.
• the rate of glucose consumption as calculated using amount of glucose remaining (FIG. 8) can be negative.
• the change in the rate of glucose consumption is calculated for both treated and untreated samples.
• the percent change in the rate of glucose consumption is ((rate of glucose consumption of treated - rate of glucose consumption of untreated) / rate glucose consumption of untreated) * 100. If the rate of glucose consumption of treated and the rate of glucose consumption of untreated are negative, and the percent change in rate of glucose consumption is positive, the rate of glucose consumption is decreased by adding mitochondrial enhancer. The decrease in the rate of glucose consumption is the percent change in rate of glucose consumption. When the rate of glucose consumption is decreased, more glucose is left in the fluid at a selected time point.
• the rate of glucose consumption can be preferably decreased by at least about 5%, by at least about 25%, at least about 75%, and up to at least about 100%. In an embodiment of this invention, the rate of glucose consumption can be decreased by an amount between about 25% and about 150%.
• the amount of glucose consumed, as measured by the amount of glucose remaining can be decreased by at least about 10%, by at least about 25%, and up to at least about 80%. In an embodiment of this invention, the amount of glucose consumed, as measured by the amount of glucose remaining, can be decreased by an amount between about 10% and about 150%, or between about 25% and about 100% on Day 3.
• the percent change in ESC is the amount of increase in ESC, in percent, at that time point.
• the extent of cell shape change is increased, as compared to the ESC of a fluid containing platelets to which mitochondrial enhancer has not been added.
• the ESC can be increased by an amount between about 5% and about 125%.
• the ESC can be increased by at least about 5%, by at least about 25%, by at least about 75%, and up to at least about 100%.
• the ESC can be increased by at least about 75% on Day 5.
• the amount and/or the rate of oxygen consumption of the cellular blood component are/is increased, as compared to the amount and/or rate of oxygen consumption of a cellular blood component in a fluid to which mitochondrial enhancer has not been added.
• Amount of oxygen present in a gas above a fluid can be measured by any method known in the art. Glycolysis does not require oxygen, but the citric acid cycle does, therefore, when cells are utilizing mitochondrial biochemistry to generate energy (citric acid cycle), more oxygen is consumed and consequently less is left in the gas above the fluid containing the cells.
• Amount of oxygen is optionally measured before adding mitochondrial enhancer and then at one or more time points after adding mitochondrial enhancer.
• amount of oxygen is measured for both treated and untreated samples.
• the percent change of oxygen consumption is also the percent change in oxygen remaining in the gas above the fluid.
• the percent change of oxygen consumption is the ((amount of oxygen in treated samples - amount of oxygen in untreated samples) / amount of oxygen in untreated samples) * 100. If the percent change of oxygen consumption is negative, the percent increase in oxygen consumption due to adding mitochondrial enhancer is the absolute value of the percent change of oxygen consumption.
• amount of oxygen is measured at two or more selected time points, wherein at least one of the time points occurs after adding mitochondrial enhancer. Time point 2 is measured after time point 1.
• the rate of change of oxygen consumption is calculated for both treated and untreated samples.
• the percent change in the rate of oxygen consumption is ((rate of change of oxygen consumption of treated - rate of change of oxygen consumption of untreated) / rate of change of oxygen consumption of untreated) * 100. If the rate of oxygen consumption of treated and the rate of oxygen consumption of untreated are positive, and the percent change in rate of lactate production is negative, the rate of oxygen consumption is increased by adding mitochondrial enhancer.
• the increase in the rate of oxygen consumption is the absolute value of the percent change in rate of oxygen consumption.
• the oxygen consumption can be increased by an amount between about 5% and about 125%.
• the oxygen consumption can be increased by at least about 5%, by at least about 15%, by at least about 75%, by at least about 100%, and up to at least about 175%.
• the oxygen consumption can be increased by at least about 50% at twenty-four hours after adding mitochondrial enhancer.
• the rate of oxygen consumption can be increased by at least about 10% or between about 10% and about 50%.
