WO1997021346A1 - Inactivation des virus non enveloppes - Google Patents

Inactivation des virus non enveloppes Download PDF

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Publication number
WO1997021346A1
WO1997021346A1 PCT/US1996/009837 US9609837W WO9721346A1 WO 1997021346 A1 WO1997021346 A1 WO 1997021346A1 US 9609837 W US9609837 W US 9609837W WO 9721346 A1 WO9721346 A1 WO 9721346A1
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compound
psoralen
compounds
blood
blood product
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PCT/US1996/009837
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English (en)
Inventor
Susan Wollowitz
Stephen T. Isaacs
Gary Wiesehahn
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Cerus Corporation
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Priority to AU62675/96A priority Critical patent/AU6267596A/en
Publication of WO1997021346A1 publication Critical patent/WO1997021346A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
    • A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods

Definitions

  • the present invention provides methods of using new and known compounds to inactivate non-enveloped viruses in health related products to be used in vivo and in vitro.
  • the present invention particularly provides methods of inactivating human parvoviruses in blood products and blood products.
  • HCV hepatitis B
  • HCV hepatitis C
  • HCV Human Immunodeficiency Virus
  • the risk of transmission from single donor blood products is varied, generally in the range of 1 in 3- 50,000.
  • Some clinical manifestations of parvovirus include: erythema infectiosum, arthropathy, hy drops fetalis, aplastic crisis, chronic anemia, neurologic disease, and rheumatologic disease.
  • this and other viruses which are not commonly screened for still represent a significant health risk to the population receiving transfused blood products.
  • testing will not insure the safety of the blood supply against future unknown pathogens that may enter the donor population resulting in transfusion associated transmission before sensitive tests can be implemented.
  • CMV Cytomegalovirus
  • parvovirus B19 in humans are common. When they occur in healthy, immunocompetent adults, they nearly always result in asymptomatic seroconversion. Because such a large part of the population is seropositive, exclusion of positive units would result in substantial limitation of the blood supply.
  • An alternative approach to eliminate transmission of viral diseases through blood products is to develop a means to inactivate pathogens in transfusion products.
  • Psoralens are tricyclic compounds formed by the linear fusion of a furan ring with a coumarin. Psoralens can intercalate between the base pairs of double-stranded nucleic acids, forming covalent adducts to pyrimidine bases upon absorption of long wave ultraviolet light (UVA).
  • UVA long wave ultraviolet light
  • the covalently bonded psoralens act as inhibitors of DNA replication and thus have the potential to stop the replication process. Due to this DNA binding capability, psoralens are of particular interest in relation to solving the problems inherent in creating and maintaining a pathogen blood supply. Some known psoralens have been shown to inactivate viruses in some blood products. H.J. Alter et al., The Lancet (ii:1446) (1988); L. Lin et al., Blood 74:517 (1989) (decontaminating platelet concentrates); G.P. Wiesehahn et al., US Patents Nos.
  • Psoralen photoinactivation is only feasible if the ability of the psoralen to inactivate viruses is sufficient to ensure a safety margin in which complete inactivation will occur.
  • the psoralen must not be such that it will cause damage to blood products.
  • the methods just described, when applied using known psoralens, require the use of difficult and expensive procedures to avoid causing damage to blood products. For example, some compounds and protocols have necessitated the removal of molecular oxygen from the reaction before exposure to light, to prevent damage to blood products from oxygen radicals produced during irradiation. See L. Lin et al., Blood 74:517 (1989); US Patent No. 4,727,027, to Wiesehahn. This is a costly and time consuming procedure.
  • PCD photochemical decontamination
  • a method of inactivating non-enveloped viruses such as human parvoviruses, is needed which displays low mutagenicity and which does not result in significant damage to blood products for which it is used.
  • the present invention provides methods of using new and known compounds to inactivate non-enveloped viruses, such as human parvoviruses, in health related products to be used in vivo and in vitro, and particularly, in blood products and blood products in synthetic media.
  • non-enveloped viruses such as human parvoviruses
  • the present invention contemplates methods of inactivating non- enveloped viruses in blood products, comprising, in the following order: a) providing, in any order, i) a psoralen; ii) photoactivating means for photoactivating said psoralen; and iii) a blood product; b) adding said psoralen to said blood product; and c) photoactivating said psoralen, so as to inactivate any non-enveloped virus present.
  • a method is contemplated wherein said blood product comprises platelets or plasma.
  • the present invention further contemplates photoactivating means comprises a photoactivation device capable of emitting a spectrum of electromagnetic radiation comprising wavelengths between 180 nm and 400 nm and intensity between 1 and 30 mW/cm ⁇ and where said blood product is exposed to said intensity for between 1 second and thirty minutes.
  • the invention contemplates that said spectrum of electromagnetic radiation comprises wavelengths between 320 nm and 380 nm.
  • said blood product is in a synthetic media.
  • the synthetic media comprises a phosphate buffered salt solution.
  • said psoralen is added to said blood product to a final concentration of between .1 and 250 ⁇ M.
  • the invention specifically contemplates that said psoralen is added to said blood product to a final concentration of between 100 and 200 ⁇ M.
  • said aminopsoralen is a 4'- primaryamino-substituted psoralen or a 5'-primaryamino-substituted psoralen.
  • said aminopsoralen comprises: a) a substituent R] on the 4' carbon atom, selected from the group comprising: -(CH 2 ) U -NH 2 , -(CH 2 ) W -R 2 -(CH 2 ) Z -NH 2 , -(CH 2 ) w -R 2 -(CH 2 ) ⁇ - R3-(CH 2 ) Z -NH 2 , and -(CH 2 ) w -R 2 -(CH 2 ) x -R3-(CH 2 ) y -R4-(CH 2 ) z -NH 2 ; wherein R 2 , R3, and R4 are independently selected from the group comprising O and NH, in which u is
  • the invention contemplates that said compound comprises 4'-(4- amino-2-oxa)butyl-4,5',8-trimethylpsoralen.
  • the invention further contemplates the administration of the blood product by intravenous infusion to a mammal.
  • said aminopsoralen comprises: a) a substituent Ri on the 5' carbon atom, selected from the group comprising: -(CH 2 ) U -NH 2 , -(CH 2 ) W -R 2 -(CH2) Z -NH 2 , -(CH 2 ) W -R2- (CH 2 ) ⁇ -R 3 -(CH ) z -NH 2 , and -(CH2)w-R2-(CH 2 ) ⁇ -R3-(CH 2 ) y -R4-(CH2) z - NH 2 ; wherein R 2/ R3, and R4 are independently selected from the group comprising O and NH, and in which u is a whole number from 1 to 10, w is a whole number from 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2 to 5, and z is a whole number from 2 to 6; and, b) substituents R5, R ⁇ , and R7
  • the invention provides methods of inactivating human parvovirus B19 in blood products, comprising, in the following order: a) providing, in any order, i) an aminopsoralen; ii) photoactivating means for photoactivating said aminopsoralen; and iii) a blood product suspected of being contaminated with human parvovirus B19; b) adding said aminopsoralen to said blood product; and c) photoactivating said aminopsoralen, so as to inactivate any human parvovirus B19 present.
  • the invention further contemplates that said blood product comprises platelets.
  • the invention further contemplates that said blood product comprises plasma.
  • said photoactivating means comprises a photoactivation device capable of emitting a spectrum of electromagnetic radiation comprising wavelengths between 180 nm and 400 nm and intensity between 1 and 30 mW/cm ⁇ .
  • the invention further contemplates that said blood product is exposed to said intensity for between 1 second and thirty minutes.
  • said spectrum of electromagnetic radiation comprises wavelengths between 320 nm and 380 nm.
  • said blood product is in a synthetic media.
  • said synthetic media comprises a phosphate buffered salt solution.
  • said aminopsoralen is added to said blood product to a final concentration of between .1 and 250 ⁇ M.
  • said aminopsoralen is added to said blood product to a final concentration of between 100 and 150 ⁇ M.
  • the invention further contemplates that said aminopsoralen is a 4'- primaryamino-substituted psoralen.
  • the invention further contemplates that said aminopsoralen is a 5'-primaryamino-substituted psoralen.
  • said aminopsoralen comprises: a) a substituent R on the 4' carbon atom, selected from the group comprising: -(CH 2 ) U -NH 2 , -(CH 2 ) W -R2-(CH 2 ) Z -NH 2 , -(CH 2 ) w -R 2 -(CH 2 ) ⁇ - R3-(CH 2 )z-NH 2 , and -(CH2) w -R2-(CH 2 ) x -R3-(CH 2 ) y -R4-(CH 2 ) z -NH 2 ; wherein R 2/ R3, and R4 are independently selected from the group comprising O and NH, in which u is a whole number from 1 to 10, w is a whole number from 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2 to 5, and z is a whole number from 2 to 6; and b) substituents R5, R6, and
  • the invention further contemplates that said compound comprises 4'-(4-amino-2- oxa)butyl-4,5',8-trimethylpsoralen.
  • the invention further contemplates the administration of said platelet preparation by intravenous infusion to a mammal.
