WO1991016911A1 - Decontamination du sang entier et des composants cellulaires a l'aide de colorants au phenthiazine-5-ium et de lumiere - Google Patents

Decontamination du sang entier et des composants cellulaires a l'aide de colorants au phenthiazine-5-ium et de lumiere Download PDF

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WO1991016911A1
WO1991016911A1 PCT/US1991/002976 US9102976W WO9116911A1 WO 1991016911 A1 WO1991016911 A1 WO 1991016911A1 US 9102976 W US9102976 W US 9102976W WO 9116911 A1 WO9116911 A1 WO 9116911A1
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blood
dye
light
effective
composition
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PCT/US1991/002976
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Stephen J. Wagner
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The American National Red Cross
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/10—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person
    • A61K41/17—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person by ultraviolet [UV] or infrared [IR] light, X-rays or gamma rays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20061—Methods of inactivation or attenuation
    • C12N2760/20063—Methods of inactivation or attenuation by chemical treatment
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • FIELD OF INVENTION This invention is directed to methods for inactivating viruses and other pathogenic contaminants in transfusible blood and blood components.
  • HIV human immunodeficiency viruses
  • Virucidal methods including heat, solvent-detergent, and gamma irradiation have been used to produce non- infectious plasma derivatives, but such methods are either ineffective or too harsh to be used for the decontamination of whole blood, red cells or platelets. Any treatment that damages or introduces harmful or undesirable contaminants into the whole blood or blood components is unsuitable to decontaminate a product intended for transfusion.
  • red blood cells and platelets Because of the critical need for transfusible red blood cells and platelets, it is of great importance to develop methods that can be readily used to decontaminate cellular blood components and whole blood without substantially or irreversibly altering or harming them. To be acceptable for transfusion, at least 75% of the red cells must be circulating 24 hours after the transfusion. The shelf-life and suitability of red blood cells for transfusion is determined on this basis. The concentrations of ATP and 2,3 diphosphoglycerate (2,3 DPG) and the morphology of red cells serve as indicators of the suitability of such cells for transfusion. During prolonged storage and/or as a result of harsh treatments, human red blood cells undergo changes that include decreases in the cellular levels of ATP and 2,3 DPG and changes in cellular morphology.
  • the concentration of ATP after a brief initial rise, progressively declines to about 50% of its initial level.
  • the fluidity of the cell membranes of red cells which is essential for their passage through the narrow channels in the spleen and liver, correlates with their levels of ATP.
  • the level of 2,3 DPG falls rapidly after about 3 or 4 days of storage and approaches zero after about 10 days. 2,3 DPG is associated with the ability of the hemoglobin in the red cells to deliver oxygen to the tissues.
  • Solutions that prolong the shelf life of red cells are known (see e.g., Meryman et al., U.S. Patent No. 4,585,735, incorporated herein by reference).
  • Typically such solutions contain citrate, phosphate, glucose, adenine and other ingredients and function to prolong shelf life by maintaining the levels of ATP and 2,3 DPG in the cells.
  • Solutions that contain a penetrating salt, such as ammonium acetate, in addition to phosphate, glucose, and adenine and that are hypotonic with respect to molecules that are unable to penetrate the cell membrane, have been shown to maintain the levels of ATP for more than 100 days of refrigeration (see, Meryman et al., supra.).
  • Decontamination treatments that inactivate pathogens, but that do not harm the cellular fractions of blood are not readily available.
  • Presently used decontamination procedures include photosensitizers, which, in the presence of oxygen and upon exposure to wavelengths of light absorbed by the photosensitizer, inactivate viruses (see, e.g., EP 0196515, published 08.10.86., to Baxter Travenol Laboratories, Inc.).
  • photosensitizers include psoralen derivatives (see, e.g., U.S. Patent No. 4,748,120 to Wiesehahn), porphyrin derivatives (see, e.g., U.S. Patent No. 4,878,891 to Judy et al.) and other photosensitizers.
  • psoralen derivatives see, e.g., U.S. Patent No. 4,748,120 to Wiesehahn
  • porphyrin derivatives see, e.g., U.S.
  • the virucidal activity of these compounds is realized when the absorption spectrum of the photosensitizer does not significantly overlap the absorption spectrum of pigments present in the blood.