• the rate of carbon dioxide production by cells in the gas above a fluid being treated and the amount of carbon dioxide produced are increased, as compared to the rate of carbon dioxide production and amount of carbon dioxide produced by a cell-containing fluid to which mitochondrial enhancer has not been added.
• Carbon dioxide is produced during mitochondrial biochemical pathways (citric acid cycle) for producing energy.
• Amount of carbon dioxide in the gas above a fluid can be measured by any method known in the art.
• Amount of carbon dioxide is optionally measured before adding mitochondrial enhancer and then at one or more time points after adding mitochondrial enhancer.
• Amount of carbon dioxide can be measured as the concentration (e.g. partial pressure) of carbon dioxide in the fluid. At a selected time point, amount of carbon dioxide is measured for both treated and untreated samples.
• the percent change of carbon dioxide production is percent change of carbon dioxide in the gas above a treated sample compared to an untreated sample.
• the percent change of carbon dioxide production is the ((amount of carbon dioxide in treated samples - amount of carbon dioxide in untreated samples) / amount of carbon dioxide in untreated samples) * 100. If the percent change of carbon dioxide production is positive, it is the percent increase in carbon dioxide production due to adding mitochondrial enhancer.
• amount of carbon dioxide is measured at two or more selected time points, wherein at least one of the time points occurs after adding mitochondrial enhancer. Time point 2 is measured after time point 1.
• the rate of change of carbon dioxide production is calculated for both treated and untreated samples.
• the percent change in the rate of carbon dioxide production is ((rate of change of carbon dioxide production of treated - rate of change of carbon dioxide production of untreated) / rate of change of carbon dioxide production of untreated) * 100. If the rate of carbon dioxide production of treated and the rate of carbon dioxide production of untreated are positive, and the percent change in rate of carbon dioxide production is positive, the rate of carbon dioxide production is increased by adding mitochondrial enhancer.
• Potential for aggregation is another vital quality that indicates whether blood platelets have maintained their functional integrity during storage. This potential is measured by using ADP and collagen to induce aggregation.
• An agonist is an agent that binds to a receptor and initiates a certain response. In an agonist-induced aggregation, aggregation or clumping is the response to the agonist.
• the agonists ADP and collagen are used to induce aggregation to determine if platelets have retained their ability to aggregate.
• Vital cell qualities that determine allowed storage life of blood components are determined by the U.S. FDA (See Circular of Information for the Use of Human Blood and Blood Components or http://www.fda.gov/cber/gdlns/circbld.pdf). To increase storage life of a blood component, the vital cell quality that is limiting the storage life of that blood component must be improved. Additional vital cell qualities can be improved as well.
• the vital quality of cellular blood component activation can be decreased by at least about 3%, and/or the vital quality of HSR can be increased by at least about 5%, and/or the vital quality of platelet swirl can be increased by at least about 5%, and/or the vital quality of pH of the fluid containing the cellular blood component can be decreased by at least about 0.1 pH units, and/or the vital quality of rate of lactate production can be decreased by at least about 5%, and/or the vital quality of rate of glucose consumption can be decreased by at least about 5%, and/or the vital quality of rate of oxygen consumption can be decreased by at least about 5%, and/or the vital quality of carbon dioxide production can be decreased by about 5%.
• the cellular blood component is platelets and they can be stored, e.g. for a period greater than five days, for between about five days and about seven days.
• Platelets can be concentrated before treating using the methods of this invention.
• Platelets can be concentrated by any method known in the art, such as using apheresis devices while collecting blood or from previously collected samples of whole blood.
• platelets are hyperconcentrated to form hyperconcentrated platelets (HCP) by centrifugation at 3000 times gravity (3000xG) for fifteen minutes and allowed to rest for one hour.
• HCP are resuspended in autologous plasma, resulting in approximately five trillion platelets per milliliter.
• Fluid can consist essentially of platelets in plasma or cell culture media, e.g. comprising platelets and between about 5% and about 95% plasma or media.
• the methods provided by this invention optionally further comprise photoradiating, adding photoactivator, adding nitric oxide, adding quencher, adding glycolysis inhibitor, adding oxygen, and/or adding process enhancers to the fluid being treated.
