MXPA00002800A - Method and apparatus for inactivation of biological contaminants using photosensitizers - Google Patents

Abstract

Methods and apparatuses are provided for inactivation of microorganisms in fluids or on surfaces. Preferably the fluids contain blood or blood products and comprise biologically active proteins. Preferred methods include the steps of adding an effective, non-toxic amount of an endogenous photosensitizer to a fluid and exposing the fluid to photoradiation sufficient to activate the endogenous photosensitizer whereby microorganisms are inactivated. Other fluids, including juices, water and the like, may also be decontaminated by these methods as may surfaces of foods, animal carcasses, wounds, food preparation surfaces and bathing and washing vessel surfaces. Alloxazines and K- and L- vitamins are among the preferred photosensitizers. Systems and apparatuses for flow-through and batch processes are also provided for decontamination of such fluids using photosensitizers.

Description

METHOD AND APPARATUS FOR THE INACTIVATION OF BIOLOGICAL POLLUTANTS THROUGH THE USE OF PHOTOSENSITIZERS CROSS REFERENCE WITH RELATED APPLICATIONS This application is a partial continuation of US Application No. 09/119666, filed on July 21, 1998, which is fully incorporated in this document to an extent that is not incompatible with what is It is described. BACKGROUND Contamination of blood supplies with infectious organisms such as HIV, hepatitis and other viruses, and with bacteria, presents a serious health hazard to those who must receive full blood transfusions or administration of various blood components, such as such as platelets, red blood cells, blood plasma, Factor VIII, plasminogen, fibronectin, anti-thrombin III, cryoprecipitate, human plasma protein fraction, albumin, immune serum globulin, growth hormones of the plasma prothrombin complex and other components isolated from the blood. Blood screening procedures may not detect contaminants, and there are still no sterilization procedures that do not damage the cellular components of the blood but inactivate effectively all infectious viruses and other microorganisms. Solvent detergent methods for the decontamination of blood components work by dissolving the membranes of phospholipids that surround viruses such as HIV, and do not damage the protein components of the blood. However, if there are blood cells present, no Such methods can be used since they also cause damage to cell membranes. The use of photosensitizers, compounds that absorb light of a given wavelength and transfer the absorbed energy to an energy acceptor, has been proposed for the sterilization of blood components. For example, European Patent Application 196515, published on October 8, 1986, suggests the use of non-endogenous photosensitizers such as porphyrins, psoralens, acridine, toluidines, flavin (acriflavine hydrochloride), phenothiazine derivatives and stains such as red neutral and methylene blue, as aggregates for blood. Protoporphyrin, which occurs naturally in the body, can be metabolized to form a photosensitizer. However, its utility is limited since it degrades the biological activities of desired proteins. Chlorpromazine is also exemplified as one such photosensitizer. However, its utility is limited by the fact that it should be removed from any fluid administered to a patient after the decontamination procedure since it has a sedative effect. Goodrich, RP et al (1997) "The Design and Development of Selective, Photoactivated Drugs for Sterilization of Blood Products", Drugs of the Future 22: 159-171 provides a review of some photosensitizers including psoralens, and some of the factors of importance in choosing photosensitizers for the decontamination of blood products. The use of texaphyrins for DNA photoseparation is described in U.S. Patent No. 5607924, published March 4, 1997, and 5714328, published February 3, 1998 by Magda et al. The use of safirins for viral inactivation is described in US Patent No. 5041078, published August 20, 1991 by Matthews et al. Inactivation of extracellular enveloped viruses in blood and blood components by Fentiazin-5-inium stains plus light is described in US Patent No. 5545516, published August 13, 1996 by Wagner. The use of porphyrin, hematoporphyrin and merocyanin stains as photosensitizer feels to eradicate infectious contaminants such as viruses and protozoa from body tissues such as body fluids is described in US Patent No. 4915683, published April 10, 1990. and U.S. Patent No. 530413 related, published on April 19, 1994 by Sieber et al. The mechanism of action of such photosensitizers is described as involving the peripheral binding to domains of lipid bilayers, for example, in enveloped viruses and in some cells infected by viruses. Photoexcitation of membrane-bound agent molecules leads to the formation of reactive oxygen species such as atomic oxygen, which causes lipid peroxidation. A problem with the use of such photosensitizers is that they attack the cell membranes of the desired components of the fluids to be decontaminated, such as the red blood cells, and the atomic oxygen also attacks the desired protein components of the fluids to be treated. US Patent No. 4727027, published February 23, 1998 by Wieselah, GP et al, describes the use of furocoumarins, including psoralep and its derivatives, for the decontamination of blood and blood products, but instructs that the steps should be taken in such a way as to reduce the availability of dissolved oxygen and other reactive species to inhibit the denaturation of the biologically active proteins. Photoinactivation of viral and bacterial contaminants of blood using halogenated coumarins is described in US Patent No. 5516629, published May 14, 1996 by Park et al. U.S. Patent No. 5587490, published December 24, 1996 by Goodrich Jr, RP et al, and U.S. Patent No. 5418130 to Platz et al, which describe the use of substituted psoralen for the inactivation of contaminants viral and bacterial blood. The latter patent also indicates the need to control the damage by free radicals to other components of the blood. U.S. Patent No. 5654443, published August 5, 1997 by Wollowitz et al, shows new compositions of psoralen used for the photodecontamination of blood. U.S. Patent No. 5709991, published January 20, 1998 by Lin et al, teaches the use of psoralen for photodecontamination of platelet preparations and the subsequent removal of psoralen. US Patent No. 5120649, published June 9, 1993, and related US Patent No. 5232844, published August 3, 1993 by Horowitz et al, also discloses the need for the use of "forgers" in combination with photosepsibilizers that attack the lipid membranes, and US Patent No. 5360734, published on November 1, 1994 by Chapmap et al, also describes this problem of preventing damage to other blood components. Photosensitizers that attack nucleic acids are known in the art. U.S. Patent No. 5342752, published August 30, 1994 by Platz et al, discloses the use of compounds based on acridine stains to reduce parasitic contamination in blood, which encompasses red blood cells, platelets. and protein fractions of blood plasma. These materials, although they present a quite low toxicity, still present a certain toxicity, for example, for the red blood cells. This patent fails to describe an apparatus for decontaminating blood on a flow basis. U.S. Patent No. 5798238 to Goodrich Jr et al, describes the use of quinolone and quinolone compounds for the inactivation of viral and bacterial contaminants. The binding of DNA with photoactive agents has been exploited in processes to reduce lymphocyte populations in the blood, as indicated by US Patent No. 4612007, published September 16, 1986 and US Patent No. 4683889 related, published on August 4, 1987 by Edelson. It has been reported that riboflavin (7,8-dimethyl-10-ribityl isoaloxazine) attacks nucleic acids. Photoalteration of nucleic acids in the presence of riboflavin is reported in Tsugita et al (1965), "Photosensitized inactivation of ribonucleic acids in the presence of rlboflavin", Biochim ica et Biophysica Acta 103: 360-363 and in Speck, W T et al (1976), "Further Observations on the Photooxidation of DNA in the Presence of Riboflavin", Biochimica et Bíophysica Acta 435: 39-44. The binding of lumiflavine (7,8,10-trimethylisoaloxazine) to DNA is given in Kuratomi, K et al (1977), "Studies in the Intecations Between DNA and Flavins", Biochimica et Biophysica Acta 476: 207, 217. Hoffman, ME et al (1979), "DNA Strand Breaks ip Mammalian Cells Exposed to Light in the Presence of Riboflavin and Tryptophan," Photochemistry and Photobiology 29: 299-203 describe the use of riboflavin and truptophan to induce breaks in cell DNA of mammals after exposure to fluorescent visible light or near ultraviolet light. The article indicates that these effects did not occur if riboflavin or tryptophan was omitted from the medium. Breakthroughs in DNA strands are reported when exposed to proflavin and light in Piette, J et al (1979), "Production of Breaks in Single - and Double - Stranded Forms of Bacteriophage FX174 DNA by Proflavine an Light Treatmenf, Photochemistry and Photochemistry Photobiology 30: 369-378, and the alteration of guanine residues during proflavin-mediated photosensitization of DNA is discussed in Piette, J et al (1981), "Alteration of Guanine Residues during Proflavine Mediated Photosensitization of DNA" Photochemistry and Photobiology 33 : 325-333 J Cadet et al (1983), "Mechanisms and Products of Photosensitized Degradation of Nucleic Acids and Related Model Compoupds", Israel J Chem 23: 420-429, describe the mechanism of action by means of oxygen production atomic from bengal rose, methylene blue, thionin and other stains, in comparison with other mechanisms that do not involve the production of atomic oxygen by means of which attack comes the attack to the nucleic acids by the flavin or the pteron derivatives. Riboflavin is exemplified in this study as having the ability to degrade nucleic acids. Korycka - Dahl, M et al (1980), "Photodegradation of DNA with Fluorescent Light in the Presence of Riboflavin, and Photoprotection by Flavin Triplet - State Quenchers", Biochimica et Biophysica Acta 610: 229-234, also describe that species of Active oxygen are not directly involved in DNA cleavage by riboflavin. Peak, JG et al (1984), "DNA Breakage Caused by 334 mm Ultraviolet Light is Enhanced by Naturally Occurring Nucleic Acid Compcnents and Nucleotide Conezymes", Photochemistry and Photobiology 39: 713-716 further explore the mechanism of action of riboflavin and other photosensitizers. However, no suggestion is made that such photosensitizers can be used for the decontamination of medical fluids. Devices for blood decontamination have been described in US Patent No. 5290221, published on March 1, 1994 by Wolfe Jr et al, and in US Patent No. 5536238, published July 16. of 1996 by Bischof. U.S. Patent No. 5290221 describes the irradiation of fluid in a relatively narrow arcuate gap. U.S. Patent No. 5536238 describes devices that use optical fibers that extend in a filtration medium. Both patents recommend photosensitizers derived from benzoporphyrins, which have an affinity for cell walls. All publications mentioned in this documentation are incorporated into it for reference in an extension not inconsistent with what is described. SUMMARY Methods and apparatus are provided to treat a fluid or other material so as to inactivate at least some of the microorganisms and white cells that may be present in or within it. Such fluids may also contain one or more components selected from the group consisting of proteins, for example, biologically active proteins such as a therapeutic proteins., blood and constituents of the blood, without destroying the biological activity of such components. The methods comprise: (a) Mixing a non-toxic effective amount of an endogenous photosensitizer or a photosensitizer derivative based on an endogenous element with the fluid, (b) Expose the fluid to a photoradiation sufficient to activate the photosensitizer, where inactivates at least one of the microorganisms. One mechanism by which these photosensitizers can inactivate microorganisms is by interfering with their nucleic acids, so as to avoid the replication of such nucleic acid. As used herein, the term "inactivation of a microorganism" means to totally or partially avoid the replication of the microorganism, either by killing such a microorganism or by interfering otherwise with its ability to reproduce. Microorganisms include viruses (both extracellular and intracellular), bacteria, bacteriophages, fungi, blood-borne parasites and protozoa. Exemplary viruses include acquired immunodeficiency virus (HIV), hepatitis A, B and C viruses, Sindbis fever virus, cytomegalovirus, vesicular stomatitis virus, herpes simplex virus, Examples are types I and II, human retroviruses T-lymphotropic, HTLV-III, LAV / IDAV virus of lymphadenopathy, parvovirus, transfusion-transmitted viruses (TT), Epstein-Barr virus, and others known in the art. Bacteriophages include FX174, F6,?, R17, T4 and T2. Exemplary bacteria include P aeruginosa, $ aureus, S epidermidis, L monocytogenes, Coli, K pneumonia and S marcescens.