  • said aminopsoralen comprises: a) a substituent R on the 5' carbon atom, selected from the group comprising: -(CH 2 ) U -NH 2/ -(CH 2 ) w -R 2 -(CH 2 )z- NH 2 , -(CH 2 )w-R2-(CH 2 ) x -R 3 -(CH 2 ) z -NH 2 , and -(CH 2 ) W -R 2 -(CH 2 ) X -R 3 - (CH 2 ) -R4-(CH 2 ) z -NH2; wherein R 2/ R3, and R4 are independent ⁇ selected from the group comprising O and NH, and in which u is a whole number from 1 to 10, w is a whole number from 1 to 5, x is a whole number from 2 to 5, y is a whole number from 2 to 5, and z is a whole number from 2 to 6; and, b)
  • the invention provides methods of inactivating human parvovirus B19 in a blood product, comprising, in the following order: a) providing, in any order, i) 4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen, ii) photoactivating means for photoactivating said 4'-(4-amino-2-oxa)butyl- 4,5',8-trimethylpsoralen, and iii) a platelet preparation suspected of being contaminated with said virus; b) adding said 4'-(4-amino-2-oxa)butyl-4,5',8- trimethylpsoralen to said platelet preparation; and c) photoactivating said 4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen, so as to inactivate said virus, without causing significant damage to said platelet preparation.
  • FIG. 1 details the compound s y nthesis scheme of two isomers of bromomethyl-trialkvlpsoralen.
  • FIG. 2 details the compound synthesis scheme of several 4 -primary amino substituted psoralens.
  • FIG. 3 details the compound synthesis scheme of several 4'- primar y amino substituted psoralens.
  • FIG. 4 details the compound synthesis scheme of several psoralens having a terminal amine linked to the psoralen at the 4 -position by an alkyl chain.
  • FIG. 5 details the compound synthesis scheme of several 5'- primaryamino-substituted psoralens.
  • FIG. 6 is a perspective view of one embodiment of the device of the present invention.
  • FIG. 7 shows the impact of concentration on the log kill of R17 when Compounds 1 - 3 of the present invention are photoactivated.
  • FIG. 8 shows the impact of concentration on the log kill of R17 when
  • FIG. 9 shows the impact of concentration on the log kill of R17 when Compounds 2 and 6 of the present invention are photoactivated.
  • FIG. 10 shows the impact of concentration on the log kill of R17 when Compounds 6 and 18 of the present invention are photoactivated.
  • FIG. 11 shows the impact of concentration on the log kill of R17 when Compound 16 of the present invention is photoactivated.
  • FIG. 12 shows the impact of varying Joules/cm ⁇ (Watt second /cm ⁇ ) of irradiation on the log titer of R17 for Compound 6 of the present invention.
  • FIG. 13 shows the impact of varying Joule/cm ⁇ of irradiation on the log titer of R17 for Compounds 7, 9 and 10 of the present invention.
  • FIG. 14 shows the impact of var y ing Joules/cm ⁇ of irradiation on the log titer of R17 for Compounds 7 and 12 of the present invention.
  • FIG. 15 shows the impact of varying Joules/cm ⁇ of irradiation on the log titer of R17 for Compound 15 of the present invention.
  • FIG. 16 shows the impact of varying Joules /cm ⁇ of irradiation on the log titer of R17 for Compound 17 of the present invention.
  • FIG. 17 shows the impact of var y ing Joules/cm ⁇ of irradiation on the log titer of R17 for Compounds 6 and 17 of the present invention.
  • FIG. 18 shows the impact of varying Joules/cm ⁇ of irradiation on the log titer of R17 for Compounds 6 and 15 of the present invention.
  • FIG. 19 shows the effect of varying the concentration of Compounds 2 and 6 of the present invention, in plasma.
  • FIG. 20 shows the effect of varying the concentration of Compounds 2 and 6 of the present invention, in synthetic medium.
  • FIG. 21A schematically shows the standard blood product separation approach used presently in blood banks.
  • FIG. 21 B schematically shows an embodiment of the present invention whereby synthetic media is introduced to platelet concentrate prepared as in FIG. 25A.
  • FIG. 21C schematically shows one embodiment of the decontamination approach of the present invention applied specifically to platelet concentrate diluted with synthetic media as in FIG. 25B.
  • FIG. 22A is a graph comparing a day one control (Cl), a control stored for 5 da s (D5), ultraviolet light alone (UV) and treatment with ultraviolet light and Compound 2 at 100 ⁇ M (PCD) by their effects on platelet function as measured by platelet count, "n" represents the number of experiments represented by the data point.
  • FIG. 22B is a graph comparing a day one control (Cl), a control stored for 5 days (D5), ultraviolet light alone (UV) and treatment with ultraviolet light and Compound 2 at 100 ⁇ M (PCD) by their effects on platelet function as measured by platelet aggregation, "n" represents the number of experiments represented by the data point.
  • FIG. 22C is a graph comparing a day one control (Cl), a control stored for 5 days (D5), ultraviolet light alone (UV) and treatment with ultraviolet light and Compound 2 at 100 ⁇ M (PCD) by their effects on platelet function as measured by GMP-140 expression, "n" represents the number of experiments represented by the data point.
  • FIG. 22D is a graph comparing a day one control (Cl), a control stored for 5 days (D5), ultraviolet light alone (UV) and treatment with ultraviolet light and Compound 2 at 100 ⁇ M (PCD) by their effects on platelet function as measured by pH. "n” represents the number of experiments represented by the data point.
  • FIG. 23A is a graph comparing a day one control (Cl), a control stored for 5 days (D5), ultraviolet light alone (UV) and treatment with ultraviolet light and Compound 6 at 100 ⁇ M (PCD) by their effects on platelet function as measured by platelet count, "n" represents the number of experiments represented by the data point.
  • FIG. 23B is a graph comparing a day one control (Cl), a control stored for 5 days (D5), ultraviolet light alone (UV) and treatment with ultraviolet light and Compound 6 at 100 ⁇ M (PCD) by their effects on platelet function as measured by platelet aggregation, "n" represents the number of experiments represented by the data point.
  • FIG. 23C is a graph comparing a day one control (Cl), a control stored for 5 days (D5), ultraviolet light alone (UV) and treatment with ultraviolet light and Compound 6 at 100 ⁇ M (PCD) by their effects on platelet function as measured by GMP-140 expression, "n" represents the number of experiments represented by the data point.
  • FIG. 23D is a graph comparing a day one control (Cl), a control stored for 5 days (D5), ultraviolet light alone (UV) and treatment with ultraviolet light and Compound 6 at 100 ⁇ M (PCD) by their effects on platelet function as measured by pH. "n” represents the number of experiments represented by the data point.
  • FIG. 24A is a graph comparing a day one control (Cl), a control stored for 5 days (D5), ultraviolet light alone (UV) and treatment with ultraviolet light and Compound 17 at 100 ⁇ M (PCD) by their effects on platelet function as measured by platelet count, "n" represents the number of experiments represented by the data point.
  • FIG. 24B is a graph comparing a day one control (Cl), a control stored for 5 days (D5), ultraviolet light alone (UV) and treatment with ultraviolet light and Compound 17 at 100 ⁇ M (PCD) by their effects on platelet function as measured by platelet aggregation, "n" represents the number of experiments represented by the data point.
  • FIG. 24C is a graph comparing a day one control (Cl), a control stored for 5 days (D5), ultraviolet light alone (UV) and treatment with ultraviolet light and Compound 17 at 100 ⁇ M (PCD) by their effects on platelet function as measured by GMP-140 expression, "n” represents the number of experiments represented by the data point.
  • FIG. 24D is a graph comparing a day one control (Cl), a control stored for 5 days (D5), ultraviolet light alone (UV) and treatment with ultraviolet light and Compound 17 at 100 ⁇ M (PCD) by their effects on platelet function as measured by pH. "n” represents the number of experiments represented by the data point.
  • FIG. 25A is a graph comparing a day one control (Cl), a control stored for 5 days (D5), ultraviolet light alone (UV) and treatment with ultraviolet light and Compound 18 at 100 ⁇ M (PCD) by their effects on platelet function as measured by platelet count, "n” represents the number of experiments represented by the data point.
  • FIG. 25B is a graph comparing a day one control (Cl), a control stored for 5 days (D5), ultraviolet light alone (UV) and treatment with ultraviolet light and Compound 18 at 100 ⁇ M (PCD) by their effects on platelet function as measured by platelet aggregation, "n” represents the number of experiments represented by the data point.
  • FIG. 25B is a graph comparing a day one control (Cl), a control stored for 5 days (D5), ultraviolet light alone (UV) and treatment with ultraviolet light and Compound 18 at 100 ⁇ M (PCD) by their effects on platelet function as measured by platelet aggregation, "n” represents the number of experiments represented by the data
  • FIG. 25C is a graph comparing a day one control (Cl), a control stored for 5 days (D5), ultraviolet light alone (UV) and treatment with ultraviolet light and Compound 18 at 100 ⁇ M (PCD) by their effects on platelet function as measured by GMP-140 expression, "n” represents the number of experiments represented by the data point.
  • FIG. 25D is a graph comparing a day one control (Cl), a control stored for 5 days (D5), ultraviolet light alone (UV) and treatment with ultraviolet light and Compound 18 at 100 ⁇ M (PCD) b y their effects on platelet function as measured by pH. "n” represents the number of experiments represented by the data point.
  • FIG 26 is a graph showing the kinetics of HIV expression with and without TNF-alphf*.
  • FIG 27 is a bar graph showing the inhibition of HIV p24 expression in psoralen treated OM-10.1 cells.
  • FIG 28 is a bar graph showing Compound 2 inactivation of proviral HIV-l in OM-10.1 cells.
  • FIG. 29 is a graph showing Compound 2 inactivation of parvovirus B19.
  • FIG. 30 is a graph showing psoralen inactivation of parvovirus B19. DESCRIPTION OF THE INVENTION
  • the present invention provides methods of inactivating non- enveloped viruses in health related products to be used in vivo and in vitro, and in particular, blood products. Specifically, the present invention provides methods of inactivating human parvoviruses in blood products.