• Pathogen reduction using photoradiation requires mixing a photosensitizer with the material to be decontaminated. Mixing may be done by simply adding the photosensitizer or a solution containing the photosensitizer to the fluid to be decontaminated.
• the material to be decontaminated to which the photosensitizer has been added is flowed past a photoradiation source, and the flow of the material provides sufficient turbulence to distribute the photosensitizer throughout the fluid to be decontaminated.
• the fluid and photosensitizer are placed in a photopermeable container and irradiated in batch mode, preferably while agitating the container to fully distribute the photosensitizer and expose all the fluid to the radiation.
• the amount of photosensitizer to be mixed with the fluid will be an amount sufficient to adequately reduce microorganisms therein, but less than a toxic (to humans or other mammals) or insoluble amount.
• Optimal concentrations of desired photosensitizers may be readily determined by those skilled in the art without undue experimentation.
• the fluid containing the photosensitizer is exposed to photoradiation of the appropriate wavelength to activate the photosensitizer, using an amount of photoradiation of sufficient energy to activate the photosensitizer as described above, but less than that which would cause non-specific damage to the biological components or substantially interfere with biological activity of other proteins present in the fluid.
• the wavelength used will depend on the photosensitizer selected, as is known in the art or readily determinable without undue experimentation following the teachings hereof.
• the pathogen reduced cellular blood component may be kept in the pathogen reduction solution, or may be transferred to a storage solution.
• Photoradiation to reduce pathogens is performed by methods known in the art or by methods described in references included herein.
• An amount of energy is supplied to the fluid using photoradiation.
• the amount of energy supplied is sufficient to reduce pathogens which may exist in the fluid, but also does not substantially interfere with the biological activity of the blood component(s) contained in the fluid.
• the biological activity of blood component(s) in the fluid at least meets minimum standards for medical and veterinary use for standard storage times for the specific blood component, e.g., five or preferably seven days for platelets.
• Pathogen reduction methods of this invention are described as using flux (energy) in units of joules (J) per unit area (cm 2 ) per unit time (min).
• a time length of photoradiation is selected to accomplish delivering a total amount of energy selected to substantially reduce pathogens in the fluid being treated.
• Some lamps useful for providing the required energy are VHO lights with a mercron ballast, T8 lights with an icecap ballast, or T8 lights with an icecap ballast and quartz attenuator.
• Photoradiation can be delivered continuously or in a segmented (interrupted) fashion. The photoradiation is preferably within the ultraviolet range or the visible range.
• a photoradiation light source can be selected that is capable of providing light of about 300 nm to about 700 nm, or between about 340 nm to about 650 nm of radiation.
• the energy delivered to the fluid is an amount sufficient to activate a photosensitizer, e.g., between about 5
• the total time of photoradiation is sufficient to substantially reduce pathogens, e.g., between about three and about thirty minutes.
• substantially inactive pathogens means to reduce their ability to reproduce, preferably by killing them, to levels in a blood component such that the blood component may be safely administered to a patient.
• Ultraviolet wavelength of 320 nm is useful in the practice of this invention.
• This invention provides methods for improved pathogen reduction. Pathogen reduction processes using photoradiation are improved by the addition of mitochondrial enhancer. Addition of mitochondrial enhancer to a fluid containing a blood product containing mitochondria prevents damage to the blood product from photoradiation, which allows the use of high photoradiation energies, which are more effective at reducing pathogens. Prevention of damage to the blood product is indicated by improvement to vital cell qualities in fluids treated with mitochondrial enhancer compared to fluids treated at equivalent energies without addition of mitochondrial enhancer, before, after, or during photoradiation. High energies useful in the practice of this invention include more than about 30 J/cm , more than about 50 9 9 9 9
• This invention provides an improved method for treating a fluid comprising a cellular blood component to reduce pathogens which may be present therein, comprising the steps of:
• the photoradiation is preferably applied at an energy more than about 25 J/cm , wherein the photoradiation is substantially non-toxic to the cellular blood component.