Inactivation of white blood cells may be desirable when the suppression of the immune or autoimmune response is sought, for example, in a process involving the transfusion of red cells, platelets or plasma when there may be white donor cells present. Materials that can be treated by the methods of this invention include any material that is adequately permeable to a photoradiation that provides sufficient light for viral inactivation, or that can be suspended or dissolved in fluids having sufficient permeability to photoradiation . Examples of such materials are whole blood and aqueous compositions containing biologically active proteins derived from blood or blood constituents. Packaged red cells from blood, platelets and plasma (fresh plasma or fresh frozen plasma) are examples of such blood constituents, In addition, therapeutic compositions of proteins containing blood-derived proteins, such as fluids that contain biologically active proteins useful for the treatment of medical conditions, for example, factor VIII, von Willebrand factor, factor IX, factor X, factor XI, factor of Hegeman, prothrombin, anti-thrombin III, fibronectin, plasminogen, protein fraction of the plasma, immune serum globulin, modified immune globulin, albumin, plasma growth hormone, plasminogen streptokinase plasmid, plasminogen, plasminogen, plasminogen, hemoglobin, aptitripsin and precalicrein, may be treated by the decontamination methods of the invention. Other fluids that could benefit from the treatment of this invention are the peritoneal solutions used for peritoneal dialysis, which are sometimes contaminated during connection, leading to peritoneal infections. The term "biologically active" means capable of effecting a change in a living organism or in a component thereof, "biologically active", with respect to "biologically active protein", as used herein, does not refer to proteins that be part of the microorganisms to inactivate. Similarly, "non-toxic", with respect to the photosensitizers, means low or no toxicity towards humans or towards other animals, and does not mean non-toxic for the microorganisms to be inactivated. "Substantial destruction" of the biological activity means at least as much destruction as may be caused by porphyrins and by derivatives, metabolites and porphyrin precursors which are known to have a harmful effect on the biologically active proteins and on the cells of humans and mammals . Similarly, "substantially non-toxic" means less toxic than porphyrins, derivatives, metabolites, and porphyria precursors known for blood sterilization. The term "blood product", as used herein, includes blood constituents and therapeutic protein compositions containing blood derived proteins, as defined above. Fluids containing biologically active proteins other than those derived from blood can also be treated by the methods of this invention. The methods of this invention using endogenous photosensitizers and derivatives d on endogenous photosensitizers do not substantially destroy the biological activity of the fluid components. different from microorganisms. As much biological activity is retained as possible from these components, although, in some instances, when the methods are optimized, some loss of biological activity can be balanced, for example, denaturation of protein components, in favor of an effective decontamination of the fluid. As long as the fluid components retain enough biological activity to be useful for their intended or natural purposes, their biological activities will not be considered "substantially destroyed". The photosensitizers useful in the present invention include any photosensitizer useful for inactivating microorganisms. A "photosensitizer" is defined as any compound that absorbs radiation of one or more defined wavelengths and subsequently uses the energy absorbed to carry out a chemical process. Examples of such photosensitizers include porphyrins, psoralens, stains such as neutral red, methylene blue, acridine, toluidines, flavin (acriflavine hydrochloride) and phenothiazine derivatives, coumarins, quinolones, quinones and antroquinones. The photosensitizers of this invention may include compounds which are preferably adsorbed to nucleic acids, then focusing their photodynamic effect on microorganisms and on viruses, with little or no effect on the cells or the accompanying proteins. Other photosensitizers are also useful in this invention, such as those using mechanisms that depend on atomic oxygen. Endogenous photosensitizers are especially preferred. The term "endogenous" means naturally found in the body of a human or a mammal, either as the result of a synthesis by the body or due to the ingestion of an essential food (for example, vitamins), or by the formation of metabolites and / or subprouts in vivo. Examples of such endogenous photosensitizers are alloxazines such as 7,8-dimethyl-10-ribityl isoalozazine (riboflavin), 7,8,10-trimethylisoaloxazine (lumiflavine), 7,8-dimethyloxazine (lumicromo), isoaloxazine - adenine dipucleotide (flavin adenine dinucleotide [FAD]), mononucleotide alloxazine (also known as flavin mononucleotide [FMN] and riboflavin -5-phosphate), vitamin K5, vitamin L, its metabolites and precursors, and naphthoquinones, naphthalenes, naphthols and their derivatives that have flat molecular conformations. The term "alloxazine" includes isoaloxazines. Derivatives based on endogenous photosensitizers include analogs and homologs derived synthetically from endogenous photosensitizers that may have or lack substituents of lower alkyl (1 to 5) or halogen of the photosensitizers from which they are derived, and which may preserve function and substantial non-toxicity thereof. When endogenous photosensitizers are used, particularly when such photosensitizers are not inherently toxic or do not provide toxic photoproducts after photoradiation, no removal or purification step is required after decontamination, and the treated product can be returned directly to the patient's body. or administer it to a patient who needs its therapeutic effect. The preferred endogenous photosensitizers are:, 8-dimethyl-10-ribityl isoaloxazine Isoaloxazine - adenine dinucleotide 'n, HOCH HOCH HOCH I Aloxazine mononucleotide VITAMIN K1 VITAMIN K2 VITAMIN OXIDE K1 NH2 VITAMIN K5 VITAMIN K-S (ll) NH2 VITAMIN K6 NH2 VITAMIN K7 VITAMIN L The method of this invention requires the mixture of the photosensitizer with the material to be decontaminated. The mixture can be made by simply adding the photosensitizer or a solution containing the photosensitizer to a fluid to be decontaminated. In one embodiment, the material to be decontaminated to which the photosensitizer is added is flowed through a photoradiation source, and the flow of the material generally provides sufficient turbulence to distribute the photosensitizer through the fluid to be decontaminated. In another embodiment, the fluid and the photosensitizer in a photopermeable container and irradiated in a batch mode, preferably while stirring the container to completely distribute the photosensitizer and to expose the fluid to the radiation. The amount of photosensitizer to be mixed with the fluid will be in an amount sufficient to adequately inactivate the microorganisms present therein, but less than a toxic amount (for humans or other mammals) or insoluble. As taught herein, the optimal concentrations of the desired photosensitizers can be readily determined by those trained in the art without undue experimentation. Preferably, the photosensitizer is used in a concentration of at least about 1 μM until the solubility of the photosensitizer in the fluid, and, preferably, of about 10 μM. Preferred for the 7,8-dimethyl-10-ribltyl isoaloxazine is a concentration range between about 1 μM and about 160 μM, preferably about 10 μM. The fluid containing the photosensitizer is exposed to the photoradiation of the appropriate wavelength to activate the photosensitizer, using a sufficient amount of photoradiation to activate the photosensitizer, as described above, but less than that which would cause non-specific damage to the biological components or that would substantially interfere with the biological activity of other proteins present in the fluid. The wavelength used will depend on the selected photosensitizer, as is known in the art, or it can be easily determined without undue experimentation in accordance with the teachings in this documentation. Preferably, the light source is a light source fluorescent or luminescent that provides light of about 300 nm to about 700 nm and, more preferably, about 340 nm to about 650 nm of radiation. Wavelengths in the range from ultraviolet to visible are useful in this invention. The source or light sources can provide light in the visible range, light in the ultraviolet range or, preferably, a mixture of light in the visible and ultraviolet ranges, more preferably in about half the spectrum in the visible and ultraviolet range. another half in the ultraviolet, although other relationships can also be used. One benefit of light mezcal is that the visible spectrum does not damage platelets but reduces the amount of ultraviolet light required, which is more damaging. The activated photosensitizer is able to inactivate the microorganisms present, in ways such as by interfering with their replication. The specificity of the action of the photosensitizer is conferred by the close proximity of the photosensitizer to the nucleic acid of the microorganism, and this may result from the binding of the photosensitizer to the nucleic acid. "Nucleic acid" includes ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Other photosensitizers can act by binding to cell membranes by other mechanisms. The photosensitizer can be directed to the microorganism to be inactivated by binding it covalently to an antibody, preferably to a specific monoclonal antibody against the microorganism. The fluid containing the photosensitizer can be poured into a photopermeable container for irradiation. The term "container" refers to a closed or open space, which can be made of a flexible or rigid material, for example, it can be a bag or a box. It may be closed or open at the top or may have openings at both ends, for example, it may be a pipe or a spout, to allow the flow of fluid therethrough. A cuvette has been used to exemplify an embodiment of this invention that involves a flow system. The collection bags, such as those used with the Trlma ™ Spectra ™ and apheresis systems of Cobe Laboratories, Inc, have been used to exemplify another embodiment involving batch treatment of the fluid. The term "photopermeable" means that the material of the container is suitably transparent to the photoradiation of the appropriate wavelength to activate the photosensitizer. In the flow system, the container has a depth (dimension measured in the direction of the radiation from the photoradiation source) sufficient to allow the photoradiation to properly penetrate the container so that it comes in contact with the molecules of the radiation. photosensitizer at all distances from the light source and ensure the inactivation of microorganisms in the fluid to be decontaminated, and a length (dimension in the direction of fluid flow) sufficient to ensure a sufficient exposure time of the fluid to the photoradiation . The materials for making such containers, the depths and the lengths of the containers can be easily determined by those trained in the art without undue experimentation following the teachings in this documentation, and together with the flow rate through the container, the intensity of the photoradiation and the absorptions of the components of the fluid, for example, plasma, platelets, red blood cells, will determine the amount of time that the fluid needs to be exposed to the photoradiation.

For 7,8-dimethyl-10-ribityl isoaloxazine, a preferred amount of radiation is between about 1 J / cm2 to about 120 J / cm2. In another embodiment involving a batch treatment, the fluid to be treated is placed in a photopermeable container that is agitated and exposed to the photoradiation for a sufficient time to substantially inactivate the microorganisms. The photopermeable container is preferably a blood bag made of transparent or semi-transparent plastic, and the agitation is preferably on a shaking table. The photosensitizer can be added to the container in the form of a liquid or a powder, and the container can be shaken to mix the photosensitizer with the fluid and adequately expose all the fluid to the photoradiation in order to ensure the inactivation of the microorganisms. The photosensitizer can be added or flowed in the photopermeable container separately from the fluid to be treated, or it can be added to the fluid before placing the fluid in the container. In one embodiment, the photosensitizer is added to the anticoagulant, and the mixture of photosensitizer and anticoagulant is added to the fluid. Breeders can also be added to the fluid to make the process more efficient and selective. Such enhancers include antioxidants or other agents that prevent damage to the desired components of the fluid or that improve the inactivation rate of the microorganisms. Examples of enhancers are adenine, histidine, cysteine, tyrosine, triprophane, ascorbate, N-acetyl-L-cysteine, propyl gallate, glutathione, mercaptopropionylglycine, dithiothrethol, nicotinamide, BHR, the BHA, ia .? lysine, serine, methionine, glucose, mannitol, trolox, glyceroi and mixtures thereof. This invention also comprises fluids comprising biologically active proteins, blood or blood constituents, and contain endogenous photosensitizers, derivatives based on endogenous photosensitizers or photoproducts thereof made by the method of claim 1. The fluid may also contain inactivated microorganisms. In addition to decontaminating whole blood, fluids containing blood products and biologically active proteins, this method is useful for treating other fluids including fluids that are used for the nutrition of humans or animals, such as like water, fruits, juices, milk, drinks, soups and the like. The method is also useful for treating peritoneal or parenteral solutions. This invention also includes methods for treating surfaces for the purpose of inactivating microorganisms that may be present therein, comprising applying to said surfaces a non-toxic amount effective for inactivation of an endogenous photosepsibilizer or a derivative based on endogenous photosensitizers, and exposing the surface to the photoradiation sufficient to activate the photosensitizer. The surface can be the surface of a fruit, a vegetable or an animal rest, the surface or the surfaces of meals cut or processed. The particulate materials such as ground meats can be treated by mixing the photosensitizer with the material and continuing the mixing while irradiating, so as to expose fresh surfaces to the photoradiation.