  • the photosensitizer is not toxic to red cells and platelets and selectively binds to a component of the virus that is either not present in the red cells or platelets or, if present therein, that is not essential to the red cells' or platelets' function. It is also preferable if the photodynamic treatment inactivates extracellular and intracellular virus as well as cells containing provirus. It is beneficial if the virucidal activity of the photosensitizer is not inhibited by the presence of plasma proteins.
  • Photosensitizers such as the psoralens (see, U.S. Patent No. 4,748,120 to Wiesehahn) damage nucleic acids in the presence of light while the porphyrins (see, e.g., U.S. Patent No. 4,878,891 to Judy et al . ) and merocyanine 540 (MC540) (see, e.g., U.S. Patent No. 4,775,625 to Sieber) cause membrane damage in the presence of light and oxygen and thereby inactivate viruses and bacteriophages.
  • the porphyrins see, e.g., U.S. Patent No. 4,878,891 to Judy et al .
  • MC540 merocyanine 540
  • the phenthiazin-5-ium dyes which include methylene blue, toluidine blue O, thionin, azure A, azure B, and azure C, are useful for inactivating animal viruses (see, e.g., U.S. Patent Nos 4, 407,282, 4,402,318, 4,305,390 and 4,181,128 to Swartz).
  • these dyes have not been used to inactivate pathogens in whole blood or in cellular blood components because red cells readily take up or bind su ⁇ h dyes (see, e.g., Sass et al . , J. Lab. Clin. Med. 73:744-752 (1969)).
  • This invention significantly improves the procedure for decontaminating blood and cellular blood components by providing a method that produces non- infectious blood or blood components that can be transfused without the need for diluting or removing the photosensitizing dye.
  • At least one phenthiazin-5-ium dye said dye such as methylene blue, toluidine 0 thionin, azure A, azure B, azure C, and any other phenthiazin-5-ium dye known to those of skill in the art, is added to the blood, blood component, plasma, platelet concentrate or composition that contains blood, a blood component, or platelets.
  • the mixture is then treated with an effective wavelength of light, such as red light, and any pathogenic contaminant, such as a viral pathogen, is inactivated.
  • FIGURES Figure 1 presents the dependence of inactivation of bacteriophage ⁇ 6 in 16% solution of plasma in Unisol as a function of methylene blue concentration in the sample. Samples containing the bacteriophage, plasma and increasing concentrations of methylene blue were exposed for 4 minutes at a fluence rate of 2 mW/cm 2 of sample delivered by General Electric F15T8-R bulbs.
  • Figure 2 presents the dependence of the inactivation of bacteriophage ⁇ 6 by 40 ⁇ g/ml amino- methyl-trimethyl psoralen (AMT) as a function of plasma concentration in Unisol. Samples containing the bacteriophage, AMT and increasing concentrations of plasma were exposed to UVA irradiation (wavelength of 365 nm) at a fluence rate 42 mW/cm 2 for 90 seconds.
  • Figure 3 depicts the dependence of bacteriophage ⁇ 6 inactivation in platelet concentrates as a function of'leukocyte concentration.
  • a pathogenic contaminant is a contaminant that, upon transfusion or handling of blood or a component thereof, may cause disease in the recipient or handler thereof.
  • pathogens include, but are not limited to, retroviruses, such as HIV, and hepatitis viruses.
  • a blood component is a component that is separated from blood and includes, but is not limited to red blood cells, platelets, blood clotting factors, plasma, enzymes, plasminogen, and immunoglobulins.
  • a cellular blood component is a component of blood, such as a red blood cell, that is a cell.
  • a blood protein is a protein that is normally found in blood. Examples of such proteins are blood factors VII, VIII. Such proteins and components are well-known to those of skill in the art.
  • a composition containing a cellular blood component or a blood protein is a composition that contains a biologically compatible diluent and a blood component, blood protein, or mixtures thereof. Such compositions may also contain plasma and leukocytes. If such compositions are leukodepleted, the concentration of leukocytes is reduced by a specified amount.
  • a transfusible composition is a composition that can be transfused into the blood stream and that contains blood, at least one cellular blood component, concentrated plasma, or mixtures of blood, cellular blood components, and plasma.