  • the inactivation methods of the present invention provide methods of inactivating pathogens, and, in particular, viruses, including several forms of cytomegalovirus (CMV), bovine viral diarrhea virus (BVDV), proviral human immunodeficiency virus (proviral HIV), and parvovirus, such as human parvovirus B19 (B19), in blood products prior to use in vitro or in vivo.
  • viruses including several forms of cytomegalovirus (CMV), bovine viral diarrhea virus (BVDV), proviral human immunodeficiency virus (proviral HIV), and parvovirus, such as human parvovirus B19 (B19)
  • CMV cytomegalovirus
  • BVDV bovine viral diarrhea virus
  • proviral HIV proviral human immunodeficiency virus
  • parvovirus such as human parvovirus B19 (B19)
  • Methods of the present invention use devices for photoactivation and specifically, for photoactivation of photoreactive nucleic acid binding compounds.
  • the specific devices used in the examples described below are either described in detail in issued U.S. Patent No. 5,399,719 (the entire contents of which is hereby incorporated by reference) or have been tested (results not shown) and prove to have approximately equivalent properties to the devices described.
  • the present invention contemplates a photoactivation device for treating photoreactive compounds, comprising: a) means for providing appropriate wavelengths of electromagnetic radiation to cause photoactivation of at least one photoreactive compound; b) means for supporting a plurality of samples in a fixed relationship with the radiation providing means during photoactivation; and c) means for maintaining the temperature of the samples within a desired temperature range during photoactivation.
  • the present invention also contemplates methods, comprising: a) supporting a plurality of sample containers, containing one or more photoreactive compounds, in a fixed relationship with a fluorescent ' ource of electromagnetic radiation; b) irradiating the plurality of sample containers simultaneously with electromagnetic radiation to cause photoactivation of at least one photoreactive compound; and c) maintaining the temperature of the sample within a desired temperature range during photoactivation.
  • the present application is not limited to any particular light sources described here or in U.S. Patent No. 5,399,719 or similar thereto.
  • the present invention contemplates that any light source which delivers comparable intensity, wavelength and distribution of light in a temperature controlled fashion is additionally appropriate for use in the present invention.
  • the major features of one embodiment of the device of the present invention involve: A) an inexpensive source of ultraviolet radiation in a fixed relationship with the means for supporting the sample containers, B) rapid photoactivation, C) large sample processing, D) temperature control of the irradiated samples, and E) inherent safety.
  • a preferred photoactivation device of the present invention has an inexpensive source of ultraviolet radiation in a fixed relationship with the means for supporting the sample vessels.
  • wavelength is herein described in terms of nanometers ("nm”; 10 " 9 meters)
  • ultraviolet radiation extends from approximately 180 nm to 400 nm.
  • a radiation source by virtue of filters or other means, does not allow radiation below a particular wavelength (e.g. 320 nm), it is said to have a low end “cutoff” at that wavelength (e.g. "a wavelength cutoff at 300 nanometers”).
  • a radiation source allows only radiation below a particular wavelength (e.g. 360 nm), it is said to have a high end “cutoff” at that wavelength (e.g. "a wavelength cutoff at 360 nanometers”).
  • any photochemical reaction it is desired to eliminate or least minimize any deleterious side reactions.
  • Some of these side reactions can be caused by the excitation of endogenous chromophores that may be present during the photoactivation procedure.
  • the endogenous chromophores are the nucleic acid bases themselves. Restricting the photoactivation process to wavelengths greaier than 320 nm minimizes direct nucleic acid damage since there is very little absorption by nucleic acids above 313 nm.
  • the nucleic acid is typically present together with additional biological constituents. If the biological fluid is just protein, the 320 nm cutoff will be adequate for minimizing side reactions (aromatic amino acids do not absorb above 320 nm). If the biological fluid includes other analytes, there may be constituents that are sensitive to particular wavelengths of light. In view of the presence of these endogenous constituents, it is intended that the device of the present invention be designed to allow for irradiation within a small range of specific and desirable wavelengths, and thus avoid damage blood components. The preferred range of desirable wavelengths is between 320 and 350 nm. Some selectivity can be achieved by choice of commercial irradiation sources.
  • the device of the present invention comprises an additional filtering means, such as a glass cut off filter or a liquid filter solution that transmits onl a specific region of the electromagnetic spectrum, which yields a transmission window of 320-400 nm . It is not intended that the present invention be limited by the particular filter employed. Several inorganic salts and glasses satisfy the necessary requirements.
  • UV radiation When ultraviolet radiation is herein described in terms of irradiation, it is expressed in terms of intensity flux (milliwatts per square centimeter or “mW cm-2" or J/cm ⁇ sec). "Output" is herein defined to encompass both t > emission of radiation (yes or no; on oi off) as well as the level of irradiation. In a preferred embodiment, intensity is monitored at 4 locations: 2 for each side of the plane of irradiation.
  • a preferred source of ultraviolet radiation is a fluorescent source.
  • fixed relationship is defined as comprising a fixed distance and geometry between the sample and the light source during the sample irradiation. Distance relates to the distance between the source and the sample as it is supported. It is known that light intensity from a point source is inversely related to the square of the distance from the point source. Thus, small changes in the distance from the source can have a drastic impact on intensity. Since changes in intensity can impact photoactivation results, changes in distance are avoided in the devices of the present invention. This provides reproducibility and repeatability.
  • Geometry relates to the positioning of the light source. For example, it can be imagined that light sources could be placed around the sample holder in many ways (on the sides, on the bottom, in a circle, etc.).
  • the geometry used in a preferred embodiment of the present invention allows for uniform light exposure of appropriate intensity for rapid photoactivation.
  • the geometry of a preferred device of the present invention involves multiple sources of linear lamps as opposed to single point sources. In addition, there are several reflective surfaces and several absorptive surfaces. Because of this complicated geometry, changes in the location or number of the lamps relative to the position of the samples to be irradiated are to be avoided in that such changes will result in intensity changes.
  • the light source of the preferred embodiment of the present invention allows for rapid photoactivation.
  • the intensity characteristics of the irradiation device have been selected to be convenient with the anticipation that many sets of multiple samples may need to be processed. With this anticipation, a fifteen minute exposure time or less is a practical goal.
  • relative position of the elements of the preferred device have been optimized to allow for approximately fifteen minutes of irradiation time, so that, when measured for the wavelengths between 320 and 350 nanometers, an intensity flux greater than approximately 1 mW cm-2 (.001 J/cm ⁇ sec.) is provided to the sample vessels.
  • one element of the de ces of the present invention is a means for supporting a plurality of blood bags.
  • the supporting means comprises a blood bag support placed between two banks of lights.
  • Temperature control is important because the temperature of the sample at the time of exposure to light can dramatically impact the results.
  • irradiation at low temperatures enhances the covalent addition of HMT to 5S rRNA by two fold at 4°C compared to 20°C.
  • Thompson et al. J. Mol. Biol. 147:417 (1981).
  • Even further temperature induced enhancements of psoralen binding have been reported with synthetic polynucleotides. Thompson et al., Biochemistry 21 :1363 (1982).
  • Ultraviolet radiation can cause severe burns. Depending on the nature of the exposure, it may also be carcinogenic.
  • the light source of a preferred embodiment of the present invention is shielded from the user. This is in contrast to the commercial hand-held ultraviolet sources as well as the large, high intensity sources.
  • the irradiation source is contained within a housing made of material that obstructs the transmission of radiant energy (i.e. an opaque housing). No irradiation is allowed to pass to the user. This allows for inherent safety for the user.
  • Photoactivation compounds in General
  • Photoactivation compounds defines a family of compounds that undergo chemical change in response to electromagnetic radiation.
  • Table 1 is a partial list of photoactivation compounds.
  • the preferred speci.es of photoreactive compounds described herein is commonly referred to as the furocoumarins.
  • the present invention contemplates those compounds described as psoralens: [7H- furo(3,2-g)-(l)-benzopyran-7-one, or ⁇ -lactone of 6-hydroxy-5- benzofuranacrylic acid], which are linear:
  • Psoralen derivatives are derived from substitution of the linear furocoumarin at the 3, 4, 5, 8, 4', or 5' positions.
  • 8-Methoxypooralen (known in the literature under various named, e.g., xanthotoxin, methoxsalen, 8-MOP) is a naturally occurring psoralen with relatively low photoactivated binding to nucleic acids and low mutagenicity in the Ames assay, which is described in the following experimental section.
  • 4'-Aminomethyl-4,5',8-trimethylpsoralen (AMT) is one of most reactive nucleic acid binding psoralen derivatives, providing up to 1 AMT adduct per 3.5 DNA base pairs.
  • AMT also exhibits significant levels of mutagenicity.
  • a new group of psoralens was desired which would have the best characteristics of both 8-MOP and AMT: low mutagenicity and high nucleic acid binding affinity, to ensure safe and thorough inactivation of pathogens.
  • the compounds of the present invention were designed to be such compounds.
  • 4'-primaryamino-substituted psoralen shall herein be defined as a psoralen compound which has an NH 2 group linked to the 4'- position of the psoralen by a hydrocarbon chain having a total length of 2 to 20 carbons, where 0 to 6 of those carbons are independently replaced by NH or O, and each point of replacement is separated from each other point of replacement by at least two carbons, and is separated from the psoralen by at least one carbon.
  • 5'-primaryamino-substituted psoralen shall herein be defined as a psoralen compound which has an NH 2 group linked to the 5 -position of the psoralen by a hydrocarbon chain having a total length of 1 to 20 carbons, where 0 to 6 of those carbons are independently replaced by NH or O, and each point of replacement is separated from each other point of replacement by at least two carbons, and is separated from the psoralen by at least one carbon.