The surface may alternatively be the surface of a food preparation such as a back cover or a storage shelf, or it may be the surface of a bath or wash container, such as a kitchen sink, of a bath tub. or a lavatory, or a swimming pool or similar. In addition, the surface may be the surface of an animal or a living plant, or it may be the surface of a wound. The photosepsibilizer can also be applied in a suitable vehicle, such as water, or in a solution containing other treatment aggregates, by spraying, dipping, rubbing or using other means known in the art. The amount of photosensitizer and the photoradiation energy required for the treatment will be readily determined by someone skilled in the art without undue experimentation, according to the level of contamination and the material to be treated. This invention also provides a method for treating a fluid or other material as indicated above so as to inactivate microorganisms that may be present therein, which comprises adding a non-toxic amount effective for inactivation of vitamin K5 to said fluid or other material. . Preferably, but not necessarily, the fluid or other material is irradiated to improve the inactivation of the microorganisms. In some cases, the use of inactivation by vitamin K5 occurs under ambient light or in the dark, as described in the Examples below. Fluids containing red blood cells are preferred for treatment with vitamin K5 in the absence of a photoradiation step. Compound K5 can also cover surfaces such as pipe equipment for dialysis of blood or peritoneal, in order to ensure sterile connections and sterile approaches. In the decontamination systems of this invention, the photoradiation source may be connected to the photopermeable container of the fluid by means of a light guide such as a light channel or a fiber optic tube which prevents the scattering of light between the source and the fluid container and, more importantly, prevent substantial heating of the fluid within the container. Direct exposure to the light source can increase temperatures as much as 10 to 15 ° C, especially when the amount of fluid exposed to light is small, which can cause the denaturation of the components. The use of the light guide maintains any heating at less than 2 ° C. The method may also include the use of temperature sensors and cooling mechanisms, when necessary, to maintain the temperature below the temperatures at which the desired proteins in the fluid are damaged. Preferably, the temperature will be maintained between about 0 ° C and about 45 ° C, more preferably, between about 4 ° C and about 37 ° C, and, more preferably, about 22 ° C. This invention also provides a system for treating a fluid so as to inactivate microorganisms that may be present therein, comprising: (a) A container comprising said fluid and an endogenous photosensitizer or a derivative based on endogenous photosensitizers, said container equipped with inlet means and having a photopermeable surface sufficient to allow exposure of the fluid therein to the amount of photoradiation sufficient to activate the photosensitizer, (b) At least one source of photoradiation that provides sufficient photoradiation to the fluid in said container, of a type and in a selected amount to activate the photosensitizer, the microorganisms present being substantially inactivated. The photoradiation source can be a source of visible radiation or ultraviolet radiation, or both. Preferably sources of visible and ultraviolet radiation are provided, and, preferably, the photoradiation has a ratio of about half the ultraviolet radiation and the other half visible radiation, although other ratios may be used. The photoradiation in both the visible and ultraviolet spectra can be supplied concurrently or sequentially, with the visible portion being preferably supplied first. The photoradiation source can be a simple lamp or it can consist of multiple lamps that radiate at different wavelengths. The photoradiation source should be able to deliver between about 1 and at least about 120 J / cm2. It is especially preferred to use ultraviolet and visible lights mixed together when the photosensitizer is one that loses its ability to absorb visible light after a period of exposure, such as 7,8-dimethyl-10-ribityl-isoaloxazine. Any means known in the art can be used to add the photosensitizer to the fluid to be decontaminated and to place the fluid in the photopermeable container, typically including flow conduits, ports, reservoirs, valves and the like. Preferably, the system includes a means, such as pumps or adjustable valves, for controlling the flow of photosensitizer in the fluid to be decontaminated so that its concentration can be controlled at effective levels as described above. In one embodiment, the photosensitizer is mixed with the anticoagulant that is delivered in a blood apheresis system. For endogenous photosensitizers and derivatives having sugar portions, the pH of the solution is preferably kept low enough, as is known in the art, to avoid separation of the sugar portion. Preferably, the photosensitizer is added to the fluid to be decontaminated in an aqueous solution previously mixed, for example, in a water solution or storage buffer. The poutopermeable container for the flow system can be a transparent cuvette made of polycarbonate, glass, quartz, polyvinyl chloride, polyolefin or other transparent material. The cuvette can be contained in a radiation chamber with mirrored walls. A photoradiation enhancer such as a second photoradiation source or a reflective surface in a position adjacent to the cuvette can be placed to increase the amount of photoradiation that comes into contact with the fluid within the cuvette. The system preferably includes a pump for adjusting the flow rate of the fluid to the desired levels in order to ensure the substantial decontamination described above. The cuvette has a length, coordinated with the flow rate along it, sufficient to expose the fluid in it to the sufficient amount of photoradiation in order to effect substantial decontamination thereof.

Also preferably, the cuvette is placed away from the light source, at a sufficient distance so that the heating of the fluid in the cuvette does not occur and that the light is transmitted from the light source in the cuvette by means of a guide light. In another embodiment, the fluid is placed in a photopermeable container such as a blood bag, for example, used with the apheresis system described in US Patent No. 5653887, and is agitated while being exposed to the photoradiation. Suitable bags include the collection bags described in this documentation. The collection bags used in the Spectra ™ system or in the Trima ™ apheresis system from Cobe Laboratories, Inc are especially suitable. Stirring tables are known in the art, for example, as described in US Patent No. 4880788. The bag is equipped with at least one port for adding fluid therein. In one embodiment, the photosensitizer, preferably 7,8 * dimethyl-10-ribytyl-isoaloxazine, is added to the fluid-filled bag in the form of a powder. The bag is then placed on the shaker table or shaken under photoradiation until all the fluid has been substantially exposed to the photoradiation. Alternatively, the bag can be pre-packaged with the powder photosensitizer contained therein. The fluid to be decontaminated can then be added through the appropriate port. The decontamination systems described above can be designed as individual units or can be easily incorporated into existing apparatuses known in the art to separate or treat blood that is being withdrawn or administered to a patient. For example, such blood handling apparatuses include the COBE Spectra ™ or TRIMA® apheresis systems, available from Cobe Laboratories, Inc., Lakewood, CO, or the apparatus described in US Patent No. 5653887 and in the US Patent No. US Serial No. 08/924519, filed September 5, 1997 (PCT Publication No. WO 99/11305) of Cobe Laboratories, Ipc, as well as apheresis systems of other manufacturers. The decontamination system can be inserted just downstream from the point at which the blood is withdrawn from the patient or donor, just before the insertion of the blood product into the patient, or at any point before or after the separation of the constituents. The photosensitizer is added to the blood components together with the anticoagulant in a preferred embodiment, and separate sources of irradiation and cuvettes are placed downstream from the collection points for platelets, for plasma and for red blood cells. The use of three different blood decontamination systems is preferred over the use of a single decontamination system upstream of the blood separation vessel of an apheresis system since the lower rates of looseness in the lines of the separate components they allow a greater facility of irradiation. In other embodiments, the decontamination systems of this invention can be used to process previously collected and stored blood products. When all the red blood cells are present in the fluid to be treated, as those skilled in the art will appreciate, to compensate for the absorption of light by the cells, the fluid can be refined, exposed to high radiation energies for periods longer or present to photoradiation in containers or in shallower ducts than those needed for use with other blood components.

The endogenous photosensitizers and derivatives based on endogenous photosensitizers described in this documentation can be used in decontamination systems of pre-existing blood components as well as in the decontamination system described in this documentation. For example, the endogenous photosensitizers and derivatives based on endogenous photosensitizers of this invention can be used in the decontamination systems described in US Patent Nos. 5290221, 5536238, 5290221 and 5536238. The platelet aggregate solutions comprising the photosensitizers endogenous and derivatives based on endogenous photosensitizers that were described above are also provided in this documentation. Platelet aggregate solutions known in the art can be used for this purpose and include those described in US Patent Nos. 5908742, 5482828, 5569579, 5236716, 5089146 and 5459030. Such solutions of platelet aggregates can contain physiological saline , buffer, preferably sodium phosphate, and other components including magnesium chloride and sodium gluconate. The pH of such solutions is preferably between about 7.0 and 7.4. These solutions are useful as vehicles for platelet concentrates that allow the maintenance of the quality and metabolism of the cells during storage, reduce the plasma content and extend the storage duration. The photosensitizer may be present in such solutions in any desired concentration from about 1 μM to the solubility of the photosensitizer in the solution, and, preferably, between about 10 μM and about 100 μM, more preferably, about 10 μM. In a preferred embodiment, the platelet aggregate solution also comprises enhancers as described above. A preferred solution of platelet aggregates comprises sodium acetate, sodium chloride, sodium gluconate, magnesium chloride 1.5 mM, sodium phosphate 1 mM, 7,8-dimethyl-10-ribitylyl isoaloxazine 14 μM, and preferably also, 6 mM ascorbate. BRIEF DESCRIPTION OF THE FIGURES Figure 1 illustrates the absorption spectrum of riboflavin. Figure 2 illustrates a correlation between light absorption and hematocrit observed and predicted for red blood cells, and predicted for platelets. Figure 3 illustrates the photodecomposition over time of riboflavin in an anti-swallowing solution of Dextrose Acid Citrate (ACD). The solid line with circles indicates the percentage of initial riboflavin remaining at 373 nm. The dotted line with squares indicates the percentage of initial riboflavin remaining at 447 nm. Figure 4 illustrates the transmission profile of several plastic cuvettes as a function of wavelength. The solid line represents a 3.2 mm acrylic tray. The dotted line (-) represents a 3.2 mm UV acrylic cuvette. The striped line (-) represents a 3.2 mm polystyrene (PS) cuvette, and the crossed line indicates a 3.2 mm polycarbonate (PC) cuvette. Figure 5 illustrates the required light flux in mW per cm2 as a function of the flow rate, that is, the flow required to deliver a joule / cm2 to a sample in the cuvette.

Figure 6 illustrates a blood separation apparatus incorporating the photoradiation device of the present invention. Figure 7 illustrates the decontamination assembly of the present invention. Figure 8 illustrates the inactivation of bacteria in platelet preparations using vitamin K5 as the photosensitizer as a function of the irradiation energy. Figure 9 illustrates the inactivation of bacteria as a function of platelet preparation and irradiation energy, using 90% platelets and 10% platelet aggregate solution (90: 10), and 30% of platelets with 70% aggregate solution (30: 70). Figure 10 shows the effect on the inactivation of a virus, a bacteriophage and a bacterium from the addition of antioxidants to the platelet concentrate. Figure 11 shows the inactivation curve for the herpes simplex virus type II as a function of the concentration of photosensitizer at an irradiation energy of 20 J / cm2 using half the ultraviolet light and the other half visible light. Figure 12 shows the inactivation of S epidermidis at varying concentrations of photosensitizer and at different irradiation energies. Figure 13 shows the inactivation of FX174 at various concentrations of photosensitizer and at different irradiation energies. Figure 14 shows the inactivation of S aureus and FX174 at various irradiation energies using a 50:50 mixture of ultraviolet light and visible light.