• Amounts of photoradiation energy and mitochondrial enhancer are selected such that extent of pathogen reduction and vital cell quality immediately after photoradiation and at about 24 hours after photoradiation are about the same or better than an equivalent process without mitochondrial enhancer at a lower energy. Examples of lower energies are about five to about 15 J/cm .
• photoradiation is preferably delivered at more than about 25 J/cm 2 or more than about 40 J/cm 2 .
• photoradiation is
• Fluids may optionally be mixed before or during photoradiation. Mixing may enhance dissolution of components. Before or during photoradiation, the fluid may be mixed by mixing and/or shaking at a speed between about 70cpm and about 150cpm, or between about 120cpm and about 135cpm. When performed before photoradiation, mixing can be performed for about one to about ten minutes. The mixing and shaking may be performed in any motion known to the art, including mixing and shaking using a to-and-fro motion.
• One or more of the light sources may move in a coordinated manner with the movement of the mixing.
• Mixing enables the majority of the photosensitizer and fluid contained within the container to be exposed to the light emitted from each of the discrete radiation sources by continually replacing the exposed fluid at the light- fluid interface with fluid from other parts of the bag not yet exposed to the light.
• Such mixing continually brings to the surface new fluid to be exposed to light.
• Photoradiation can be performed at a temperature that allows for reduction of pathogens and does not interfere with the biological activity of cellular blood components.
• Photoradiated fluids containing cellular blood components can be stored at temperatures known in the art for storing blood products.
• Materials which may be treated by methods of this invention that involve photoradiation greater than ambient light include any materials which are adequately permeable to photoradiation to provide sufficient light to achieve pathogen reduction, or which can be suspended or dissolved in fluids which have such permeability to photoradiation. Plasma and cell culture media containing cellular blood components are permeable to photoradiation.
• Decontamination methods of this invention do not destroy the biological activity of fluid components other than microorganisms. As much biological activity of these components as possible is retained, although in certain instances, when the methods are optimized, some loss of biological activity, e.g., denaturization of protein components, must be balanced against effective decontamination of the fluid.
• this method is useful for treating other cell- containing fluids including fluids which are meant for nourishment of humans or animals such as fruit and vegetable juices.
• Platelets were collected using standard collection methods using a COBE® SpectraTM apheresis machine (manufactured by Gambro BCT, Lakewood, CO, USA). Fresh platelets were less than 24 hours old after collection via apheresis. Other apheresis machines useful for collecting transfusion-quality platelets are useful in the practice of this invention. Collected platelets were diluted in a solution containing 0.9% sodium chloride with either 0 or 10 ⁇ M riboflavin. Samples were saturated with air via vigorous mixing. Platelets were exposed to no photoradiation greater than ambient light. Results are shown in Table 1.
• Platelets were collected using standard collection methods using a COBE® SpectraTM apheresis system (manufactured by Gambro BCT, Lakewood, CO, USA) and TRIMA® apheresis system (available from Gambro BCT, Lakewood, CO, USA) . Fresh platelets were less than 24 hours old after collection via apheresis. Other apheresis machines useful for collecting transfusion-quality platelets are useful in the practice of this invention. Collected platelets were diluted in a solution containing 0.9% sodium chloride with either 0 or 10 ⁇ M riboflavin. Samples were saturated with air via vigorous mixing. The samples were irradiated with a Dymax light source using one bulb at 15.5 mW/cm for varying times. The UV component of the light was filtered out using a polycarbonate filter. Exposure time was 31 minutes. Results are shown in Table 2.
• Platelets were collected using standard collection methods using a COBE® SpectraTM apheresis machine (manufactured by Gambro BCT, Lakewood, CO, USA). Fresh platelets were less than 24 hours old after collection via apheresis. Other apheresis machines useful for collecting transfusion-quality platelets are useful in the practice of this invention. Collected platelets were diluted in a solution containing 0.9% sodium chloride with either 0 or 10 ⁇ M riboflavin. Samples were saturated with air via vigorous mixing. The samples were irradiated with a Dymax light source using one bulb at 15.5 mW/cm for varying times. Results are shown in Tables 3 and 4.