  • Table 2 sets forth the nomenclature used for the psoralen derivatives discussed herein. Note that in this section (entitled “B. Synthesis of the Psoralens”) the roman numerals used to identify compounds correlate with Schematics 1-6 only, and do not correlate with the compound numbers listed in Table 2.
  • the 5'-chloromethyl-4,4',8- trimethylpsoralen (5'-CMT) and 5'-bromomethyl-4,4',8-tr ⁇ methylpsoralen (5'-BrMT) are prepared similarly, using the isomeric starting compound, 4,4',8-trimethylpsoralen (4'-TMP) [U.S. Patent No. 4,294,822, to Kaufman; McLeod, et al., "Synthesis of Benzofuranoid Systems. I. Furocoumarins, Benzofurans and Dibenzofurans," Tetrahedron Letters 237 (1972)].
  • Ai and A 2 are independently selected from the group comprising H and an alkyl chain having 1-6 carbon atoms. Reaction of the 4,8-dialkyl-7-hydr. >xycoumarin with 2-chloro-3-butanone under typical basic conditions, provides 4,8-dialkyl-7-(l-methyl-2- oxo)propyloxycoumarin (I).
  • This material is cyclized by heating in aqueous NaOH to provide 4,8-dialkyl-4',5'-dimethylpsoralen (II).
  • Treatment of the tetrasubstituted psoralen and N-bromosuccinimide (NBS) in a solvent at room temperature up to 150°C leads to bromination at the 4'- or 5'- position, depending upon the conditions used.
  • a catalyst such as dibenzoyl peroxide may be added, but is not necessary. If the solvent used is carbon tetrachloride at reflux, 4,8-dialkyl-5'-bromomethyl-4'- methylpsoralen (IV) is obtained in yields of 50% or greater.
  • the 4,8-dialkylcoumarins are prepared from 2-alkylresorcinols and a 3-oxoalkanoate ester by the Pechmann reaction (Organic Reactions Vol VII, Chap 1, ed. Adams et al, Wiley, NY, (1953)).
  • Claisen rearrangement of the resultant allyl ether gives 4,8-dialkyl-6- allyl-7-hydroxycoumarin.
  • the coumarins are converted to the 4,5',8- trialkylpsoralens using procedures similar to one of several previously described procedures (i.e., see, Bender et al, J. Org. Chem. 44:2176 (1979); Kaufman, US Patent Nos. 4,235,781 and 4,216,154, hereby incorporated by reference).
  • 4,5',8-Irimethylpsoralen is a natural product and is commercially available (Aldrich Chemical Co., Milwaukee, WI).
  • General Scheme of Synthesis of 5'-Substituted Psoralens The 4,4',8-trialkylpsoralens can be prepared in two steps also starting from the 4,8-dialkyl-7-hydroxycoumarins discussed above.
  • the coumarin is treated with an alpha-chloro ketone under basic conditions to give the 4,8-dialkyl-7-(2-oxoalkoxy)coumarin. Cyclization of this intermadiate to the 4,4',8-trialkylcoumarin occurs by heating in aqueous base.
  • Longer chain 4'-( ⁇ -haloalkyl)trialkylpsoralens (herein referred to as longer chain 4'-HATP) where the alkyl groups are selected from the group (CH 2 ) 2 to (CH 2 ) ⁇ o can be prepared under Freidel-Crafts conditions as discussed elsewhere (Olah and Kuhn, J. Org.
  • the terminal hydroxy group can be transformed to an amino group under a variety of conditions (for example see Larock, 'Comprehensive Organic Transformations," VCH Publishers, NY, 1989). Particularly, the hydroxy group can be converted to the ester of methanesulfonic acid (structure VI) in the presence of methanesulphonyl chloride (CH3SO3CI). This can subsequently be converted to the azide in refluxing ethanol and the azide reduced to the final amine, structure VII (examples are Compounds 2, 4 and 7).
  • the method described herein utilizes triphenylphosphine and water in tetrahydrofuran (THF) for the reduction but other methods are contemplated.
  • a preferred method of preparation of structure VII uses the reaction of 4'-HATP with a primary linear alcohol containing a protected amine (e.g., a phthalimido group) at the terminal position in a suitable solvent such as DMF at 25 - 150°C to give V.
  • the amine is then deprotected under standard conditions (e.g., hydrazine or aqueous MeNH 2 to deprotect a phthalimido group [higher alkyl hydrazines, such as benzyl hydrazines, are also contemplated]) to give VII.
  • structure VI can be reacted with diamines, H N-(B')- NH 2 where B' is an alkyl chain (e.g., 1,4,-butanediamine), a monoether (e.g., 3-oxa-l,5-pentanediamine) or a polyether (e.g., 3,6-dioxa-l,8- octanediamine) to give the final product, compound VIII (examples of compounds in this structure group are Compounds 8, 13 and 14).
  • This reaction is carried out with an excess of diamine in acetonitrile at reflux, but other solvents and temperatures are equally possible.
  • This method is also applicable to final products that contain more than two nitrogens in the chain (structure XIII) (examples are Compounds 12 and 15) starting from polyamines of structure XII (e.g., norspermidine or spermine [commercially available from Aldrich, Milwaukee, WI]), however, in this case isomeric structures are also formed in considerable amounts.
  • the preferred method for the preparation of structure XIII is reductive amination of the psoralen-4'-alkanal (XI) with a polyamine of structure XII and a reducing agent such as sodium cyanoborohydride. This reductive amination is applicable to the synthesis of compounds X as well.
  • these compounds (structures XIV) (an example is Compound 3) are prepared either 1) by reaction of the 4'- HATP with potassium phthalimide or azide and subsequent liberation of the desired amine as before, for example, with hydrazine, or 2) conversion of the 4'-HATP to the cyanide compound, followed by reduction, for example with NaBH4-CF3C0 H.
  • the 4,4',8- trialkylpsoralens or the 4,4',8-trialkyl-5'-methylpsoralens can be converted to the 5'-( ⁇ -haloalkyl)-4,4',8-trialkylpsoralens, (herein called 5'-HATP), as detailed in Schematic 5, below. (See Kaufman, U.S. Patent No. 4,294,822 and 4,298,614 for modified version).
  • the present invention contemplates binding new and known compounds to nucleic acid, including (but not limited to) viral nucleic acid and bacterial nucleic acid.
  • One approach of the present invention to binding photoactivation compounds to nucleic acid is photobinding.
  • Photobinding is defined as the binding of photobinding compounds in the presence of photoactivating wavelengths of light.
  • Photobinding compounds are compounds that bind to nucleic acid in the presence of photoactivating wavelengths of light.
  • the present invention contemplates methods of photobinding with photobinding compounds of the present invention.
  • One embodiment of the method of the present invention for photobinding involves the steps: a) providing a photobinding compound of the present invention; and b) mixing the photobinding compound with nucleic acid in the presence of photoactivation wavelengths of electromagnetic radiation.
  • the invention further contemplates a method for modifying nucleic acid, comprising the steps: a) providing photobinding compound of the present invention and nucleic acid; and b) photobinding the photobinding compound to the nucleic acid, so that a compound:nucleic acid complex is formed.
  • a method for modifying nucleic acid comprising the steps: a) providing photobinding compound of the present invention and nucleic acid; and b) photobinding the photobinding compound to the nucleic acid, so that a compound:nucleic acid complex is formed.
  • the present invention contemplates treating a blood product with a photoactivation compound and irradiating to inactivate contaminating pathogen nucleic acid sequences before using the blood product.
  • activation is here defined as the altering of the nucleic acid of a unit of pathogen so as to render the unit of pathogen incapable of replication. This is distinct from “total inactivation”, where all pathogen units present in a given sample are rendered incapable of replication, or “substantial inactivation,” where most of the pathogen units present are rendered incapable of replication. "Inactivation efficiency" of a compound is defined as the level of inactivation the compound can achieve at a given concentration of compound or dose of irradiation.
  • an “inactivation” method may or may not achieve “total inactivation,” it is useful to consider a specific example.
  • a bacterial culture is said to be inactivated if an aliquot of the culture, when transferred to a fresh culture plate and permitted to grow, is undetectable after a certain time period.
  • a minimal number of viable bacteria must be applied to the plate for a signal to be detectable. With the optimum detection method, this minimal number is 1 bacterial cell. With a sub optimal detection method, the minimal number of bacterial cells applied so that a signal is observed may be much greater than 1.
  • the detection method determines a "threshold" below which the "inactivation method” appears to be completely effective (and above which "inactivation” is, in fact, only partially effective).
  • the detection method can theoretically be taken to be the measurement of the level of infection with a disease as a result of exposure to the material.
  • the threshold below which the inactivation method is complete is then taken to be the level of inactivation which is sufficient to prevent disease from occurring due to contact with the material. It is recognized that in this practical scenario, it is not essential that the methods of the present invention result in “total inactivation”. That is to say, “substantial inactivation” will be adequate as long as the viable portion is insufficient to cause disease. Thus “substantially all” oi a pathogen is inactivated when any viable portion of the pathogen which remaining is insufficient to cause disease.
  • the inactivation method of the present invention renders nucleic acid in pathogens substantially inactivated.
  • the inactivation method renders pathogen nucleic acid in blood preparations substantially inactivated. Without intending to be limited to any method by which the compounds of the present invention inactivate pathogens, it is believed that inactivation results from light induced binding of psoralens to pathogen nucleic acid. Further, while it is not intended that the inactivation method of the present invention be limited by the nature of the nucleic acid; it is contemplated that the inactivation method render all forms of nucleic acid (whether DNA, mRNA, etc.) substantially inactivated.