Figure 15 shows the inactivation of S epidermidis and HSV-II at various irradiation energies using a 50:50 mixture of ultraviolet light and visible light. Figure 16 shows the inactivation of HSV2 virus in agitated and irradiated blood bags at different energy levels. Figure 17 compares the results of the inactivation of several smallpox viruses in various fluids using ultraviolet light alone or 50"50 ultraviolet light and visible light Figure 18 compares the inactivation results with and without a smallpox virus sensitizer. Several times of irradiation Figure 19 compares the results of the inactivation of extracellular HIV-1 at 5 and at 50 μM of photosensitizer and at different irradiation energies Figure 20 compares the results of intracellular HIV-1 inactivation at 5 and 50 μM of photosensitizer and at different irradiation energies Figure 21 compares the results of the inactivation of intracellular HIV-1 to 5 and 50 μM of photosensitizer and at different irradiation energies, using levels of p24 antigen Figure 22 shows the inactivation of HSV-II at various levels of irradiation using platelet concentrates and platelet concentrates in media containing solution n platelet aggregate ascorbate. Figure 23 shows an embodiment of this invention that employs a blood bag to contain the fluid to be treated and a photosensitizer, and a shaker table to agitate the fluid while it is exposed to the photoradiation of a light source. DETAILED DESCRIPTION The decontamination method of this invention using endogenous photosensitizers and derivatives based on endogenous photosensitizers is exemplified herein using 7,8-dimethyl-10-ribityl-isoaloxazine. However, any photosensitizer that is capable of being activated by photoradiation and causes the inactivation of microorganisms can be used. The photosensitizer must be one that does not destroy the desired components of the fluid to be decontaminated, and that also, preferably, is not destroyed as a result of photoradiation, resulting in products that significantly destroy the desired components or have significant toxicity. The wavelength at which the photosensitizer is activated is determined as described in this documentation, using literature sources or direct measurements. Its solubility in the fluid to be decontaminated or in a combination of vehicle fluid and fluid to be decontaminated is determined in the same way. The ability of the photoradiation to the activating wavelength to penetrate the fluid to be decontaminated should also be determined as indicated in this documentation. The appropriate temperatures for the reaction of the photosensitizer with its substrate are determined, as well as the temperature ranges, the intensity and the duration of the photoradiation, and the concentration of the photosensitizer, so as to optimize the microbial inactivation and minimize the damage to the photosensitizer. the desired proteins and / or cellular components in the fluid. Examples 1 to 7 and Figures 1 to 5 illustrate the determination of information required to develop a flow decontamination system of the present invention. One of the requirements of such a system for flow systems has been determined. The devices must be designed to provide the correct flow rates, photopermeabilities and light intensities to cause inactivation of the microorganisms present in the fluid, as indicated in this documentation. The fluid to be decontaminated is mixed with photosensitizer and then irradiated with a sufficient amoof photoradiation to activate the photosensitizer so that it reacts with the microorganisms in the fluid and these are inactivated. The amoof photoradiation that reaches the microorganisms in the fluid is controlled through the selection of an appropriate source of photoradiation, of an appropriate distance between the photoradiation source and the fluid to be decontaminated, which can be increased through the use of light guides to bring the photoradiation directly to the fluid container, of a photopermeable material suitable for the fluid container, an appropriate depth to allow a complete penetration of the photoradiation in the container, of photoradioration enhancers such as one or more additional sources of photoradiation, preferably on the side opposite the container of the first, or reflectors that reflect the light of the source of radiation. radiation back to the container, of appropriate flow rates for the fluid in the container, and of an appropriate length of container to allow sufficient time for the inactivation of the microorganisms present to occur. Temperature monitors and controllers may also be required to maintain the fluid at an optimum temperature. Figure 6 illustrates a decontamination system of this invention as part of an apparatus for separating blood components, and Figure 7 provides details of a preferred decontamination system. For batch systems, it is preferable to place the fluid to be decontaminated together with the photosensitizer in bags that are photopermeable or, at least, sufficiently photopermeable to allow enough radiation to reach their contents and activate the photosensitizer. Sufficient photosensitizer is added to each bag to provide inactivation, preferably to provide a photosensitizer concentration of at least 10 μM, and the bag is shaken while irradiating, preferably at about 1 to about 120 J / cm2 per a period of between about 6 and about 36 minutes so as to ensure exposure of substantially all the fluid to the radiation. Preferably, a combination of visible light and ultraviolet light is used concurrently. The photosensitizer can be added in powder form. The method preferably uses endogenous photosensitizers, including endogenous photosensitizers that work by interfering with the replication of nucleic acids. 7,8-Dimethyl-10-ribityl-soaloxazine is the preferred photosensitizer for use in this invention. The chemical reaction that is believed to occur between 7,8-dimethyl-10-ribityl isoaloxazine and nucleic acids does not proceed by means of an atomic oxygen-dependent process (ie, by means of a Type II mechanism), but rather by rather, it does so through direct interactions between sensitizer and substrate (Type I mechanisms). Cadet et al (1983) J Chem, 23: 420-429, clearly demonstrate that the effects of 7,8-dimethyl-10-ribityl isoaloxazine are not due to oxidation by atomic oxygen of the guanosine residues. In addition, adenosine bases appear to be sensitive to the effects of 7,8-dimethyl-10-ribityl isoaloxazine plus UV light. This is important since adenosine residues are relatively insensitive to processes that depend on atomic oxygen. 7,8-Dimethyl-10-ribityl isoaloxazine does not appear to produce large amounts of atomic oxygen when exposed to UV light, but rather exerts its effects through direct interactions with the substrate (eg, nucleic acids) by means of electron transfer reactions with sensitizer species in the excited state. Since the indiscriminate damage to cells and proteins arises primarily from sources of atomic oxygen, this mechanistic pathway for the action of 7,8-dimethyl-10-ribityl isoaloxazine allows a greater selectivity in its action than in the case of compounds such as psoralen, which possess a significantly type II chemistry, Figure 6 shows a blood device apparatus and an afresh system incorporating the photoradiation devices of the present invention. Whole blood is removed from a patient / donor 4 is provided to an apheresis system or a blood component separation device 8, where the blood is separated into the various types of components and at least one of these types of components is removed from the device 8. These blood components can then be provided for subsequent use for another or they can undergo therapeutic treatment and be returned to the donor / patient 4.

In the device for separating blood components 8, the blood is withdrawn from the patient / donor 4 and is directed through a circuit of extracorporeal tubes 10 and a blood processing vessel 12, defining a completely closed system and sterile. The blood component separation device 8 is connected to a pump (not shown). Blood flows from donor / patient 4 through the extracorporeal tube circuit 10 into the rotary blood processing vessel 12, Blood within the blood processing vessel 12 separates into the various types of blood components , and these types of components (platelets, plasma, red blood cells) are constantly removed from the blood processing vessel 12. Blood components that are not being retained for collection or for therapeutic treatment (eg , red blood cells, white blood cells, plasma) are also removed from the blood processing vessel 12 and returned to the donor / patient 4 cia the extracorporeal tube circuit 10. The operation of the blood vessel of the Blood is preferably controlled by one or more computer processors included in this documentation. The extracorporeal tube circuit 10 comprises a cassette assembly 14 and a number of tube assemblies 20, 50, 60, 80, 90, 100 connected together. The blood removal / return assembly 20 provides a unique needle interface between the donor / patient 4 and the cassette assembly 14, and the blood / blood components sub-assembly 60 provides the interface between the cassette assembly 14 and the blood processing vessel 12. An assembly of anticoagulant tubes 50, an assembly of plate collector tubes 80, an assembly of plasma collection tubes 90, an assembly of collection tubes are also connected to the cassette assembly 14. of red cells 70 and a subassembly of ventilation tubes of the bag 100. The blood removal / return assembly 20 includes a needle subassembly 30 connected therewith and an anticoagulant tube system 26 which connects it to the tube assembly anticoagulants 50 through the cassette assembly 14, the cassette assembly 14 includes molded plastic plates anterior and posterior which are welded together to define a rectangular cassette member having integral fluid passageways. The cassette assembly 14 also includes a number of external tube loops that interconnect several integral channels. The integral tracks are also interconnected to the various tube assemblies. Specifically, the cassette assembly 14 connects to a system of anticoagulant tubes 26 of the blood removal / return assembly 20 and to the assembly of anticoagulant tubes 50. The anticoagulant tube assembly 50 includes a needle drip chamber 52 connectable to the source of anticoagulant and photosensitizer 53, and with a sterilizing filter 56. During use, the assembly of anticoagulant tubes 50 supplies the anticoagulant mixed with the photosensitizer to the blood withdrawn from the donor / patient 4 to reduce or prevent any coagulation in the extracorporeal tube circuit 10. Many anticoagulants are known in the art, for example, those described in Chapter 3 of the AABB Technical Manual, 11th edition, 1993, including ACD-A, CPD, CP2D, CPDA-1 and heparin. These, as well as the cell storage solutions, AS-1, AS-3 and AS-5, are all compatible with the endogenous photosensitizers and the derivatives based on endogenous photosensitizers described in this documentation. The cassette assembly 14 also includes a connection to the blood removal / return assembly 20. Blood passes through the pressure sensors and an internal filter in the cassette assembly 14 and, thus, towards the inlet pipe system 62. The inlet pipe system 62 is also connected to the blood processing vessel 12 in order to provide it with whole blood for its operation. In order to return the separated blood components to the cassette assembly 14, the blood / blood component 60 input assembly further includes an output tube circuit for red blood cells (RBC) / plasma, for platelets and for plasma connected to the corresponding output ports. , ßQ. the blood processing vessel of \ SL. 12. The output tube circuit for red blood cells (RBC) / plasma channels the separate components of red blood cells (RBC) / plasma through! assembly of cassettes 14 up to the assembly of red cell collection tubes 70 through a first decontamination system 72. The circuit of exit tubes for platelets channels the separated platelets through the assembly of cassettes 14 up to the assembly of collection tubes platelets 80 through a second decontamination system 82. The outlet tube circuit for ? _ plasma channels the separated plasma through the assembly of cassettes 14 to the assembly of plasma collecting tubes 90 through a third decontamination system 92. After irradiation in decontamination systems 72, 82 and 92, to activate the photosensitizer and inactivating the microorganisms present, the blood components are collected in the red cell collection bag 74, in the platelet collection bag 84 and in the plasma collection bag 94. The ventilation bag 104 can be used to ventilate the gases within the system. Figure 7 illustrates an individual version of the decontamination assembly of this invention. The product of blood 180 (which can be freshly collected blood, blood components or stored blood) is collected in the blood product line 186, which leads through a pump 184 to a decontamination cuvette 164. The photosensitizer reservoir 166 is connected to the photosensitizer input line 168 equipped with an input pump 170, and leads to the blood product line 186 upstream of the decontamination cuvette 164. The decontamination cuvette 164 is a cuvette depth photopermeable (d) and length (I) selected to ensure decontamination. The cooling system 190, combined with the temperature monitor 192, is connected to the decontamination cuvette 164 to control the temperature of the fluid. The decontamination cuvette 164 is connected via a light guide 162 to a photoradiation source 160. A photoradio enhancer 163 is placed adjacent to (either in contact with or separate from) the decontamination cuvette 164 to increase the amount of Photo that reaches the product of the blood in the bucket.

The decontaminated blood product line 188 leads from the decontamination cuvette 164 to the collection of decontaminated blood products 182. In the operation, the blood product 180 is led to the blood product line 186 where it is stored. meets with the photosensitizer of the photosensitizer reservoir 166 flowing at a rate controlled by a photosensitizer input pump 170 at the photosensitizer entry line 68, which meets the blood product line 186. The flow rate in the blood product line 186 is controlled by pump 184 at a selected rate to ensure decontamination in decontamination cuvette 164. The temperature monitor checks the temperature in decontamination cuvette 164 and controls the cooling system 190 , which maintains the temperature in the cuvette within a range required for its optimal operation. The product of the blood in the decontamination cuvette 164 is irradiated by the photoradiation source 160 and conducted in the light guide 162. The photoradiation source can in fact comprise two or more lights. The arrows indicate the photoradiation from one end of the light guide 162 which propagates in the blood product inside the clear decontamination cuvette 164. Adjacent to the decontamination cuvette 164 is a photoradiation enhancer 163, which can be an additional source of photoradiation or a reflective surface. The arrows from the photoradiator enhancer 163 pointing to the decontamination cuvette 164 indicate the photoradiation from the photoradiator enhancer 163 which shines in the blood product in the decontamination cuvette 164. The decontaminated product from the blood it leaves the decontamination cuvette 164 via the decontaminated blood product line 188 and is collected in the collection of blood decontaminated products 182. In an embodiment employing 7,8-dimethyl-10-ribityl isoaloxazine from Sigma Chemical Co as the photosensitizer, a light guide is used from EFOS Corporation, Williamsville, NY composed of optical fibers. The system is capable of delivering a focused beam of light with an intensity of 6200 mW / cm2 in the region of 355 to 380 nm. It is also possible to use interchangeable filters with the system to achieve outputs of 4700 mW / cm2 in the spectral region from 400 to 500 nm. In both cases, the light output in the region of 320 nm is minor and negligible. Several light guides of different dimensions (3m 5 and 8mm) are available for this system. The light comes out from the tip of the light guide with a dispersion of 21 degrees. The 8 mm light guide is suitable, if placed correctly, to adequately illuminate the face of the preferred decontamination cuvette, which is a common cuvette used in disposable Cobe Spectra® assemblies from Industrial Plastics, Forest Grove, OR: The flow velocity is variable and is determined by the amount of light energy that is intended to be delivered to the sample. The flow rate is controlled by means of a peristaltic pump from the Colé - Parmer Instrument Company, Vernon Hills, IL. The flow rates and the type of input current can be controlled by means of a computer processor, as is known in the art.