• lactate production rate was decreased by about 82%
• glucose consumption rate was decreased by about 66%
• oxygen consumption rate was increased by about 175%.
• Other cell quality indicators were improved as well.
• the energy of both ultraviolet and visible light combined was 40 J/cm .
• FIG. 5 is a graph showing the effect of mitochondrial enhancer on lactate production (lactate concentration mM/1000 cells) by platelets as a function of storage time (days). The amount of lactate produced decreased about 70% with riboflavin.
• Figure 6 is a graph showing the effect of mitochondrial enhancer on the rate of lactate production by platelets as a function of mitochondrial enhancer concentration. The rate of lactate production decreased about 80% when 10 micromolar riboflavin was added compared to no riboflavin. The rate of lactate production decreased with increasing amounts of riboflavin.
• Example 4 Treatments of Platelets with NaCN to Simulate Electron Transport Chain Damage
• the platelets were 6-day-old apheresis platelets which had been stored under standard conditions at a local blood bank. Products were placed into 30 mL bags for irradiation and 24 hour storage. Results are shown in Table 6.
• lactate production decreased by about 91%. Other cell quality indicators were improved as well.
• Example 6 Treatment of Stored Platelets with Riboflavin and Visible Light
• the platelets were 6-day-old apheresis platelets which had been stored under standard conditions at a local blood bank. Products were placed into 30 mL bags for irradiation and 24 hour storage.
• the light source used was a Dymax light source with a single bulb. UN light was filtered out using a polycarbonate sheet. Exposure time was adjusted to deliver 40 J/cm . Results are shown in Table 7.
• the platelets were 6-day-old apheresis platelets which had been stored under standard conditions at a local blood bank. Products were placed into 30 mL bags for irradiation and 24 hour storage.
• the light source used was a Dymax light source with a single bulb. UV light was filtered out using a polycarbonate sheet. Exposure time was adjusted to deliver 40 J/cm combined visible light and ultraviolet light. Results are shown in Table 8.
• FIG. 1 is a graph showing the effect of mitochondrial enhancer on platelet swirl (0-4 units) of cellular blood components as a function of storage time (days). Platelet swirl increased on Day 1 with the addition of riboflavin.
• Figure 2 is a graph showing the effect of mitochondrial enhancer on hypotonic shock response (HSR), % reversal, of platelets as a function of storage time (days). HSR increased with the addition of riboflavin.
• Figure 3 is a graph showing the effect of mitochondrial enhancer on pH of the stored fluid as a function of storage time (days).
• FIG. 9 is a graph showing the effect of mitochondrial enhancer on p-selectin expression (GMP-140 (granule membrane protein- 140) expression, also called % activation, by platelets as a function of storage time (days). Percent activation is the percentage of cells expressing p- selectin. P-selectin expression was decreased with riboflavin.
• Figure 4 is a graph showing the effect of mitochondrial enhancer on percent extent of shape change (ESC) of platelets as a function of storage time (days). ESC% was increased with riboflavin.
• ESC percent extent of shape change
• FIG. 7 is a graph showing the effect of mitochondrial enhancer on lactate production •by platelets as a function of storage time (days). Lactate production was decreased with riboflavin by between about 20% and about 30%. The rate of lactate production was decreased by about 25%.
• Figure 8 is a graph showing the effect of mitochondrial enhancer on glucose consumption by platelets as a function of storage time (days).
• Glucose consumption was decreased by riboflavin by between about 15% and about 85%. Rate of glucose consumption was decreased by about 36%.
• Figure 9 is a graph showing the effect of mitochondrial enhancer on p-selectin expression (% activation), by platelets as a function of storage time (days). Activation was reduced by riboflavin by about 10%.
• Figure 10 is a graph showing the effect of mitochondrial enhancer on oxygen consumption by platelets as a function of storage time (days).
• Figure 10 is alternatively labeled as a graph of oxygen concentration which is indicative of consumption because the lower the oxygen concentration, the higher the oxygen consumption. Oxygen consumption was increased by about 30%. The rate of oxygen consumption was increased by about 37.5%.