  • the interaction of the pathogen nucleic acid (whether DNA, mRNA, etc.) with the photoacti ation compound preferably prevents replication of the pathogen, such that, if a human is exposed to the treated pathogen, infection will not result.
  • Synthetic media is herein defined as an aqueous synthetic blood or blood product storage media. In one embodiment, the present invention completely effective (and above which "inactivation” is, in fact, only partially effective).
  • the detection method can theoretically be taken to be the measurement of the level of infection with a disease as a result of exposure to the material.
  • the threshold below which the inactivation method is complete is then taken to be the level of inactivation which is sufficient to prevent disease from occurring due to contact with the material. It is recognized that in this practical scenario, it is not essential that the methods of the present invention result in “total inactivation”. That is to sa y , “substantial inactivation” will be adequate as long as the viable portion is insufficient to cause disease. Thus “substantially all" of a pathogen is inactivated when any viable portion of the pathogen which remaining is insufficient to cause disease.
  • the inactivation method of the present invention renders nucleic acid in pathogens substantially inactivated.
  • the inactivation method renders pathogen nucleic acid in blood preparations substantially inactivated. Without intending to be limited to any method by which the compounds of the present invention inactivate pathogens, it is believed that inactivation results from light induced binding of psoralens to pathogen nucleic acid. Further, while it is not intended that the inactivation method of the present invention be limited by the nature of the nucleic acid; it is contemplated that the inactivation method render all forms of nucleic acid (whether DNA, mRNA, etc.) substantially inactivated.
  • the interaction of the pathogen nucleic acid (whether DNA, mRNA, etc.) with the photoactivation compound preferably prevents replication of the pathogen, such that, if a human is exposed to the treated pathogen, infection will not result.
  • Synthetic media is herein defined as an aqueous synthetic blood or blood product storage media.
  • synthetic media comprising a buffered saline solution. This method reduces harm to blood products and permits the use of much lower concentrations of photoactivation compounds.
  • the psoralen photoinactivation method inactivates nucleic acid based pathogens present in blood through a single procedure. Thus, it has the potential to eliminate bacteria, protozoa, and viruses as well. Had an effective decontamination method been available prior to the advent of the AIDS pandemic, no transfusion associated HIV transmission would have occurred. Psoralen-based decontamination has the potential to eliminate all infectious agents from the blood supply, regardless of the pathogen involved. Additionally, psoralen-based decontamination has the ability to sterilize blood products after collection and processing, which in the case of platelet concentrates could solve the problem of low level bacterial contamination and result in extended storage life. Morrow J.F., et al., JAMA 266: 555-558 (1991); Bertolini F., et al., Transfusion 32: 152-156 (1992).
  • Non-enveloped viruses are defined as viruses which do not have a lipid envelope surrounding the nucleic acid. This list is not exhaustive, and is merely representative of the great variety of pathogens psoralens can inactivate.
  • the present invention contemplates the inactivation of these and other viruses b the compounds described herein, including both enveloped and non-enveloped viruses.
  • the compound's ability to inactivate pathogens may be determined by several methods.
  • One technique is to perform a bacteriophage screen; an assay which determines nucleic acid binding of test compounds.
  • a screen of this type, an R17 screen is described in detail in EXAMPLE 12, below. If the R17 screen shows inactivation activity, it is useful to directly test the compound's ability to inactivate a virus.
  • a model pathogen assay may be found below, or in the published literature, which is particularly appropriate to test a compound's inactivation efficiency against the target pathogen or class of pathogens.
  • pathogen models can be identified and selected by finding a model which shares with the target pathogen the following elements: as close a match as possible between model and target pathogen in nucleic acid structure, composition and size (for example, RNA, DNA, single stranded or double stranded); and similar packaging, including the presence or absence of an envelope, and the composition and structure of any protein coat.
  • a final element that must be present in a model is relative ease of use, which encompasses the following factors: ability of the model pathogen to grow in culture and relative safety, which includes considerations of the facilities and skills available to use the model. These elements can be used to determine a predictive model of target pathogen inactivation.
  • the R17 bacteriophage screen is believed to be predictive of the efficiency of compounds against many viruses.
  • R17 was chosen because it was expected to be a very difficult pathogen to inactivate. It is a small, single stranded RNA phage. Without intending to be limited to any means by which the present invention operates, it is expected that shorter pieces of nucleic acid are harder to inactivate because they require a higher frequency of formation of psoralen adducts than do longer pieces of nucleic acid. Further, single stranded RNA pathogens are more difficult to inactivate because psoralens can neither intercalate between base pairs, as with double- stranded nucleic acids, nor form diadducts which function as interstrand crosslinks. Thus it is expected that when inactivation of R17 is achieved, these same conditions will cause the inactivation of many viruses and bacteria.
  • the B19 screen complements the R17 screen by affirming that a given compound which has tested positive in R17 will actually work effectively to inactivate human parvoviruses. Thus, if a compound shows activity in the R17 screen, it is next tested in the viral inactivation screen.
  • the second property that is important in testing a compound for use in methods of the present invention is mutagenicity.
  • the most widely used mutagen/ carcinogen screening assay is the Ames test. This assay is described by D.M. Maron and B.N. Ames in Mutation Research 113: 173 ( 1983) and a specific screen is described in detail in Example 17, below.
  • the Ames test utilizes several unique strains of Salmonella typhimurium that are histidine- dependent for growth and that lack the usual DNA repair enzymes. The frequency of normal mutations that render the bacteria independent of histidine (i.e., the frequency of spontaneous revertants) is low. The test allows one to evaluate the impact of a compound on this revertant frequency.
  • the compound to be tested is mixed with the bacteria on agar plates along with the liver extract.
  • the liver extract serves to mimic metabolic action in an animal. Control plates have only the bacteria and the extract.
  • a new compound (X) can be evaluated as a potential blood photodecontamination compound for B19, as shown in Table 4, below.
  • X is initially evaluated in Step I.
  • X is screened in the R17 assay at several different concentrations between 4 and 320 ⁇ M, as explained in EXAMPLE 12. If the compound shows inactivation activity greater than 1 log inactivation of R17 (log kill) in the R17 screen at any concentration, the compound is then screened in the B19 assay, as explained in Example 27. If the compound shows suppression of infection by allowing the appearance of BFU-E, the compound and AMT are then screened in the Ames assay. Finally, if the compound shows lower mutagenicity in the Ames assay than does AMT, the new compound is identified as a useful agent for inactivation of pathogens. TABLE 4
  • the present invention contemplates several different formulations and routes by which the compounds described herein can be delivered in an inactivation method. This section is merely illustrative, and not intended to limit the invention to any form or method of introducing the compound.
  • the compounds of the present invention may be introduced in an inactivation method in several forms.
  • the compounds may be introduced as an aqueous solution in water, saline, a synthetic media such as
  • “SterilyteTM 3.0” (contents set forth at the beginning of the Experimental section, below) or a variety of other media.
  • the compounds can further be provided as dry formulations, with or without adjuvants.
  • the new compounds may also be provided by many different routes.
  • the compound may be introduced to the reaction vessel, such as a blood bag, at the point of manufacture.
  • the compound may be added to the material to be sterilized after the material has been placed in the reaction vessel.
  • the compounds may be introduced alone, or in a "cocktail" or mixture of several different compounds.
  • Psoralens are useful in inactivation procedures, because the reaction can be carried out at temperatures compatible with retaining biochemical properties of blood and blood products. Hanson, C.V., Blood Cells 18:7 (1992). But not all psoralens or methods will decontaminate without significantly lowering the biological activity of the decontaminated material. Previous compounds and protocols have necessitated the removal of molecular oxygen from the reaction before and during exposure to light, to prevent damage to blood products from oxygen radicals produced during irradiation. See L. Lin et al., Blood 74:517 (1989); US Patent No. 4,727,027, to Wiesehahn.
  • the present invention may be used to decontaminate blood products, in the presence of oxygen, without destroying the activity for which the products are prepared.
  • the present invention contemplates that in vivo activity of a blood product is not destroyed or significantly lowered if a sample of blood product which is decontaminated by methods of the present invention tests as would a normall functioning sample of blood product in known assays for blood product function.
  • in vivo activity is not destroyed or significantly lowered if pH of the platelets are substantially the same in platelets treated by the methods of the present invention and stored 5 days as they are in untreated samples stored for 5 days.
  • “Substantially the same" pH means that the values fall within the range of error surrounding that particular data point.
  • the present invention contemplates a preferred way of protecting blood and blood products during irradiation by introducing a synthetic media comprising a buffered saline solution for the period of irradiation and any storage thereafter.
  • dibasic phosphate is used as a buffer.
  • Dibasic phosphate contains both singly charged ions and a doubly charged ions.
  • the second factor is whether the compounds used are toxic or mutagenic to the patient treated.
  • a "compound displaying low mutagenicity" is defined as a compound which is less mutagenic than AMT when it is tested at concentrations below 250 ⁇ M in the Ames assay, described in the Experimental section, below.
  • the inactivation compounds and methods of the present invention are especially useful because they display the unlinking of pathogen inactivation efficiency from mutagenicity.
  • the compounds exhibit powerful pathogenic inactivation without a concomitant rise in mutagenicity.
  • nanomoles g (grams); mg (milligrams); ⁇ g (micrograms); Kg (kilograms); L (liters); mL (mil li liters); ⁇ L(microliters); cm (centimeters); mm (millimeters); ⁇ m (micrometers); nm (nanometers); J (Joules, also watt second, note that in FIGS.