Figure 23 illustrates an embodiment of the present invention in which the fluid to be decontaminated is placed in a blood bag 284 equipped with an inlet port 282, through which the photosensitizer powder of 284 is added from the bottle 286 via the outlet 288. The shaker table 280 is activated to agitate the blood bag 284 and thus dissolve the photosensitizer 290, while activating the photoradiation source 260 to irradiate the fluid and the photosensitizer in the blood bag 284. As an alternative , the bag can be provided pre-packaged with photosensitizer, then the fluid is added to the bag. The methods of the present invention do not require the use of improvers such as "setters" or oxygen scavengers. However, these can be used to improve the process in terms of reducing the extent of chemical reactions that damage non-specific cells or proteins, or to improve the rate of inactivation of pathogens. Other preferred methods using endogenous non-toxic photosensitizers and derivatives based on endogenous photosensitizers do not require the removal of photosensitizers from the fluid after photoradiation. The results of the tests show little or no damage to other components of the blood, for example, the platelets remain biologically active five days after treatment. EXAMPLES Example 1: Absorption profile of 7.8-dimethyl-10-ribidyl isoaloxazipa A sample of 7,8-dimethyl-10-ribityl isoaloxazine (98% purity) was obtained from Sigma Chemical Company. A portion of this sample was subjected to analysis using a UV explorer spectrophotometer. The range analyzed covered the region from 200 to 900 nm. For the analysis, the sample was dissolved in distilled water. In Figure 1 a sample spectrum of this analysis is shown. The results were compatible with those that appear in the literature for the maximum absorption values and the extinction coefficients for 7,8-dimethyl-10-ribityl-isoaloxazine.

The wavelengths suitable for irradiation are 373 and 445 nm. The extinction coefficients observed in these maximum absorption values are sufficient to ensure adequate activation of the sensitizer in the solution. Example 2. Solubility of 7.8-dimethyl-10-ribityl isoaloxazine Solubility in a medium of Isolvte S. PH 7.4 The maximum solubility of 7,8-dimethyl-10-ribityl-soaloxazine in an Isolyte S medium was determined as follows : 7,8-D-methyl-10-ribityl isoaloxazine was mixed with Isolyte S until a precipitate formed. The mixture was stirred at room temperature for one hour and vortexed to ensure complete dissolution of the suspended material. Additional 7,8-dimethyl-10-ribityl isoaloxazine was added until a solid suspension remained despite vortex mixing additional. This suspension was then centrifuged to remove the undissolved material. The supernatant of this preparation was removed and analyzed using a spectrophotometer. The absorption values of the solution were determined at 447 nm and 373 nm. From the extinction coefficients that had been previously determined, it was possible to estimate the concentration of the saturated solution. Concentration (373) = 100 μM = 42 μg / ml Concentration (447) = 109 μM = 40.9 μg / ml Solubility in anticoaqulant ACD-A The same procedure previously described was repeated using an anticoagulant. The values obtained from these measurements were the following: Concentration (373) = 166 μM = 63 μg / ML Concentration (447) = 160 μM = 60.3 μg / ML The values obtained from these studies indicate an upper limit of solubility of the compound that could be expected. Example 3. Photodecomposition of 7.8-dimetiM 0-ribityl-soaloxazine in aqueous media A solution of 7,8-dimethyl-10-ribityl isoaloxazine was prepared in Sigma ACD-A at a concentration of 63 μg / ml. This preparation was collected in a glass pipette and placed in the path of a UV light source (365 nm? Max with filters to remove light below 320 nm). The suspension was irradiated during specific intervals in which aliquots were removed for spectroscopic analysis. The absorption of dissolved 7, 8-dimethyl-10-ribityl-soaloxazine was monitored at 373 nm and 447 nm in each interval of weather. The results are shown in Figure 3 and Table 1. Table 1. Photodecomposition of 7,8-dimethyl-10-ribityl isoaloxazine before the The absorption profile for the solution at 373 nm indicates that no significant decomposition of the reagent occurred during the entire irradiation period. The absorption of light at this wavelength corresponds to electronic transitions n-p *. The absence of a decrease in the intensity of this peak over time indicates that the ring structure of the molecule is intact despite prolonged irradiation under these conditions. The absorption of the molecule at 447 n is due to the electronic state transitions p-p *. The decrease in the absorption of the molecule at this wavelength with higher irradiation times is indicative of the subtle alterations in the resonance structure of the molecule. This change is most likely due to the loss of ribose from the ring structure of the main structure of 7,8-dimeti! isoaloxazine and the formation of 7,8-dimethyl isoaloxazine as a result. These changes are compatible with reports in the literature regarding the behavior of the molecule before irradiation with UV light. The apparent lack of decomposition of the ring structure of the molecule is in stark contrast to observations with compounds in base to psoralen in similar conditions. During the irradiation, a significant fluorescence of the molecule in solution was observed. This behavior of the molecule is compatible with the resonance characteristics of the ring structure and provides means for the dissipation of the energy in the excited state molecule in a non-destructive way. Example 4. Evaluation of the flow system concept Transmission properties of the existing Spectra tank The existing Spectra tank is made of polycarbonate. The light transmission properties of this cuvette were measured at 373 and 447 n, placing the cuvette in the light path of a UV spectrophotometer. The values obtained were the following: Wavelength light% of transmittance 373 nm 66% 447 nm 80% These results are compatible with those indicated in the literature for polycarbonate plastics (see Figure 4). The values of the literature indicate a pronounced projection for the transmission of light through polycarbonates in the 300 nm region. For the region above 350 nm, the properties of light transmission were adequate for this case. Light flux requirements calculated as a function of flow rates For a flow system to be viable, the sample must have adequate light flux during its presence in the path of the beam. If the proposed Spectra cuvette is appropriate for this purpose, then it is possible to estimate the light flow requirements as a function of flow rates through the cuvette as follows: the volume of the solution present in the irradiation zone of the cuvette is approximately 0.375 ml. The transit time for a cell in this region of the cuvette can be determined from the following equation: Cuvette volume (ml) T = flow rate (ml / min) At 100 ml per minute, the transit time (T ) would be 0.00375 min = 0.225 seconds. The energy to which the sample is exposed depends on the flow according to the following equation: Energy (E, Joules / cm2) = Flow (, mW / cm2) * Time (T. sec) 100 If we consider that 1 Joule / cm2 is needed to activate the sensitizer properly and the transit time is (T) of 0.22 seconds (ie flow rate of 100 ml / min through the cuvette), then the Flow required during the transit of the sample through the bucket is 4,545 mW / cm2. In Figure 5 a graph is provided showing the ratio of the required flow from the light source to the flow rates through the cuvette. These results indicate that for a flow system to work properly, UV sources with outputs in the watts / cm2 region are necessary.

Figure 2 shows how the absorption should vary with the concentration of platelets. Example 5. Absorption of red blood cells In order to evaluate the extent to which UV light can penetrate a sample of red blood cells and the effects of the thickness of the sample and hematocrit on the extent of light penetration were carried out Several preliminary experiments using chemical actinometry, a method to determine the actual amount of light intensity emanating from a source by measuring the capacity and extent to which the absorbed light can perform a chemical reaction. For these studies, a ferrioxalate solution was used in order to measure the intensity of the relative source with that observed for water. The details of the chemical reaction as well as the methods used for the preparation of the sample are taught in Gordop, AJ and Ford, RA (1972) "The Chemist's Companion: A Handbook of Practical Data, Techniques and References" (Joh Wiley & Sons), pages 362 to 368. Samples of iron (III) oxalate were prepared in the test material (water or blood in different hematocrits of red blood cells) at a concentration of 0.15 M. Then these samples were loaded in a cuvette. Spectra and were placed in the irradiation assembly. Samples were exposed for pre-determined time intervals corresponding to the desired energy dose level (1 J / cm2). The samples were then removed and the amount of conversion of Fe3 + to Fe2 + was determined by reading the absorption of the test article in a solution of 1.10-phenanthroline at 510 nm, as described in Gordon, AJ and Ford, RA, supra. . The higher absorption values are indicative of a greater penetration of light in the sample. The observed absorption value for water after exposure to 1 J / cm2 of UV radiation was used as the transmittance level of 100%. All values of red blood cell samples were determined in relation to this pattern. Table 2. Absorption readings after exposure of the samples at 1 J / cm2 of UV light. All the average values represent the average of six experiments. The% transmittance values were calculated with respect to the water samples.