• Vaccinia virus was used to innoculate Isolyte S media (Halpern, et al. (1997) Grit Care Med. 25(12):2031-8). Samples also contained 10 micromolar riboflavin. Vaccinia virus was titered before and after exposure and measured as TCID 50 (tissue culture infection dose for 50% of the tissue culture cells). UV photoradiation was continued for a total of 30 minutes (40 J/cm ), with vaccinia virus titered at ten minute intervals. More energy was delivered with longer exposure times.
• Figure 11 is a graph showing the effect of mitochondrial enhancer on reduction kinetics of vaccinia virus as a function of photoradiation exposure time (delivered energy). Vaccinia virus reduction increased with increasing exposure time (energy). There was no reduction without riboflavin. Results are shown in Table 9.
• HSV-2 was used to innoculate 90% PCO, wherein the balance was Isolyte S media. HSV-2 was titered before and after exposure to photoradiation from a DYMAX 2000 irradiator.
• Figure 12 is a graph showing the effect of various concentrations of mitochondrial enhancer on reduction of Herpes Virus 2 as a function of photoradiation exposure time (delivered energy). Photoradiation included both visible and ultraviolet wavelengths. The four arrows on figure 12 indicate that the actual log inactivation data points might be higher than those shown because these samples were at the detection limit of the assay.
• S. epidermidis was used to innoculate 90% PCO, wherein the balance was Isolyte S media.
• S. epidermidis was titered before and after exposure to photoradiation from a DYMAX 2000 i ⁇ adiator. Photoradiation was delivered at 40
• FIG. 13 is a graph showing the effect of various energy doses on reduction of S. epidermidis as a function of concentration of mitochondrial enhancer. Reduction was greatest with higher energy delivery and with 10 micromolar riboflavin. Results are shown in Table 11.
• Photoradiation was delivered at 40 J/cm , 80 J/cm , and 120 J/cm . Photoradiation included both visible and ultraviolet wavelengths. Riboflavin was at 0, 50, or 100 micromolar.
• Figure 14 is a graph showing the effect of various concentrations of mitochondrial enhancer on reduction of ⁇ X174 as a function of delivered photoradiation energy. Reduction was greatest at higher riboflavin concentrations (100 micromolar) and higher energies (120 J/cm ). Results are shown in Table 12.
• Non-endogenous alloxazine is added to a fluid containing platelets and photosensitizer.
• the fluid is exposed to about 30 J/cm 2 ultraviolet light. After five days of storage, cell quality indicators are improved compared to an equivalent process not using non-endogenous alloxazine.
• Example 15 Treatment of Platelets with Endogenously Based Derivative Alloxazine and Ultraviolet Light
• Endogenously based derivative alloxazine is added to a fluid containing platelets and photosensitizer.
• the fluid is exposed to about 30 J/cm 2 ultraviolet light. After five days of storage, cell quality indicators are improved compared to an equivalent process not using endogenously based derivative alloxazine.
• Vitamin K is added to a fluid containing platelets and photosensitizer.
• the fluid is exposed to about 30 J/cm ultraviolet light. After five days of storage, cell quality indicators are improved compared to an equivalent process not using vitamin K.
• Vitamin L is added to a fluid containing platelets and photosensitizer.
• the fluid is exposed to about 30 J/cm ultraviolet light. After five days of storage, cell quality indicators are improved compared to an equivalent process not using vitamin L.
• Peritoneal solution is removed from a body, mitochondrial enhancer is added, and the peritoneal solution with mitochondrial enhancer is administered to the peritoneal space of a body.
• the body may be the same body from which the peritoneal solution was removed.
• mitochondrial enhancer is administered directly to the peritoneal space of a body.
• Cells within the peritoneal solution and/or cells that are in contact with the mitochondrial enhancer containing peritoneal space are mitochondrially enhanced.
• a wound is treated by administration of mitochondrial enhancer.
• Cells on and near the surface of the wound including but not limited to white blood cells, red blood cells, and fibroblasts, are mitochondrially enhanced.

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