  • Joules or J refers to Joules/cm ⁇ ); °C (degrees Centigrade); TLC (Thin Layer Chromatograp vy); EAA (ethyl- acetoacetate); EtOH (ethanol); HOAc (acetic acid); W (watts); mW (milliwatts); NMR (Nuclear Magnetic Resonance; spectra obtained at room temperature on a Varian Gemini 200 MHz Fourier Transform Spectrometer); m.p.
  • the acid is preferably selected so as to contain an anion which is non-toxic and pharmacologically acceptable, at least in usual therapeutic doses.
  • Representative salts which are included in this preferred group are the hydrochlorides, hydrobromides, sulphates, acetates, phosphates, nitrates, methanesulphonates, ethanesulphonates, lactates, citrates, tartrates or bitartrates, and maleates.
  • Other acids are likewise suitable and may be employed as desired.
  • fumaric, benzoic, ascorbic, succinic, salicylic, bismethylenesalicylic, propionic, gluconic, malic, malonic, mandelic, cinnamic, citraconic, stearic, palmitic, itaconic, glycolic, benzenesulphonic, and sulphamic acids may also be employed as acid addition salt-forming acids.
  • HEPES buffer contains 8.0 g of 137 mM NaCl, 0.2 g of 2.7 mM KCl, 0.203 g of 1 mM MgCl 2 (6H 2 0), l.Og of 5.6 mM glucose, 1.0 g of lmg/ml Bovine Serum Albumin (BSA) (available from Sigma, St. Louis, MO), and 4.8 g of 20 mM HEPES (available from Sigma, St. Louis, MO).
  • BSA Bovine Serum Albumin
  • phosphate buffered synthetic media is formulated for platelet treatment. This can be formulated in one step, resulting in a pH balanced solution (e.g. pH 7.2), by combining the following reagents in 2 liters of distilled water:
  • a pH balanced solution e.g. pH 7.2
  • the solution is then mixed, sterile filtered (0.2 micron filter) and refrigerated.
  • Synthetic media plus phosphate contains the following reagents:
  • PCR Polymerase Chain Reaction
  • the two primers are complementary to their respective strands of the double stranded target sequence.
  • the mixture is denatured and the primers then to annealed to their complementary sequences within the target molecule.
  • the primers are extended with a polymerase so as to form a new pair of complementary strands.
  • the steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e. denaturation, annealing and extension constitute one "cycle;” there can be numerous "cycles”) to obtain a high concentration of an amplified segment of the desired target sequence.
  • the length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter.
  • the method is referred to by the inventors as the "Polymerase Chain Reaction”. Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified”.
  • PCR it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g. hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32p labeled deoxynucleotide triphosphates, e.g. dCTP or dATP, into the amplified segment).
  • any oligonucleotide sequence can be amplified with the appropriate set of primer molecules.
  • the PCR amplification process is known to reach a plateau concentration of specific target sequences of approximately 10 " 8 M.
  • a typical reaction volume is 100 ⁇ l, which corresponds to a yield of 6 x 10 ⁇ double stranded product molecules.
  • PCR is a polynucleotide amplification protocol.
  • the amplification factor that is observed is related to the number (n) of cycles of PCR that have occurred and the efficiency of replication at each cycle (E), which in turn is a function of the priming and extension efficiencies during each cycle.
  • E the efficiency of replication at each cycle.
  • Amplification has been observed to follow the form E n , until high concentrations of PCR product are made. At these high concentrations (approximately 10 " ⁇ M/l) the efficiency of replication falls off drastically. This is probably due to the displacement of the short oligonucleotide primers by the longer complementary strands of PCR product.
  • sequences of the polynucleotide primers used in this experimental section are as follows:
  • DCD03 as a common forward primer, the pairs generate amplicons of length 127, 327, and 1072 bp. These oligos were selected from regions that are absolutely conserved between 5 different dHBV isolates (DHBV1,
  • DHBV3, DHBV16, DHBV22, and DHBV26 as well as from heron HBV
  • a photoactivation device for decontaminating blood products according to the method of the present invention.
  • This device comprises: a) means for providing appropriate wavelengths of electromagnetic radiation to cause photoactivation of at least one photoreactive compound; b) means for supporting a plurality of blood products in a fixed relationship with the radiation providing means during photoactivation; and c) means for maintaining the temperature of the blood products within a desired temperature range during photoactivation.
  • FIG. 6 is a perspective view of one embodiment of the device integrating the above-named features.
  • the figure shows an opaque housing (100) with a portion of it removed, containing an array of bulbs (101) above and below a plurality of representative blood product containing means (102) placed between plate assemblies (103, 104) which filter certain wavelengths of light.
  • the plate assemblies (103, 104) are described more fully, subsequently.
  • the housing (100) can be opened via a latch (105) so that the blood product can be placed appropriately. As shown in FIG. 6, the housing (100), when closed, completely contains the irradiation from the bulbs (101). During irradiation, the user can confirm that the device is operating by looking through a safety viewport (106) which does not allow transmission of ultraviolet light to the user.
  • the housing (100) also serves as a mount for several electronic components on a control board (107), including, by way of example, a main power switch, a count down timer, and an hour meter.
  • the power switch can be wired to the count down timer which in turn is wired in parallel to an hour meter and to the source of the electromagnetic radiation.
  • the count down timer permits a user to preset the irradiation time to a desired level of exposure.
  • the hour meter maintains a record of the total number of radiation hours that are provided by the source of electromagnetic radiation. This feature permits the bulbs (101) to be monitored and changed before their output diminishes below a minimum level necessary for rapid photoactivation.
  • a reflector completely surrounds each array of bulbs.
  • Blood product containing means are placed between upper and lower plate assemblies (e.g. BK-7 glass, Shott Glass Technologies, Inc., Duryea, PA).
  • Each plate assembly is comprised of an upper and lower plates.
  • the plate assemblies are connected via a hinge which is designed to accommodate the space created by the blood product containing means.
  • the upper plate assembly is brought to rest just above the top of the blood product containing means supported by the lower plate of the lower plate assembly.
  • Detectors may be conveniently placed between the plates of the plate assemblies. They can be wired to a printed circuit board which in turn is wired to the control board.
  • Six blood product containing means may be placed in a fixed relationship above an array of bulbs.
  • the temperature of the blood product can be controlled via a fan alone or, more preferably, by employing a heat exchanger having cooling inlet and outlet ports connected to a cooling source.
  • Upper plate assembly plates and lower plate assembly plates each create a temperature control chamber.
  • the fan can circulate air within and between the chambers. When the heat exchanger is employed, the circulating air is cooled and passed between the plates.
  • Step 1 3-Chloro-2-butanone (29.2 mL, 0.289 mol) was added to a mechanically stirred suspension of 7-hydroxy-4,8-dimethylcoumarin
  • Step 2 A suspension of 4,8-dimethyl-7-(l-methyl-2-oxo)propyloxy- coumarin (67.5g, 0.260 mol), 10% aqueous NaOH (114 mL, 0.286 mol) and water (900 mL) was heated for 2-4 hours at 70-85 ° C. The mixture was then allowed to cool to room temperature. The solid was filtered, and then rinsed with chilled water (1.5 L) until the mother liquor became colorless and pH was neutral (pH 5-7). The product was air and vacuum dried to give 4, 4',5',8-tetra ⁇ nethylpsoralen (56.3 g, 89.5%) as a white solid, mp 197- 199°C. NMR: d 2.19 (s, 3H), 2.42 (s, 3H), 2.51 (s, 3H), 2.56 (s, 3H), 6.23 (s, IH), 7.40 (s, IH).
  • Step 3 Dry 4,4',5',8-tetramethylpsoralen (10. OOg, 41.3mmol) was dissolved in methylene chloride (180 mL) at room temperature. N-
  • Step 1 4'-Bromomethyl-4,5',8-trimethylpsoralen (3.09 g, 9.61 mmol), (synthesis described in Example 2), and N-(2-hydroxyethyl)phthalimide (4.05 g, 21.2 mmol) were stirred in dry dimethylformamide (65 mL). Dry N 2 gas was bubbled gently into the reaction mixture. The reaction mixture was heated to 100 °C for 4.5 hours then allowed to cool to room temperature and put in the freezer for several hours. The crystalline product was filtered and washed with MeOH followed by H 2 0. The solid was further tritutrated with MeOH (100 mL) to remove the impurities. The crude product was air dried and dissolved in CHCI3 (150 mL).
  • Step 2 4'-[4-(N-phthalimido)-2-oxa]butyl-4,5',8-trimethylpsoralen (1.56 g, 3.61 mmol) was suspended in tetrahydrofuran (75 mL) at room temperature. Methylamine (40 % aqueous solution, 25 mL, 290 mmol) was added to the suspension and stirred overnight. The solvent and methylamine were completely removed. The resulting solid was taken up in 0.3 N HCl aqueous solution (25 mL). The acid suspension was rinsed three times with 40 mL CHCI3 then taken to pH 11 with 20 % aqueous NaOH.
  • CHCI3 (3x60 mL) was used to extract the product (i.e. 4'-(4-amino-2- oxa)butyl-4,5',8-trimethylpsoralen) from the basified layer.
  • the combined CHCI3 layers were washed with H 2 0 (100 mL) followed by brine (100 mL) then dried over anhydrous Na 2 S0 4 and concentrated to give 4'-(4-amino-2- oxa)butyl-4,5',8-trimethylpsoralen, mp 139-141 °C. Purity was greater than 99 % by NMR.
  • Step 2 may be performed using either hydrazine or butylamine rather than methylamine.
  • the method which uses butylamine is preferred for larger scale syntheses because, while an excess of methylamine is needed due to volatization, the same is not true for butylamine.