Using these values, it is possible to calculate the penetration depth of UV light using Beer's law (A = eb C): From Lambert's law, Absorption = logarithm (1 / transmittance) If we allow the concentration ( C) is equal to the hematocrit of the sample, and as b = 0.3 cm (the length of the path of the Spectra cuvette), it is possible to determine a pseudo-extinction coefficient for the samples (e ') by plotting the values of absorption for the red blood cell samples versus the product of the hematocrit by the length of the trajectory. The extinction coefficient for the samples is represented by the inclination of this line. Table 3. Determination of the extinction coefficient for red blood cell samples Using the obtained values as previously described, it was with 7,8-dimethyl-10-ribityl isoaloxazine in combination with UV light. Several in vitro parameters were used as controllers of platelet function in order to determine the extent of the changes induced by the treatment conditions. Factors such as the UV light exposure energy level, the dose of 7, 7-dimetiM 0-ribityl isoaloxazine used and the conditions of sample processing were examined for the impact on post-platelet quality treatment. The results obtained in this study were used to establish an appropriate treatment window for the inactivation of HIV-1 without compromising the function of the platelets. The samples were prepared with three different concentrations of 7,8-dimethyl-10-ribityl isoaloxazine. Platelets obtained from a common LRS Spectra collection were used for these studies. The initial samples were centrifuged to concentrate the platelet pellet. The precipitate was resuspended in a 70:30 solution (Isolyte S Medium, pH 7.4; Me Graw, Inc: Plasma). In the plasma mixture: medium was present 7, 8-dimethyl-10-ribityl isoaloxazine in the specific concentration. The platelet suspension then passed through a UV irradiation chamber in one of the three specified flow rates. The flow rates were correlated with the exposure energy level for the cell / media mixture passing through the irradiation chamber. After flowing through the irradiation chamber, the samples were stored in a citrated plasticized sampling bag for subsequent analysis. After irradiation, in vitro measurements of platelet function, including hypotonic shock response (HSR), GMP-140 expression, pH, pCO2l? O2) platelet swirl and cell count, were evaluated to determine the effects of the treatment protocol on the quality of the cells. The quality of platelets was monitored as a function of irradiation conditions (sensitizer concentration and levels of flow / energy indices). The platelet quality includes parameters such as, for example, HSR response, GMP-140 activation, etc. The flow rates of the studies can be related to the energy of the exposure as follows: Transit time (T, sec) = Exposure time = 0.375 ml (Fr / 60) Fr = flow index (ml / min) 0.375 ml = volume of cuvette (ml) T (sec) = 22 Fr Flow (f.mW/cm2)*T(seg) Energy (Joules / cm2) = 1000 f * 0.022 E = Ff The effect of UV exposure and the concentration of 7,8-dimethyl-10-ribityl isoaloxazine was evaluated on the stability and feasibility of platelets It is possible to determine a pseudo-extinction coefficient for these samples at 0.08661. The value for the extinction coefficient allows the calculation of the penetration distance of UV light in the red blood cell samples as a function of the hematocrit of the sample. For this estimation, the depth of penetration of the sample in which 90% of the incident light is absorbed was determined using the following equation: A = e b C A-1 (90% absorption of incident light); e = 0.08661; C = sample hematocrit; b - path length. The values determined using actinometry were compared with the values previously calculated using the estimated figures of the spectrophotometric IV measurements of light absorption in the red blood cell and platelet samples. Figure 2 shows how the absorption and distance of the light source varies for red blood cells, compared to the predicted observed values. These results indicate that, for samples in hematocrit in the 80% region, it is possible, using the preferred configuration of this invention to obtain light in the sample to a depth of 0.14 cm, This represents a flow path width that is less than half the width of the current Spectra cuvette, Example 6. Effects of virus inactivation treatment on in vitro platelet parameters. The effects of virus inactivation treatment on in vitro platelet parameters were evaluated. The platelet preparations were treated treated, The three energy levels and the three concentration levels were evaluated as follows: Energy levels: 1.5.9 J / cm2 * 7,8-dimethyl-10-ribityl isoaloxazine Concentrations: 1, 50, 100 μM ** * The total energy exposure levels were determined by the flow rate of the suspension through the irradiation chamber according to the conversion chart in Table 4. ** As the medium is diluted 70:30 (medium: plasma) the concentration of 7,8-dimethyl-10-ribityl isoaloxazine in the medium alone was adjusted appropriately before mixing with the plasma. This required initial concentrations in Isolyte of 1.43; 71.4 and 143 μM. Table 4. Energy exposure levels as a function of flow rate through the irradiation chamber Flow: 3640 mW / cm2; chamber volume = 0.117 ml. The values of the treated samples were compared with the control groups. Control samples included the following: Untreated plasma sample (historical control) + f I u] oU V-7, 8-d imethyl-10-ribityl isoaloxazine Procedure: A platelet donor apheresis product was obtained normal to from a blood bank of an installation accredited by AABB. The sample was collected using the common LRS Spectra procedures. All manipulations or procedures described below were performed with common laboratory safety procedures and methods. The unit number and blood type were recorded. All samples were used within twenty-four hours of collection. An aseptic procedure was followed for all demonstration transfers and process steps. The sample was transferred to a 500 ml PVC transfer pack and centrifuged at 5000 x g for five minutes to package the platelets. The plasma was then removed from the platelet pellet using a common plasma press. The plasma was retained for additional use. The plasma removed from the cell pellet was then mixed with a stock solution of Isolyte S, pH 7.4; Me Graw, Inc. This stock solution was prepared by adding a predetermined amount of 7,8-dimethy1-10-ribityl isoaloxazine to Isolyte S to give final concentrations of 1.43.; 71, 4 and 143 μM. After addition of 7,8-dlmethyl-10-ribityl-soaloxazine, the stock solution was filtered through a sterile 0.22 μM filter. The stock solution was then mixed with plasma derived from itself at a ratio of 70:30 (volume by volume) to give final concentrations of 7,8-dimethyl-10-ribityl isoaloxazine of 1.50 and 100 μM, respectively. Preparation of the stock solutions of 7, 8-dimethyl-10-ribityl-soaloxazine, special care was taken to avoid exposure to light. Samples were prepared according to the following: 1 μM 2 samples 100 μM 2 samples 50 μM 1 sample The platelet pellet was then resuspended in the plasma mixture: medium to the original volume of the initial sample. The sample was connected to a flow apparatus with a cell container and a sensitizer, a container for the medium, said containers being connected through tubes with valves to a single tube for the mixed cells / sensitizer and the medium equipped with a bomb. The mixed cells / sensitizer and medium were flowed in a cuvette supported on a support with a mirrored wall, irradiated by a light source. This irradiation chamber was equipped with a temperature probe. Once the fluid passed through the bucket, it was collected in a bag for products. The tube kit was initially primed with the medium of Isolyte S. Five minutes before the start of the test sample flow, the light source was activated. The temperature was controlled during this interval and kept below 32 ° C in the irradiation chamber. The flow rate of the sample through the irradiation chamber was determined by means of the graph in Table 4. The flow rates that provide Total irradiance energy levels of 1, 5 and 9 J / cm2 were used according to the following test matrix: Sample use # 1: concentration of 7,8-dlmethyl-l 0-ribidyl isoaloxazine = 1μM A. + 7, 8-dimethyl-10-ribityl isoaloxazine + 1 J / cm2 B. + 7,8-dimethyl-10-ribidyl isoaloxazine + 9 J / cm2 Sample use # 2: 7,8-dimetiM 0-ribidyl isoaloxazine = 100 μM A, +7, 8-dimethyl-10-ribityl isoaloxazine + 1 J / cm2 B. + 7,8-dimethyl-10-ribityl isoaloxazine + 9 J / cm2 Use of sample # 3: 7,8-dimethyl-IO -ribityl isoaloxazine = 50 μM A. + 7,8-dimethyl-10-ribityl isoaloxazine + 5 J / cm2 Sample use # 3: 7,8-dimethyl-10-ribitii isoaloxazine = 0 μM A. + UV-Flow- 7,8-dimethyl-10-ribidyl isoaloxazine All samples were identified by the use number and the sample letter designation corresponding to the treatment condition (ie, 1 A). Each sample set was used for a total of 2 replicates. The order in which the samples were treated was determined by assignments according to a random number generator. A sample volume of 20 ml was collected per condition of use for each sample. These samples were collected in citrated plastic sample bags (53 ml total volume) and stored for analysis. The temperature of the sample and the irradiation chamber were indicated at the beginning, middle and end of each use. After the treatment, an initial aliquot of each preparation was removed for analysis. The parameters for the analysis included the total irradiance energy levels of 1, 5 and 9 J / cm2 were used according to the following test matrix: Sample use # 1: concentration of 7,8-dimetiM 0-ribityl isoaloxazine = 1μM A. + 7,8-d¡methyl-10-ribityl isoaloxazine + 1 J / cm2 B. + 7,8-dimethyl-10-ribityl isoaloxazine + 9 J / cm2 Sample use # 2: 7,8- dimetiM 0-ribityl isoaloxazine = 100 μM A. + 7,8-dimethyl-10-ribityl isoaloxazine + 1 J / cm2 B. +7, 8-dimethyl-10-ribityl isoaloxazine + 9 J / cm2 Sample use # 3: 7,8-dimetiM 0-ribityl isoaloxazine - 50 μM A. +7, 8-dimethyl-10-ribityl isoaloxazine + 5 J / cm2 Use of sample # 3: 7, 8-dimethyl-10-ribityl isoaloxazine - 0 μM A . + Flow-UV-7,8-dimethyl-10-ribityl isoaloxazine All samples were identified by the use number and the sample letter designation corresponding to the treatment condition (ie, 1 A). Each sample set was used for a total of 2 replicates. The order in which the samples were treated was determined by assignments according to a random number generator. A sample volume of 20 ml was collected per condition of use for each sample. These samples were collected in citrated plastic sample bags (53 ml total volume) and stored for analysis. The temperature of the sample and the irradiation chamber were indicated at the beginning, middle and end of each use. After the treatment, an initial aliquot of each preparation was removed for analysis. The parameters for the analysis included cell count, pH, pCO2, pO2, platelet swirl, HSR and GMP-140 analysis. The remaining portion of the sample was placed on a rotary shaker for a +22 incubator and stored for five days after treatment. On day five, a second aliquot was removed and analyzed for the same parameters in vitro. The following equipment was used: Nikon Labophot microscope; hematological analyzer Serono - Baker System 900; analytical balance; platelet incubator (+22 Celsius) and rotator; laboratory refrigerator (+4 Celsius); Mistral 3000 centrifuge; Corning blood gas analyzer; flow cytometer Becton - Díckinson FACSCALIBUR; irradiation chamber IV; UV radiometer (UVX Radiometer, UVP Inc.); Ultracure 100SS Plus EFOS (365 nm maximum output and 340 nm bandpass filters) and a temperature probe (thermocouple). The results of each set of test variables were compared with the defined energy conditions of exposure and concentration of 7,8-dimethyl-ribityl isoaloxazipa. The direct comparison with the untreated control sample was carried out and significant differences were defined. by a probability of p <0.05 of a matched single-tail analysis, Student's T-Test. The results of these studies are summarized as follows. 1. At sensitizer concentrations in excess of 10 μM and platelet concentrations above 1.5E + 06 / μL, there was a drop in the pH of the sample on day 2. The pH dropped regularly after two days of storage, reaching unacceptable levels (< 6.5) on day 3 of storage. All other in vitro parameters followed the pattern observed with the pH of the sample. 2. This decrease in the pH of the sample occurred regardless of whether the sample was exposed to UV light or not. 3. At platelet concentrations of 5.4E + 05 / μL, there was no drop in the pH of the sample after long storage at any concentration of sensitizer studied up to 100 μM. 4. At sensitizer concentrations up to 10 μM, platelet concentrations above 1.5E + 06 / μL, and UVA levels up to 10 J / cm2, the measured platelet properties were compared with those of the cells without trying control. These remained comparable with control levels after five or more days of storage after treatment. These studies of post-treatment platelet function provided a clear window where the cell properties were maintained at levels comparable to those of untreated cells. The results also indicate that by changing the storage or treatment conditions for the cells, this window can be expanded. The observed effect of 7,8-dimethyl-10-ribityl isoaloxazine with or without UV light on the pH of the sample suggests a metabolic effect of this additive that can be moderated by changes in storage or processing conditions of the samples. Example 7. Shear stress measurements in red blood cells as a function of flow rate and sample hematocrit. Lower levels of UV light penetration in red blood cell samples in elevated hematocrits increased the need to understand the effects of passing red blood cells. through narrow openings in the path of light. The reduction in the thickness of the sample in the path of light should increase the delivery of UV doses in high sample hematocrits. In order to confirm this approach, several pressure drop measurements were made using openings of different dimensions. A pressure gauge was placed in line with a peristaltic pump both before and after the narrow openings. All blood from the different hematocrits was passed through the openings at controlled flow rates. The differences in the pressure readings in both locations allowed the direct measurement of the pressure drop across the opening. Using this value and the dimensions of the opening, it was possible to determine the shear stress experienced by red blood cells as they passed through the narrow cell using the following equation: Pressure drop 8μlQ? P Gd3w Shear force 4μQ tw = gwd2 For blood: μ = viscosity = 0.0125 (1-hematocrit) g = gravitational constant = 981 Q = flow index = ml / sec L, w, d = Opening dimensions in cm Table 5: measurement of shear stress in blood cells red as functions flow index and sample hematocrit In previous experiments, it was determined that the shear forces of 1,000-2,000 dynes / cm2 for intervals of 1-10 minutes or levels of 5,000-7,000 dynes / cm2 for intervals of approximately 10 msec were sufficient to induce the hemolysis of red blood cells. Only in the case of the highest sample hematocrit (61%) and the highest flow index (16.9) did the values exceed 1,000 dynes / cm2. This occurred only for the narrowest width openings (0.008 inches). The values for the depth of light penetration using the proposed configuration indicate that sufficient UB energy delivery to drive the virus inactivation processes can be achieved even for samples with elevated hematocrit. The results of the shear stress analysis on the red blood cell samples subjected to flow indicate that the dimensions of the flow path can be significantly reduced, and the high flow rates can be maintained without the risk of hemolysis of red blood cells. Example 8. A platelet concentrate was mixed with the Isolyte solution of platelet additive in a ratio of 20:80 platelet concentrate: Isolyte S. Here reference is made to mixtures of platelet concentrates and additive solutions of platelets as "medium". In the present reference is made to the platelet concentrate without additive solution as "plasma". Both were splashed with Listeria monocytogenes. Then K5 was added to each in the amount of 300 μg / ml B. Then each was exposed to UV light, visible light or ambient light, in the cuvette apparatus of Figure 7. Table 6 shows the results . Table 6 UV light - 365 nm VIS light - 419 nm Pathogen = Listeria monocytogenes K5 concentration = 300 μg / ml Example 9. The medium and plasma previously described with vitamin K5 were splashed with bacteria and irradiated or exposed to ambient light only (light K5) as shown in table 7 and growth was evaluated after three days of incubation. Inactivation of some species was observed in the absence of irradiation. Table 7 L. monocytogenes 3.5 logarithms E. coli 3.1 logarithms UV light - 365 nm, 40 J / cm2 + = s growth detected after three days of incubation - - no growth detected after three days of incubation K5 concentration = 300 μg / ml. Example 10. The medium made was splashed using a platelet concentrate as described in Example 8 and an Isolyte S medium at a ratio of Isolyte S: platelet concentrate of 70:30 with 300 μg / MI of vitamin K5, with several species of bacteria and irradiated at energy levels of 30 and 60 J / cm2 'The inactivation results are indicated in Table 8 and in Figure 8.