  • decontamination refers to a process whereby pathogens, such as viral contaminants, are rendered non-infectious so that blood or a composition that contains blood, a blood component or blood protein can be transfused or manipulated without harming or infecting anyone exposed thereto.
  • a pathogen includes any replicable agent that infects or occurs in blood or blood components.
  • pathogens include any virus, bacterium, or parasite known to those of skill in the art to be found in blood or products derived from blood.
  • pathogens include but are not limited to: bacteria, such as Streptococcus species, Escherichia species, and Bacillus species; viruses, such as human immunodeficiency viruses, other retroviruses, herpes viruses, paramyxoviruses, cytomegaloviruses, hepatitis viruses, including hepatitis A, hepatitis B, and hepatitis C, pox viruses, and toga viruses; and parasites, such as malarial parasites, including Plasmodium species, and trypanosomal parasites.
  • bacteria such as Streptococcus species, Escherichia species, and Bacillus species
  • viruses such as human immunodeficiency viruses, other retroviruses, herpes viruses, paramyxoviruses, cytomegaloviruses, hepatitis viruses, including hepatitis A, hepatitis B, and hepatitis C, pox viruses, and toga viruses
  • the ratio of the titer of the control sample to the titer of virus in each of the treated samples is herein called virus inactivation.
  • the log 10 of this ratio is herein called log 10 inactivation.
  • a log 10 of inactivation of at least about 5 to 6 logs indicates that the treated sample has been decontaminated.
  • a composition in which substantially all of the contaminating pathogens have been inactivated is one in which the concentration of active pathogen has been decreased by a factor of at least about 5 to 6 logs. A composition in which substantially all of the contaminating pathogens have been inactivated is, thus, decontaminated.
  • fluence is a measure of the energy per unit area of sample and is typically measured in joules/cm 2 .
  • Fluence rate is a measure of the wattage, of light that strikes a unit area of the sample. For example, it can be measured as milliwatts (mW)/ per cm 2 . Fluence rate can also be expressed as the amount of energy that strikes the sample in a given amount of time and may be measured as joules/cm 2 per unit time of exposure.
  • a phenthiazin-5-ium dye includes any dye that one having skill in the art would consider a member of that class.
  • This class includes, but is not limited to, methylene blue, toluidine blue O, thionin, and azure A, B and C.
  • plasma can be prepared by any method known to those of skill in the art. For example, it can be prepared by centrifuging blood at a force that pellets the cells and forms an interface between the red cells, the buffy coat, which contains leukocytes, and above which is the plasma. Depending on centrifugation conditions, the number of leukocytes and platelets in the plasma can vary.
  • leukocyte depleted platelets or red cells are components that have been passed through a filter that decreases the concentration of leukocytes by a factor of 10 2 to 10 5 .
  • filters are identified by the log of the factor by which the blood component is depleted of leukocytes.
  • extracellular pH is the pH of the medium in which red blood cells or other cellular blood components are stored or maintained.
  • a biologically compatible solution or a biologically compatible buffered solution is a buffered solution in which cells that are contacted therewith retain viability. Contacting includes any process in which the cells are in some manner exposed to the buffered solution and includes, but is not limited to, suspension of the cells in the buffered solution.
  • a biologically compatible buffered solution has a pH and a salt concentration that is suitable for maintaining the integrity of the cell membrane. Such a solution does not inhibit or destroy the biological and physiological reactions of the cells contacted therewith.
  • a biologically compatible buffered solution has a pH between 5 and 8.5 and is isotonic or only moderately hypotonic or hypertonic.
  • Biologically compatible buffered solutions are readily available to those of skill in the art.
  • biologically compatible buffered solutions include, but are not limited to those listed in Table I, infra.
  • an anticoagulant solution such as CDPA-1
  • a suitable, biologically compatible, buffered solution such as ARC 8 (Meryman et al., Vox Sang 18:81-98 (1991)), or any that are well-known to those of skill in the art.
  • the whole blood may then be subjected to a decontamination process in accordance with this invention.
  • the blood may be separated into its components, including, but not limited to, plasma, platelets and red blood cells, by any method known to those of skill in the art.
  • blood can be centrifuged for a sufficient time and at a sufficient centrifugal force to form a pellet containing the red blood cells.
  • Leukocytes collect primarily at the interface of the pellet and supernatant in the buffy coat region.