  • the method using butylamine was carried out as follows: 28.3 g phthalimide has been deprotected with n-butylamine in propanol. The crude reaction solution is then treated with HCl to precipitate out the product. Thus the reaction mixture in 285 mL of 1-propanol was treated with HCl gas to pH 2.
  • the method using hydrazine was carried out as follows: The phthalimide precursor (6 mol) was deprotected with hydrazine and after concentration and acid-base extractions the crude amine was obtained in 30 L of ethylene dichloride. To this was added HCl gas (0.14Kg) via dispersion tuve over 40 minutes maintaining the temperature at 15-25 °C. The resultant slurry was stirred an additional 1 hour. The solids were collected on a Buchner funnel. Upon drying in an air dryer at 80 °C for 2 hours, 0.945 kg of crude 4'-(4-amino-2-oxa)butyl-4,5',8-trimethylpsoralen was obtained.
  • the first method is a preferred embodiment of the present invention because of its high yield and purity.
  • the second method starts with the preparation of 4'-chloromethyl- 4,5',8-trimethylpsoralen from commercially available 4,5',8- trimethylpsoralen, as described above.
  • the synthesis of 4' ⁇ (4-amino-2- oxa)butyl-4,5',8-trimethylpsoralen hydrochloride is achieved in four (4) steps, as described in U.S. Patent No. 5,399,719 (incorporated by reference above) in "Example 2", starting with 4'-Chloromethyl-4,5',8- trimethylpsoralen and yielding 1.25 g of Compound 2, mp 236° C (decomp).
  • This example describes the synthesis of Compound 18.
  • N-methylformanilide (16.0 mL, 0.134 mol) in acetonitrile (130 mL) was added phosphorus oxychloride (12.5 mL, 0.134 mol), then 4,4',8- trimethylpsoralen (5.0 g, 21.9 mmol) (described in McLeod, et al., Tetrahedron Letters No. 3:237 (1972)).
  • the temperature was kept between 0- 10 °C during addition of the psoralen by use of an ice/water bath.
  • the slurry was stirred at 50°C for 2 days protected from moisture with a drierite drying tube.
  • the reaction mix was allowed to cool to room temperature, then chilled in an ice/water bath.
  • the acetonitrile was decanted off, then ice/water (150 mL) was added to the orange slurry and stirred for lh.
  • the orange solid was filtered off and rinsed with chilled water, then chilled acetonitrile.
  • the crude product was recrystallized and charcoal decolorized in dichloroethane (600 mL) to give 4,4',8-trimethyl-5'- psoralencarboxaldehyde (3.59g, 64.0%) as a pale yellow-orange solid, sublimes > 250 O C, decomp. > 300°C.
  • reaction solvents dichloroethane and water
  • dichloroethane aqueous layer was extracted three times with dichloroethane.
  • the organic layers were combined, rinsed with brine then dried (anhyd Na2S0 ) and stripped under vacuum to give the bulk of the product, 5'-bromomethyl-4,4',8-trimethylpsoralen, (13.13g, combined yield of 86.4%), as a pale yellow solid, mp 201-202 °C.
  • N-Hydroxyethylphthalimide (3.00 g, 15.5 mmol) was dissolved in DMF (5 mL) at 60-64°C while N2 was bubbled into the solution.
  • Sodium iodide (0.01 g, 0.067 mmol) and 5'-bromomethyl-4,4',8-trimethylpsoralen (1.00 g, 3.11 mmol) were added and the slurry was stirred under these conditions overnight.
  • the thick yellow reaction mixture was allowed to cool to room temperature, chilled in an ice/water bath, filtered and rinsed with ice cold MeOH to give crude product (1 g).
  • the aqueous solution was extracted with a further 2x10 mL of CHCI3 and the combined extracts were rinsed with water.
  • the product was then extracted from the CHCI3 solution with 0.3 N aqueous HCl and the acidic layer was then taken to pH 12 with concentrated NaOH solution.
  • hydrochloride salt was filtered and washed with absolute ethanol and dried under vacuum at room temperature giving 80.2 mg of product, 4'-(14-am ⁇ no-2,6,ll-triaza)tetradecyl-4,5',8-trimethylpsoralen tetrahydrochloride, as a light yellow solid.
  • EXAMPLE 12 An R17 bacteriophage assay was used in this example to predict pathogen inactivation efficiency and to determine nucleic acid binding of the photoreactive binding compounds of the present invention.
  • the bacteriophage was placed in a solution with each compound tested and was then irradiated. The ability of the phage to subsequently infect bacteria and inhibit their growth was measured. The bacteriophage was selected for its relatively accessible nucleic acid such that the culture growth inhibition would accurately reflect nucleic acid damage by the test compounds.
  • the bacteriophage assay for nucleic acid binding to test compounds offers a safe and inexpensive procedure to identify compounds likely to display efficient pathogen inactivation. Previous experiments support that the R17 assay accurately measures HIV-l sensitivity to similar compounds.
  • the R17 was grown up in Hfr 3000 bacteria, approximate titer 5 x lOH .
  • R17 and Hfr 3000 were obtained from American Tissue Culture Collection (ATCC), Washington, D.C.)
  • the R17 phage stock was added to a solution of 15% fetal bovine serum in Dulbecco's Modified Eagles Medium (DMEM) to a final phage concentration of 10 ⁇ /mL.
  • DMEM Dulbecco's Modified Eagles Medium
  • An aliquot (0.5 mL) was transferred to a 1.5 mL snap-top polyethylene tube.
  • An aliquot (0.004-0.040 mL) of the test compound stock solution prepared in water, ethanol or dimethylsulfoxide at 0.80-8.0 mM was added to the tube.
  • the plaque forming units were then counted the following morning and the titer of the phage remaining after phototreatment was calculated based on the dilution factors.
  • the following controls were run: the "phage only” in which phage was not treated with test compound and not irradiated (listed as “starting titer” in the tables below); the "UV only” in which the phage was irradiated in the absence of test compound; and the "dark” control in which the phage/test compound solution was not irradiated before it was diluted and plated.
  • Table 5 shows three different experiments which tested Compound 1 according to the R17 protocol just described.
  • a comparison of values for the control samples in runs 1 - 3 shows that neither the "UV only” nor the "dark” controls result in significant bacterial kill (at most, .3 logs killed in the "UV only” control and .1 logs killed in the "dark” control).
  • Tables 6 - 9, below, and FIGS. 7-9 show the results of the R17 assay for several of the 4'-primaryamino-substituted psoralen compounds of the present invention.
  • the data in Tables 7 and 8 appears in FIGS. 7 and 8, respectively.
  • 5'-Primaryamino-substituted psoralen compounds of the present invention which have substitutions on the 5' position similar to the 4 -primaryamino-substituted psoralen compounds, were also tested at varying concentration, as described above in this example, and are shown to exhibit comparable inactivation efficiency. The results for these compounds are shown in FIGS. 10 and 11, below.
  • FIGS. 12 - 18 The results of the assay for several 4' and 5'-primaryamino- substituted psoralen compounds are shown in FIGS. 12 - 18. This data further supports that the compounds of the present invention are comparable to AMT in their ability to inactivate R17. Further, Compounds 6 (FIG. 12), 10 (FIG. 13), 12 (FIG. 14), 15 (FIG. 15 and 18), and Compound 17 (FIG. 16), all were more efficient at inactivating R17 than was AMT.
  • HIV cell-free virus
  • Controls included HIV-l stock only, HIV-l plus UVA only, and HIV-l plus the highest concentration of each psoralen tested, with no UVA. Post irradiation, all samples were stores frozen at -70° C until assayed for infectivity by a microtiter plaque assay. Aliquots for measurement of residual HIV infectivity in the samples treated with a compound of the present invention were withdrawn and cultured.
  • Residual HIV infectivity was assayed using an MT-2 infectivity assay.
  • the assay medium was 85% DMEM (with a high glucose concentration) containing 100 ⁇ g of streptomycin, 100 U of penicillin, 50 ⁇ g of gentamicin, and 1 ⁇ g of amphotericin B per mL, 15% FBS and 2 ⁇ g of Polybrene (Sigma Chemical Co., St. Louis, Mo.) per mL.
  • Test and control samples from the inactivation procedure were diluted in 50% assay maximrw and 50% normal human pooled plasma.
  • the samples were serially diluted directly in 96-well plates (Corning Glass Works, Corning, N.Y.). The plates were mixed on an oscillatory shaker for 30 seconds and incubated at 37°C in a 5% C0 2 atmosphere for 1 to 18 hours.
  • MT-2 cells (0.025 mL) [clone alpha-4, available (catalog number 237) from the National Institutes of Health AIDS Research and Reference Reagent Program, Rockville, Md.] were added to each well to give a concentration of 80,000 cells per well.
  • the plates were incubated for 5 daws at 37°C in 5% C0 2 and stained by the addition of 0.05 mL of 50 ⁇ g/mL propidium iodide (Sigma Chemical Co.) in phosphate- buffered saline (pH 7.4) to each well. After 24 to 48 hours, the red fluorescence-stained microplaques were visualized by placing the plates on an 8,000 ⁇ W/cm 2 304 nm UV light box (Fotodyne, Inc., New Berlin, Wis.). The plaques were counted at a magnification of x20 to x25 through a stereomicroscope. The results are shown in Tables 10 and 11, below, "n" represents the number of runs for which the data point is an average.
  • HIV starting titer approximately 5.4 logs 1 minute irradiation
  • DHBV Hepatitis B Virus
  • DHBV in duck yolk was added to platelet concentrate (PC) to a final concentration of 2 x 10 7 particles per mL and mixed by gentle rocking for >15 min.