Table 8 Example 11. To the platelet concentrate as described in Example 8 and to a 70:30 medium as described in Example 10, 10 μM of 7,8-dimethyl-10-ribityl isoaloxazine was added. The platelet concentrate and medium were splashed with S. aureus or S. epidermidis and irradiated at 80 J / cm2 and 30 J / cm2 and the inactivation was measured as previously indicated. The results are shown in Figure 9. Example 12. To the plasma concentrate as described in the Example contained in a common blood bag was added 25 μM of 7,8-dimethyl-10-ribityl isoaloxazine in powder form. The bag was splashed with bacteria as shown in Table 9, shaken and exposed to a radiation of 120 J / cm2. The inactivation results are shown in Table 9. Tab a 9 Example 13. To the platelet concentrate as described in Example 8 were added 7,8-dimethyl-ribityl isoaloxazine, aloxazine mononucleotide or 7,8-dimethyl aloxazine, followed by spraying with S. aureus or S. epidermidis, and an irradiation of 80 J / cm2, The inactivation results are shown in Table 10. Table 10 7,8-dimethyl aloxazine, 7 μM 1, 6 logarithms 2.9 logarithms Example 14. To the platelet concentrate of Example 8, 10 μM of 7,8-dimethyl-ribytyl isoaloxazine was added. The aliquots did not contain additives, 10 mM ascorbate or 10 mM Kí as a "cooler" or antioxidant. The solutions were splashed with HSV-2,? X174, S. epidermidis or S. aureus and irradiated at 80 J / cm2. The results are shown in Figure 10. Example 15. To the platelet concentrates of Example 8 were added concentrations other than 7,8-dimethyl-10-ribitylyl isoaloxazine. These solutions were splashed with the type II herpes simplex virus (HSV-II) with viruses that envelop the double-stranded DNA. Irradiation was carried out at 80 J / cm2. The experiment was replicated three times. In all three trials a complete inactivation was achieved. The results are shown in Figure 11, Example 16. The protocol of Example 15 was followed using S. epidermidis instead of HSV II at irradiation energies of 40, 80 and 120 J / cm2. The inactivation results are shown in Figure 13. Example 17. The protocol of Example 15 was followed using fX174, a bacteriophage of single-stranded DNA, in various concentrations of 7,8-dimethyl-10-ribityl-isoaloxazine and energies of irradiation. The inactivation results are shown in Figure 13. Example 18 To the platelet concentrates of Example 8, 10 μm of 7,8-dimethymMO-ribityl isoaloxazine was added. They were splashed with S. aureus or fX174 and irradiated in different energies with a 50:50 mixture of visible light and ultraviolet light. The inactivation results are shown in Figure 14. Example 19 The protocol of Example 18 was followed using S. epidermidis and HSV-II as the microorganisms. A 50:50 sample of ultraviolet light and visible light was supplied by means of a DYMAX light source. The inactivation results are shown in Figure 15. Example 20 To the platelet concentrate of Example 8, 10 μM of 7,8-dimethyl-10-ribityl-isoaloxazine was added as a powder. The tests were performed with and without added ascorbate. 150 ml of the test solutions were placed in a Spectra ™ blood bag and mixed and exposed to different irradiation energies using 50:50 visible light: ultraviolet light. After receiving 40 J / cm2, the contents of each bag were transferred to a new bag to avoid errors due to microorganisms that might have remained in the port of bag splashing. The inactivation results are shown in Figure 16. The descending arrows indicate inactivation up to the possible level of detection (2.5 logarithm titration). Example 21. To the platelet concentrate of Example 8 and to the platelet concentrate in Isolyte S in 30:70 platelet concentrate: Isolyte S, 20 μM of 7,8-dimethyl-10-ribityl-isoaloxazine was added. These were splashed with smallpox virus, a virus that enveloped the double-stranded DNA and were exposed to 60 J / cm2 of visible light or mixture (50:50) visible light and ultraviolet light using a DYMAX 200 UV light source for thirty minutes, The limit of detection was 1.5 logarithms. The inactivation results are shown in Figure 17. Comparisons were made without using a photosensitizer, only in photosensitizer in Isolyte S medium, platelets in Isolyte S medium, platelets in Isolyte S medium using 8-methoxy psoralen instead of 7.8 -dimeti O-ribityl-isoaloxazine, and platelet concentrate in the medium of Isolyte (30:70). EXAMPLE 22 The platelet concentrate samples were splashed in Isolyte S 30:70 medium, with and without 10 μM of 7,8-dimethyl-ribityl-isoaloxazine, with pox virus and irradiated at 60 J / cm2 with 50:50. visible light: UV light for different periods of time, and the compared inactivation results are shown in Figure 18. Example 23 To the platelet concentrate samples as described in Example 8 were added 5 or 50 μM of 7 , 8-dimethyl-10-rlbityl-isoaloxazine. The samples were splashed with HIV-1. Using the cell flow cell shown in Figure 7, the samples were irradiated with 50:50 visible light: UV light with different energies using the EFOS light system. Figure 19 shows the inactivation results. Example 24. HIV-infected ACH-2 cells were added to the platelet concentrate samples described in Example 8. 5 or 50 μM of 7,8-dimethyl-10-ribitol-isoaloxazipa was added to the samples . The protocol of Example 23 was followed, and inactivation results appear in Figure 20. The presence of HIV was analyzed by means of its cytopathic effect on the sample cells. Example 25 The protocol of Example 24 was followed and the presence of HIV was analyzed by quantifying the production level of the P24 antigen. Figure 21 shows inactivation results. Example 26 To the platelet concentrate samples as described in Example 8 and the medium with 30% platelet concentrate and 70% PASIII ™ were added 6 mM ascorbate and 14 μM of 7,8-dimethylate. ribityl-isoaloxazine. The samples were splashed with HSV-II. The inactivation results are shown in Figure 22 and Table 11. Table 11 It will be understood by those skilled in the art that the purpose of the foregoing description has been for illustrative purposes only and that numerous changes can be made without departing from the scope of the invention. For example, other photosensitizers other than those mentioned may be used, preferably photosensitizers that bind to the nucleic acid and thereby prevent its repetition, and more preferably those which are non-toxic and which do not have toxic breakdown products. In addition, structures equivalent to those described herein for constructing a flow system for decontaminating fluids using photosensitizers can be easily devised without the need for experimentation by those skilled in the art following the teachings herein.

Claims (1)