  • the supernatant which contains plasma, platelets, and other blood components, may then be removed and centrifuged at a higher centrifugal force, whereby the platelets pellet.
  • Human blood normally contains about 7 x 10 9 leukocytes per liter.
  • the concentration of leukocytes which pellet with the red cells, can be decreased by filtering through a filter that decreases their concentration by selected orders of magnitude.
  • Leukocytes can also be removed from each of the components by filtration through an appropriate filter that removes them from the solution.
  • the concentration of dye, the light intensity, and/or the time of irradiation must be somewhat increased. It is well within the level of skill in the art to ascertain the amount by which any or all of such parameters should be adjusted. It has, however, been discovered that plasma proteins do not affect the inactivation reactions that occur when practicing the method of this invention. Thus, the values of these parameters need not be adjusted for the presence of plasma proteins.
  • composition of blood, cellular blood components, or concentrated plasma a composition containing blood, cellular blood components or mixtures of cellular blood components, plasma and leukocytes or any other composition containing blood or blood components, may be obtained or prepared as described above or by any means or method known to those of skill in the art.
  • compositions are obtained in, prepared or introduced into gas permeable blood preservation bags, which are sealed and flattened to a width sufficiently narrow to permit light to pass through and irradiate the contents, whereby any pathogen present in the bag would be irradiated.
  • Any such blood bag known to those of skill in the art may be used as long as there is sufficient oxygen present in the bag to react with the photosensitizer and the bag is transparent to the selected wavelength of light.
  • composition that is decontaminated may include any suitable biologically compatible physiological solution known to those of skill in the art.
  • suitable biologically compatible physiological solution include, but are not limited to Unisol and ARC 8 (see, TABLE 1, infra.).
  • the dyes or photosensitizer compounds of this invention include the phenthiazin-5-ium dyes. Any such dye known to those of skill in the art may be used. Examples of such dyes include, but are not limited to, methylene blue, toluidine blue O, azure A, azure B, azure C and thionin.
  • An effective amount of at least one selected dye is introduced into the composition. Ideally the selected dye is non-toxic and the effective concentration is acceptable for transfusion so that the treated blood or blood component does not require additional manipulation to remove the dye and thereby risk contamination.
  • the effective concentration of dye to be used can be determined by one of skill in the art. Generally it is in the range, but is not limited to, 0.2 to 50 ⁇ M.
  • methylene blue may be selected. Methylene blue is used therapeutically to treat methemoglobinemia at a dosage of 1 mg/kg of body weight to a maximum recommended dosage of 2 mg/kg.
  • blood or cellular blood components or other compositions treated in accordance with this invention can be directly transfused, as long as the final dosage of methylene blue is less than about 2 mg/kg of body weight.
  • methylene blue is introduced into the composition at a concentration of about 1 ⁇ M to about 25 ⁇ M .
  • the amount of methylene blue needed for inactivation is substantially less, about twenty-five fold less, than the maximum recommended dosage.
  • transfusion of ten units of red cells at a 55% hematocrit to a 70 kg individual that have been treated with 5 ⁇ M methylene blue in accordance with this invention would only provide a dose of methylene blue of 0.08 mg/kg, which is substantially less than the maximum recommended dosage.
  • the mixture of the blood or blood component composition and dye is then irradiated for a sufficient time with an appropriate wavelength or mixture of wavelengths, whereby pathogenic contaminants in the composition are inactivated.
  • wavelength is one that is absorbed by the dye, but that does not damage the blood or blood components present in the composition. It is well within the level of skill in the art to select such wavelength and to ascertain a sufficient time for inactivation.
  • the selected wavelength is based on the absorption profile of the selected dye or dyes and is one that does not substantially damage the cellular components of the composition selected for decontamination.
  • model viral systems are known to those of skill in the art. These model systems may be used to test the selected dye and light source.
  • model viral systems include, but are not limited to the enveloped bacteriophage, bacteriophage ⁇ 6, vesicular stomatitis virus (VSV), which is an animal virus that contains its genome encoded as DNA, and Sindbis virus, which is an animal virus that contains its genome encoded as RNA.
  • VSV vesicular stomatitis virus
  • Sindbis virus an animal virus that contains its genome encoded as RNA.
  • red blood cells which have been leukodepleted with a five log filter, are suspended in ARC 8 at a hematocrit of about 15 to 55% introduced into gas permeable blood preservation bags in an amount such that the filled bag has a thickness of about 4 mm, and treated with methylene blue at a concentration of about 1 ⁇ M up to about 25 ⁇ M and red light of wavelength (560 to 800 nm.) at a sufficient intensity and for a long enough time, such as for about 60 minutes at 3.6 joule/cm 2 , to inactivate pathogenic contaminants in the red blood cells.
  • the virucidal activity of the methylene blue and light treatment is not affected by the presence of up to 100% plasma but was reduced by the presence of leukocytes. Accordingly, in the presence of leukocytes, light intensity, dye concentration, and/or irradiation time must be increased in order to ensure that the sample is decontaminated.
  • compositions containing platelets and compositions containing high concentrations of plasma may be decontaminated by exposure for a sufficient time to an effective concentration of a phenthiazin-5-ium dye plus an effective amount of an appropriate wavelength of light.
  • the blood, cellular blood component or composition may be stored or transfused.
  • the composition can be centrifuged at a force sufficient to pellet the cellular components. The supernatant can then be removed following centrifugation and the cells resuspended to reduce the concentration of residual photosensitizer and any reaction products.
  • Plasma was prepared from human blood by centrifugation to pellet the red cells and to remove the platelets. Leukocytes were removed by filtration with a log filter as indicated.
  • Methylene Blue was reagent or USP grade.
  • Bacteriophage ⁇ 6 stock solution was prepared from lysates of the HB10Y strain of Pseudomonas phaseolicola. At the concentrations used, methylene blue was not harmful to virus in the absence of light. Platelet-poor human plasma was diluted to a final concentration of 16% in Unisol. Forty ⁇ l of bacteriophage ⁇ S stock solution was added to 4 ml of sample and varying amounts of 1 mg/ml methylene blue solution were added to each sample. The samples were incubated at room temperature and then exposed for 4 minutes to light delivered by General Electric F15T8-R bulbs at a fluence rate of 2 mW/cm 2 of sample.
  • the samples were diluted and the virus was titered by a plaque assay and compared with the number of plaques in a control sample that was not exposed to light.
  • the ratio of the titer of the control sample to the titer of virus in each of the treated samples, is a measure of viral inactivation.
  • the log 10 of this ratio is herein called log 10 inactivation.
  • the optimal concentration of methylene blue for maximal virus inactivation is about 5 ⁇ M.
  • bacteriophage ⁇ 6 and 7.5 ⁇ l of 1 mg/ml methylene blue were added to 4 ml samples containing varying concentrations of plasma in Unisol.
  • concentrations of plasma varied between 2.5% and 100%.
  • the final concentration of methylene blue in each sample was 5 ⁇ M.
  • the control sample contained bacteriophage, methylene blue and 100% plasma.
  • phenthiazin-5-ium dyes are unsuitable for decontaminating blood components.
  • phenthiazin-5-ium dyes are able to decontaminate in the presence of high concentrations of plasma.
  • phenthiazin-5-ium dyes were able to decontaminate in the presence of high concentrations of plasma.
  • the ineffectiveness of treatment using other dyes is most likely the result of binding of the dye to proteins and other plasma constituents so that at higher plasma concentrations virus binding is competitively inhibited.
  • this does not occur with the phenthiazin-5-ium dyes used in this invention sufficiently to interfere with viral inactivation or, as demonstrated, infra., to substantially harm or alter cellular blood components.
  • Virus Vesicular Stomatitis Virus
  • Red cells were prepared by centrifugation of whole blood to form packed red cells. The supernatant was expressed off. The remaining red cell pellet had a hematocrit of 85-95% (volume percent occupied by red cells).
  • packed red cells were diluted with 0.9% saline to a hematocrit of 55% and then the red cells were leukocyte-depleted by filtration through a three log filter that decreases the leukocyte concentration by a factor of 10 3 . Aliquots of the red cells were diluted with ARC 8 to final hematocrits of 15 or 30% and contained about 2.5% plasma.
  • the experiment was also performed with a red blood cell sample at a hematocrit of 55% with 5 ⁇ M methylene blue and 60 minutes of light exposure at a fluence rate of 0.8 mW/cm 2 .