1. CLAIMS 1. A method for treating a fluid in order to inactivate microorganisms that may be present therein, said fluid containing one or more components selected from the group consisting of proteins, blood and constituents of the blood, CHARACTERIZED BECAUSE: ) adding a substantially non-toxic amount, effective for inactivation, of an endogenous photosensitizer or a derivative based on endogenous photosensitizers to said fluid, (b) exposing the fluid from step (a) to a photoradiation sufficient to activate the photosensitizer, the microorganisms are inactivated. . The method of claim 1, characterized in that said photosensitizer is a photoactivatable compound whose photolytic products (if any) have reduced or no toxicity towards humans or towards other animals. . The method of claim 1 CHARACTERIZED BECAUSE said photosensitizer is an endogenous photosensitizer selected from the group consisting of alloxazines, vitamins K and vitamin L. The method of claim 1 wherein said photosensitizer is an endogenous photosensitizer selected from the group consisting of 7,8-dimethyl-10-ribityl-isoaloxazine, 7,8-dimethyloxazine, 7,8,10-trimethylisoxazole, alloxazine mononucleotide, isoaloxazine - adenosine dinucleotide, vitamin K1, vitamin K1 oxide, vitamin K2, vitamin K5, vitamin K6, vitamin K7, vitamin KS (l) and vitamin L. The method of claim 1 CHARACTERIZED BECAUSE said photosensitizer is 7,8-dimethyl-10-ribityl-isoaloxazine. The method of claim 1 CHARACTERIZED BECAUSE said microorganisms are selected from the group consisting of bacteria, bacteriophages and intracellular and extracellular viruses. The method of claim 1 CHARACTERIZED BECAUSE said microorganisms are bacteria. The method of claim 1 CHARACTERIZED BECAUSE said microorganisms are selected from the group consisting of HIV virus, hepatitis virus, Sindbis fever virus, smallpox virus, human T-lymphotropic retroviruses, HTLV-III, LAV virus / IDAV of lymphadenopathy, parvovirus, transfusion-transmitted virus (TT), Epstein-Barr virus, bacteriophage FX174, F6,?, R17, T and T2, P aeruginosa, S a? Reus, S epidermidis, L monocytogenes, E coli, K pneumonia and S marcescens. The method of claim 1 CHARACTERIZED BECAUSE said photoradiation is in the vis spectrum. The method of claim 1 CHARACTERIZED BECAUSE said photoradiation is in the ultraviolet spectrum. The method of claim 1 CHARACTERIZED BECAUSE said photoradiation is both in the vis and in the ultraviolet spectrum. The method of claim 1 CHARACTERIZED BECAUSE half of said photoradiation is in the ultraviolet spectrum and the other half is in the vis spectrum. . The method of claim 1 CHARACTERIZED BECAUSE said exposure step further comprises flowing the fluid containing said photosensitizer through a photoradiation source at a selected speed and depth to ensure the penetration of the photoradiation through the fluid and the inactivation of microorganisms. The method of claim 1 CHARACTERIZED BECAUSE it further comprises containing said fluid in a container transparent to said photoradiation and exposing said fluid to said photoradiation. The method of claim 14 CHARACTERIZED BECAUSE it further comprises stirring said container during photoradiation. The method of claim 1 CHARACTERIZED BECAUSE further comprises placing said fluid in a container transparent to said photoradiation, adding said photosensitizer to said fluid in powder form, shaking said container and exposing said container to said photoradiation. The method of claim 1 CHARACTERIZED BECAUSE said fluid comprises constituents of the blood. The method of claim 1 CHARACTERIZED BECAUSE said fluid comprises whole blood. The method of claim 1 CHARACTERIZED BECAUSE said fluid comprises a product separated from the blood. The method of claim 1 CHARACTERIZED BECAUSE said fluid comprises platelets separated from whole blood. The method of claim 1 CHARACTERIZED BECAUSE said fluid comprises red blood cells separated from whole blood. . The method of claim 1 CHARACTERIZED BECAUSE said fluid comprises serum separated from whole blood. . The method of claim 1 CHARACTERIZED BECAUSE said fluid comprises plasma separated from whole blood. . The method of claim 1 CHARACTERIZED BECAUSE said fluid comprises a therapeutic composition of proteins,. The method of claim 1 CHARACTERIZED BECAUSE said fluid contains a biologically active protein selected from the group consisting of: factor VIII, von Willebrand factor, factor IX, factor X, factor XI, Hegeman factor, prothrombin, anti-thrombin III, fibronectin, plasminogen, plasma protein fraction, immune serum globulin, modified immune globulin, albumin, plasma growth hormone, plasminogen streptokinase plasmid, plasmid, plasminogen, transferrin, hepatoglobin, antitrypsin, and precalicrein. The method of claim 1 CHARACTERIZED BECAUSE said photosensitizer is added to an anticoagulant and said anticoagulant is added to said fluid. A method of claim 1 CHARACTERIZED BECAUSE an improver is added to said fluid before exposing said fluid to the photoradiation. A method of claim 27 CHARACTERIZED BECAUSE said enhancer is selected from the group consisting of adenine, histidine, cysteine, tyrosine, tryptophan, ascorbate, N-acetyl-L-cysteine, propyl gallate, glutathione, mercaptopropionylglycine, dithiotreotol, nicotinamide, BHR, BHA, lysine, serine, methionine, glucose, mannitol, trolox, glycerol and mixtures thereof. . A method to treat a fluid in order to activate microorganisms that may be present in it, CHARACTERIZED BECAUSE: (a) adding a substantially non-toxic amount, effective for inactivation, of an endogenous photosensitizer or a derivative based on endogenous photosensitizers to said fluid, (b) exposing the fluid from step (a) to a photoradiation sufficient to activate the photosensitizer, resulting in the inactivation of microorganisms. The method of claim 29 CHARACTERIZED BECAUSE said fluid is a food product. The method of claim 29 CHARACTERIZED BECAUSE said fluid is a beverage intended for human or animal consumption. The method of claim 29 CHARACTERIZED BECAUSE said fluid is a peritoneal solution. A CHARACTERIZED fluid comprising a biologically active protein, blood or blood constituents, and a photosensitizer or a photoproduct thereof, created by the method of claim 1. A blood product CHARACTERIZED BECAUSE comprises a photosensitizer or a photoproduct thereof , created by the method of claim 1. A CHARACTERIZED fluid comprising a biologically active protein, blood or blood constituents, and a photosepsibilizer or a photoproduct thereof, and an enhancer, created by the method of claim 1. A system for treating a fluid in order to inactivate the microorganisms that may be present therein, CHARACTERIZED BECAUSE it comprises: (a) a container comprising said fluid and an endogenous photosensitizer or a derivative based on endogenous photosensitizers, said container being equipped with means of entry and having a photopermeable surface sufficient to allow the exposure of the fluid therein to the amount of photoradiation sufficient to activate the photosensitizer, (b) at least one source of photoradiation that provides sufficient photoradiation to the fluid in said container, of a type and in a selected amount to activate the photosensitizer, the microorganisms present being substantially inactive, The system of claim 36 CHARACTERIZED BECAUSE said photoradiation source provides light in the visible spectrum, The system of claim 36 CHARACTERIZED BECAUSE said source of fotorradiac ion provides light in the ultraviolet spectrum. The system of claim 36 CHARACTERIZED BECAUSE said photoradiation source provides light in both the visible and ultraviolet spectrum. AND! The system of claim 36 CHARACTERIZED BECAUSE it also comprises a photoradiator enhancer. The system of claim 40 CHARACTERIZED BECAUSE said photoradiation enhancer comprises a reflective surface. The system of claim 36 CHARACTERIZED BECAUSE it comprises a light guide for conducting the photoradiation from said photoradiation source towards said photopermeable container. The system of claim 36 CHARACTERIZED BECAUSE it further comprises a temperature monitor. The system of claim 36 CHARACTERIZED BECAUSE it further comprises a means for flowing said fluid in and out of said container. The system of claim 36 CHARACTERIZED BECAUSE it further comprises a means for agitating said fluid in said container. A device CHARACTERIZED BECAUSE is to separate whole blood into blood components, and because it comprises the system of claim 36. A system for the inactivation of microorganisms in a fluid containing them, CHARACTERIZED BECAUSE: (a) means to add an effective amount of an endogenous photosensitizer or a derivative based on endogenous photosensitizers to said fluid, (b) a photopermeable container for said fluid in communication with said medium for the purpose of adding the photosensitizer, which has a depth and length selected in a manner of allowing the exposure of the fluid from step (a) therein to a sufficient amount of photoradiation to activate the photosensitizer at a given flow velocity, (c) means to produce said selected flow velocity through said container, and ( d) at least one source of photoradiation to provide sufficient photoradiation to the fluid in said container. or, of a type and in a selected amount to activate the photosensitizer. A system for treating a fluid in order to inactivate the microorganisms that may be present therein, CHARACTERIZED BECAUSE it comprises: (a) a photosensitizer in the form of a powder, (b) a photopermeabie container for containing said fluid and said photosensitizer, (c) means for shaking said container, (d) at least one photoradiation source for providing sufficient photoradiation to the fluid in said container, of a type and in an amount selected to activate the photosensitizer, the microorganisms being inactivated. The system of claim 48 CHARACTERIZED BECAUSE said photopermeable container is a transparent plastic bag. The system of claim 48 CHARACTERIZED BECAUSE said means for stirring said container comprises a stirring table. The system of claim 48 CHARACTERIZED BECAUSE said photopermeable container contains said photosensitizer before the addition of said fluid. A method for inactivating the microorganisms on a surface, CHARACTERIZED BECAUSE: (a) applying to said surface UR sutastically not l '' "'"' 0, effective for the inactivation K;, of a photoser8lb, | endogenous l '' •,, ft a derivative based on ^ endogenous sensitizers, and (b) exposing that surface ^ ung efficient photoradiation com. ' I "" * activate the photosensitizer. The method of the claimed! , 52 CHARACTERIZED BECAUSE ',',, a surface is the surface of an iV ^, * t The method of the claim r ~ CHARACTERIZED BECAUSE 'surface is the surface of a, Nsto of anjma | The method of claim g CARACTERIZED? WHY surface is the surface of a * repair of comidf) The method of the claimed one 52 CARACJG¿RED because the surface is the surface of a ^. of bgñ0 0 d0 | 0Vado. The method of claiming? V 52 CAR CTERIZA £ > PORGUE i surface is the surface of the, s, of an L The method of the reivindicac \\ g2 CARACJERIZ Ü BECAUSE. { V 'surface is the surface of u? \ ^,. . The method of the claimed one 52 CARACTERIZATION POROU? In an endogenous photosensitizer, select from the group consisting of endogenous alloxazipals, vitamins, vitamins, and vitamin L. The method of the relivindicac \\ v 52 CARACTERIZED PORGUE. '' -10-photosensitizer is selected * ^ , why C0nsjste cn 7,8-dmP »'« ribityl-isoaloxazine, 7,8- <; ^ vtlla, o? azine) 7) 8I 10 -trimethyliso-ak- ^ 7 '' ^ "to alloxazine mononucleotide, 'scv ^, ^ ,. _ adenos¡pa din cleótidc. ** 'K1, vitamin K1 oxide, vitamin K2, vitamin K5, vitamin K6, vitamin K7, vitamin K-S (ll) and vitamin L. The method of claim 52 CHARACTERIZED BECAUSE said photosensitizer is 7,8-dimethyl-10-ribityl-isoaloxazine. . The method of claim 52 CHARACTERIZED BECAUSE said microorganisms are selected from the group consisting of bacteria, bacteriophages and intracellular and extracellular viruses. . A method for treating a fluid in order to inactivate the microorganisms present therein, said fluid further comprising a component selected from the group consisting of proteins, blood and blood components, without destroying the biological activity of said component, CHARACTERIZED BECAUSE adding a non-toxic amount, effective for inactivation, of vitamin K5 to said fluid in order to substantially inactivate said microorganisms. The method of claim 63 CHARACTERIZED BECAUSE it is carried out under normal ambient lighting. The method of claim 63 CHARACTERIZED BECAUSE it is carried out in the dark. The method of claim 63 CHARACTERIZED BY comprising the addition of an improver to said fluid. The method of claim 63 CHARACTERIZED BECAUSE said improver is an antioxidant. A method to treat a surface in order to inactivate the microorganisms that may be present in it or that may come into contact with it, CHARACTERIZED BECAUSE: cover said surface with a non-toxic amount, effective for inactivation, of vitamin K5 a said fluid in order to substantially inactivate said microorganisms. 69. An aqueous solution of platelet aggregates CHARACTERIZED BECAUSE it comprises an endogenous photosensitizer selected from the group consisting of endogenous alloxazines, vitamins K and vitamin L, 70. The platelet aggregate solution of claim 69 CHARACTERIZED BECAUSE it comprises: Physiological saline solution, and Buffer 71. The platelet aggregate solution of claim 69 CHARACTERIZED BECAUSE it further comprises magnesium chloride. 72. The platelet aggregate solution of claim 69 CHARACTERIZED BECAUSE it additionally comprises sodium gluconate. 73. The solution of platelet aggregates of claim 69 CHARACTERIZED BECAUSE said photosensitizer is present at a concentration between about 1 μM and its maximum solubility. 74. The solution of platelet aggregates of claim 73 CHARACTERIZED BECAUSE the concentration of said photosensitizer is around 10 μM. 75. The platelet aggregate solution of claim 69 CHARACTERIZED BECAUSE it has a pH of between about 0.0 and about 7.4. 76. The solution of platelet aggregates of claim 69, characterized in that said photosensitizer is 7,8-dimethyl-10-ribityl isoaloxazine. 77. The platelet aggregate solution of claim 69 CHARACTERIZED BECAUSE it comprises: Sodium chloride, sodium acetate, sodium gluconate, magnesium chloride, sodium phosphate, and 7,8-dimethyl-10-ribityl isoaloxazine, and has a pH of between about 7.0 and about 7.4. 78. The platelet aggregate solution of claim 69 CHARACTERIZED BECAUSE it further comprises an enhancer selected from the group consisting of adenine, histidine, cysteine, tyrosine, tryptophan, ascorbate, N-acetyl-L-cysteine, propyl gallate, glutathione, mercaptopropionylglycine , dithiothreotol, nicotinamide, BHR, BHA, lysine, serine, methionine, glucose, mannitol, trolox, glycerol and mixtures thereof. 79. A method for treating a fluid in order to inactivate the microorganisms that may be present therein, CHARACTERIZED BECAUSE it comprises: (a) adding an effective amount for the inactivation of a photosensitizer to said fluid, (b) exposing the passage fluid (a) to a mixture of ultraviolet and visible light, said microorganisms being inactivated. 80. The method of claim 79 CHARACTERIZED BECAUSE said light mixture is ultraviolet 50: 50: visible. 81. The method of claim 79 CHARACTERIZING BECAUSE said photosensitizer is a non-toxic endogenous photosensitizer or a derivative based on endogenous photosensitizers. 82. A method for treating a fluid for the purpose of inactivating white blood cells that may be present therein, characterized in that it comprises: (a) adding a substantially non-toxic amount, effective for inactivation, of an endogenous photosensitizer or of a derivative based on endogenous photosensitizers to said fluid, (b) exposing the fluid from step (a) to a photoradiation sufficient to activate the photosensitizer, said white blood cells being inactivated. 83. The method of claim 82 CHARACTERIZED BECAUSE said fluid comprises blood or a component of the blood. METHOD AND APPARATUS FOR THE INACTIVATION OF BIOLOGICAL POLLUTANTS THROUGH THE USE OF PHOTOSENSITIZERS SUMMARY Methods and devices are provided for the inactivation of microorganisms in fluids or surfaces. Preferably, the fluids contain blood or blood products, and comprise biologically active proteins. Preferred methods include the steps of adding a non-toxic amount, effective for inactivation, of an endogenous photosensitizer to a fluid and exposing the fluid to a photoradiation sufficient to activate the endogenous photosepsibilizer, resulting in the inactivation of microorganisms. Other fluids, including juices, water and the like, can be decontaminated by these methods, as well as food surfaces, animal remains, wounds, food preparation surfaces and surfaces of bath and wash containers. Aloxazines and vitamins K and L are among the preferred photosensitizers. Systems and apparatus are also provided for the purpose of being used in flow and batch processes for the decontamination of such fluids using photosensitizers.