  • the sample was placed on a reciprocating shaker during exposure to the light.
  • the log 10 inactivation was 5.7.
  • VSV is rapidly inactivated in plasma by light plus methylene blue.
  • PC platelet concentrates
  • LDPC leukodepleted platelet concentrates
  • leukocytes from the buffy coat were incubated in the presence of methylene blue. After incubation the leukocytes were spun down and the concentration of methylene blue in the supernatant was compared to the concentration of methylene blue in the absence of leukocytes. There was no difference in concentration of methylene blue, which indicated that, although leukocytes contain DNA, they do not bind or take up methylene blue.
  • LDPC leukodepleted platelet concentrate
  • the blood bags were filled with 62 ml of red blood cells, which had been leukodepleted with a 5 log filter, diluted to a hematocrit of 30%, and inoculated with bacteriophage ⁇ 6 as in the previous Examples. In order to prevent any decrease in the light intensity over the length of the bags, they were sealed with hemostats, rather than relying on the ports, which would have blocked light flux. Samples were extracted with a needle through the side of the bag. As indicated in the TABLE 8, treatment with methylene blue and light in the gas permeable bags inactivated the phage.
  • Red blood cells leukodepleted (with a 5 log filter) and suspended at a hematocrit of 30% in ARC 8.
  • thionin The ability of the phenthiazin-5-ium dye, thionin, to inactivate bacteriophage ⁇ 6 in 100% plasma in the presence of red light was studied. From the results, which are set forth in TABLE 9, it was concluded that thionin is effective for decontaminating whole blood and blood components.
  • Red blood cells leukodepleted (with a 5 log filter) and suspended at a hematocrit of 30% in ARC 8.
  • Red blood cells were leukodepleted with a 5 log filter and suspended at a hematocrit of 30% in ARC 8.
  • Red blood cells leukodepleted (with a 5 log filter) and suspended at a hematocrit of 30% in ARC 8.
  • C - n control red blood cells stored for "n” days.
  • T - n treated red blood cells stored for "n” days.
  • a 62 ml sample of cells was introduced into a blood bag as described in Example 7 and the blood bag was stored at 4°C. At the times indicated in TABLE 11, samples were removed from the bag with a needle and the in vitro properties of the cells were assessed. A second 62 ml sample of cells was introduced into a blood bag and stored at 40°C. This second bag was the control.
  • the in vitro properties that were measured included: extracellular pH; percentage of hemolysis; the concentrations of ATP and 2, 3-DPG; and morphology.
  • the in vitro properties of the treated and untreated samples were measured and compared to ascertain the effects, if any, of light and methylene blue on stored red blood cells.
  • Platelet concentrates were inoculated with VSV or bacteriophage ⁇ 6 and treated with 1 ⁇ M MB and 6.4 joules/cm 2 of red light. Treatment inactivated at least 6 logs of VSV and the phage. The effects of treatment on platelet morphology and the recovery of the treated platelets from hypotonic stress were examined.
  • a platelet concentrate in 16% plasma/Unisol was leukodepleted with a three log filter. Three hundred ⁇ l of differing concentrations of leukocytes were added back to 2.7 ml of platelet concentrate. Bacteriophage stock was added as in the previous Examples. Methylene blue was then added to each sample to a final concentration of 5 ⁇ M. Each sample was irradiated for 5 minutes by light having a fluence rate of 2 mW/cm 2 . The results are shown in Figure 3 from which it can be concluded that their presence inhibits viral inactivation by methylene blue.

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Abstract

Cette invention concerne un procédé pour décontaminer le sang et les composants cellulaires du sang en traitant le sang, le composant sanguin ou les compositions renfermant le sang ou le composant sanguin avec un colorant au phenthiazine-5-ium et de la lumière pendant une durée suffisante pour rendre inactifs tous les contaminants pathogènes. Avec le procédé de cette invention on peut décontaminer le sang ou les composants cellulaires du sang sans qu'ils subissent d'altération significative, ce qui les rend donc tout à fait aptes à la transfusion.
PCT/US1991/002976 1990-05-01 1991-05-01 Decontamination du sang entier et des composants cellulaires a l'aide de colorants au phenthiazine-5-ium et de lumiere WO1991016911A1 (fr)

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EP0587832A1 (fr) * 1992-02-10 1994-03-23 Baxter International Inc. Procede de test d'une unite de sang afin de detecter une contamination virale
EP0809433A1 (fr) * 1995-12-19 1997-12-03 Baxter International Inc. Systemes et procedes d'elimination du plasma des contaminants libres ou entraines
US6030767A (en) * 1997-01-21 2000-02-29 The American National Red Cross Intracellular and extracellular decontamination of whole blood and blood components by amphiphilic phenothiazin-5-ium dyes plus light
US6165711A (en) * 1996-04-09 2000-12-26 Baxter Aktiengesellschaft Process for disintegrating nucleic acids and preparing biological products of guaranteed quality
WO2005054217A1 (fr) * 2003-11-28 2005-06-16 Photopharmica Limited Derives ameliores (2) de bleu de methylene biologiquement actifs
US7303834B2 (en) 1998-09-11 2007-12-04 Gore Enterprise Holdings, Inc. Catalytic coatings and fuel cell electrodes and membrane electrode assemblies made therefrom
US7371744B2 (en) * 2001-05-30 2008-05-13 Photopharmica Limited Biologically active methylene blue derivatives

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US4950665A (en) * 1988-10-28 1990-08-21 Oklahoma Medical Research Foundation Phototherapy using methylene blue
WO1990013296A1 (fr) * 1989-05-11 1990-11-15 Oklahoma Medical Research Foundation Therapie antivirale utilisant des colorants a base de thiazine et de xanthene

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US4305390A (en) * 1975-11-28 1981-12-15 Massachusetts Institute Of Technology Method for generating oxygen in an excited electronic state and inactivation of microorganisms
US4727027A (en) * 1983-05-02 1988-02-23 Diamond Scientific Co. Photochemical decontamination treatment of whole blood or blood components
JPS61275228A (ja) * 1985-03-14 1986-12-05 バクスタ−、トラベノ−ル、ラボラトリ−ズ、インコ−ポレイテツド 治療用タンパク組成物中のビ−ルスの光動的不活性化
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WO1990013296A1 (fr) * 1989-05-11 1990-11-15 Oklahoma Medical Research Foundation Therapie antivirale utilisant des colorants a base de thiazine et de xanthene

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EP0587832A4 (en) * 1992-02-10 1998-07-29 Baxter Int Method for testing blood units for viral contamination
EP0587832A1 (fr) * 1992-02-10 1994-03-23 Baxter International Inc. Procede de test d'une unite de sang afin de detecter une contamination virale
EP0809433A1 (fr) * 1995-12-19 1997-12-03 Baxter International Inc. Systemes et procedes d'elimination du plasma des contaminants libres ou entraines
EP0809433A4 (fr) * 1995-12-19 2001-01-10 Baxter Int Systemes et procedes d'elimination du plasma des contaminants libres ou entraines
US6165711A (en) * 1996-04-09 2000-12-26 Baxter Aktiengesellschaft Process for disintegrating nucleic acids and preparing biological products of guaranteed quality
US6030767A (en) * 1997-01-21 2000-02-29 The American National Red Cross Intracellular and extracellular decontamination of whole blood and blood components by amphiphilic phenothiazin-5-ium dyes plus light
US7303834B2 (en) 1998-09-11 2007-12-04 Gore Enterprise Holdings, Inc. Catalytic coatings and fuel cell electrodes and membrane electrode assemblies made therefrom
US7732439B2 (en) 2001-05-30 2010-06-08 Photopharmica Limited Biologically active methylene blue derivatives
US7371744B2 (en) * 2001-05-30 2008-05-13 Photopharmica Limited Biologically active methylene blue derivatives
US7855197B2 (en) 2001-05-30 2010-12-21 Photopharmica Limited Biologically active methylene blue derivatives
US7915254B2 (en) 2001-05-30 2011-03-29 Photopharmica Limited Biologically active methylene blue derivatives
US8188074B2 (en) 2001-05-30 2012-05-29 Photopharmica Limited Biologically active methylene blue derivatives
WO2005054217A1 (fr) * 2003-11-28 2005-06-16 Photopharmica Limited Derives ameliores (2) de bleu de methylene biologiquement actifs

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