  • Psoralens Compounds 2 and 6 and AMT were added to 3 mL aliquots of PC in a TeflonTM mini-bag at concentrations of 35, 70, and 100 mM.
  • Samples, including controls without added psoralen, were irradiated with 5J/CM 2 UVA, with mixing at 1 J/cm 2 increments. After irradiation, leukocytes and platelets were separated from virus by centrifugation.
  • the supernatant containing DHBV was digested overnight with 50 ⁇ g/mL proteinase K in a buffer containing 0.5% sodium dodecyl sulphate, 20 mM Tris buffer, pH 8.0, and 5 mM EDTA at 55°C. Samples were extracted with phenol-chloroform and chloroform, followed by ethanol precipitation. Purified DNA was then used in PCR amplification reactions with a starting input of 10 ⁇ DHBV genomes from each sample. PCR amplicons were generated using primers pairs DCD03/DCD05 (127 bp), DCD03/DCD06 (327 bp) and DCD03/DCD07 (1072 bp).
  • PCR was performed in a standard PCR buffer containing 0.2 mM each deoxyribonucleoside 5'-triphosphates (dATP, dGTP, dCTP, and dTTP), 0.5 mM each primer, and 0.5 units Taq polymerase per 100 ml reaction. 30 cycles of amplification were performed with the following thermal profile: 95°C 30 sec, 60° C 30 sec, 72° C 1 min. The amplification was followed by a 7 min incubation at 72° C to yield full length products. [lambda- 32 P] dCTP was added at an amount of 10 mCi per 100 ml in order to detect and quantify the resulting products. Products were separated by electrophoresis on denaturing polyacrylamide slab gels and counted. The absence of signal in a given reaction was taken to indicate effective inactivation of DHBV.
  • dATP deoxyribonucleoside 5'-triphosphates
  • dGTP deoxyribonucleo
  • Example 13 the compounds of the present invention were tested for their ability to inactivate virus in DMEM/15% FBS.
  • the compounds are tested in both 100% plasma and predominantly synthetic media, to show that the methods of the present invention are not restricted to any particular type of medium.
  • Standard human platelet concentrates were ⁇ .-ntrifuged to separate plasma. Eighty-five percent of the plasma was then expressed off and replaced with a synthetic medium (referred to as "SterilyteTM 3.0") containing 20 mM Na acetate, 2 mM glucose, 4 mM KCl, 100 M NaCl, 10 mM Na3 Citrate, 20 M NaH 2 P0 /Na2HP0 4 , and 2 mM MgCl 2 .
  • H9 cells infected with HIV were added to either the 85% SterilyteTM 3.0 platelet concentrates or standard human platelet concentrates (2.5 x IO 7 cells per concentrate), final concentration 5xl0 5 cells/mL.
  • the platelet concentrates were placed in TeflonTM modified FL20 or TeflonTM Minibags (American Fluoroseal Co., Silver Springs, MD), treated with one of the compounds shown in FIGS. 19 and 20, at the concentrations shown, and then irradiated with 320-400 nm (20mW/cm2) for 5 J/cm 2 (for plasma samples) or 2 J/cm 2 (for 85% SterilyteTM 3.0 samples) on a device similar to the Device of Example 1.
  • the photoactivation device used here was previously tested and found to result in light exposure comparable to the Device of Example 1. (Data not shown). Aliquots for measurement of residual HIV infectivity in the samples treated with a compound of the present invention were withdrawn and cultured.
  • H9 cells infected with HIV were added to standard human platelet concentrates (2.5 x IO 7 cells per concentrate), final concentration 5xl0 5 cells /mL.
  • Aliquots of HIV contaminated platelet concentrate (5 mL) were placed in water jacketed Pyrex chambers. The chambers had previously been coated on the inside with silicon.
  • the platelet concentrates were treated with one of the compounds listed in Tables 10 and 11 at the concentrations listed in the table, and then irradiated with 320-400 nm (20mW/cm2) for 1 minute on a device similar to the Device of Example 1.
  • the photoactivation device used here was previously tested and found to result in light exposure comparable to the Device of Example 1. (Data not shown).
  • bacterial inactivation by the photoreactive nucleic acid binding compounds of the present invention was measured as a function of the ability of the bacteria to subsequently replicate.
  • a gram negative bacteria was chosen as representative of the more difficult bacterial strains to inactivate.
  • the bacteria a strain of Pseudomonus, was inoculated into LB with a sterile loop and grown overnight in a shaker at 37°C. Based on the approximation that one OD at 610 nm is equivalent to 5 x 10° colony forming units (cfu)/mL, a 1:10 dilution of the culture was measured on a spectrophotometer, (manufactured by Shimatsu).
  • the bacterial culture was added to a solution of 15% fetal bovine serum in DMEM to a final bacteria concentration of approximately 10°7mL. An aliquot (0.8 mL) was transferred to a 1.5 mL snap-top polyethylene tube. An aliquot (0.004-0.040 mL) of the test compound stock solution prepared in water, ethanol or dimethylsulfoxide at 0.80-8.0 mM was added to the tube. Compounds were tested at a concentration of 16 ⁇ M. The tubes were placed in a light device as described in EXAMPLE 1 and irradiated with 1.3 J/cm 2 , 1.2 J/cm 2 , and finally 2.5 J/cm 2 , for a total of 5 J/cm 2 .
  • each test compound required one tube with 0.4 mL of LB broth and four tubes containing 0.5 mL of LB broth.
  • a 0.050 mL aliquot of the irradiated solution of phage and test compound was added to the first dilution tube of 0.5 mL of media then 0.050 mL of this solution was added to the second tube of 0.5 mL medium (1:10). The second solution was then diluted serially (1:10) into the remaining tubes.
  • psoralens of the present invention have been demonstrated to be effective for inactivating pathogens, such as bacteria (pseudomonus), bacteriophage (R1 ) and viruses (HIV and DHBV). Without intending to be limited to any method by which the compounds of the present invention inactivate pathogens, it is believed that inactivation results from light induced binding of the psoralens to the nucleic acid of the pathogens. As discussed above, AMT is known both for its pathogen inactivation efficiency and its accompanying mutagenic action in the dark at low concentrations. In contrast, the less active psoralens, such as 8-MOP, that have been examined previously, show significantly less mutagenicity. This example establishes that photobinding and mutagenicity are not linked phenomenon in the compounds of the present invention. The psoralens of the present invention have exceptional pathogen inactivation efficiency while displaying only minimal mutagenicity.
  • pathogens such as bacteria (pseudomon
  • the compounds of the present invention are tested for their dark mutagenicity using an Ames assay.
  • the procedures used for the Salmonella mutagenicity test as described in detail by Maron and Ames were followed exactly. Maron, D.M. and B.N. Ames, Mutation Research 113: 173 ( 1983). A brief description for each procedure is given here.
  • the tester strains TA97a, TA98, TA100, TA102, TA1537 and TA1538 were obtained from Dr. Ames.
  • TA97a, TA98, TA1537 and TA1538 are frameshift tester strains.
  • TA100 and TA102 are base-substitution tester strains.
  • each strain was cultured under a variety of conditions to confirm the genotypes specific to the strains.
  • the standa ⁇ Salmonella tester strains used in this study require histidine for growth since each tester strain contains a different type of mutation in the histidine operon. In addition to the histidine mutation, these tester strains contain other mutations, described below, that greatly increase their ability to detect mutagen.
  • Histidine Dependence The requirement for histidine was tested by streaking each strain first on a minimal glucose plate supplemented only with biotin and then on a minimal glucose plate supplemented with biotin and histidine. All strains grew the lack of growth of the strains in the absence of histidine.
  • rfa Mutation A mutation which causes partial loss of the lipopolysaccharide barrier that coats the surface of the bacteria thus increasing permeability to large molecules was confirmed by exposing a streaked nutrient agar plate coated with the tester strain to crystal violet. First 100 ⁇ L of each culture was added to 2 mL of molten minimal top agar and poured onto a nutrient agar plate.
  • UV repair system (TA97a, TA98, TA100, TA1537 and TA1538). This trait was tested for by streaking the strains on a nutrient agar plate, covering half of the plate, and irradiating the exposed side of the plate with germicidal lamps. After incubation growth was only seen on the side of the plate shielded from UV irradiation.
  • the tester strains (TA97a, TA98, TA100, and TA102) contain the pKMlOl plasmid that increases their sensitivity to mutagens. The plasmid also confers resistance to ampicillin to the bacteria. This was confirmed by growing the strains in the presence of ampicillin.
  • p ⁇ Ql Strain TA102 also contains the pAQl plasmid that further enhances its sensitivity to mutagens. This plasmid also codes for tetracycline resistance. To test for the presence of this plasmid TA102 was streaked on a minimal glucose plate containing histidine, biotin, and tetracycline. The plate was incubated for 16 hours at 37°C. The strain showed normal growth indicating the presence of the pAQl plasmid.
  • the same cultures used for the genotype testing were again cultured and aliquots were frozen under controlled conditions.
  • the cultures were again tested for genotype to confirm the fidelity of the genotype upon manipulation in preparing the frozen permanents.
  • the first tests done with the strains were to determine the range of spontaneous reversion for each of the strains. With each mutagenicity experiment the spontaneous reversion of the tester strains to histidine independence was measured and expressed as the number of spontaneous revertants per platf. This served as the background controls.
  • a positive mutagenesis control was included for each tester strain by using a diagnostic mutagen suitable for that strain (2-aminofluorene at 5mg/plate for TA98 and sodium azide at 1.5 mg/plate for TA100). For all experiments, the pre-incubation procedure was used. In this procedure one vial of each tester strain was thawed and 20 ⁇ L of this culture was added to 6 mL of Oxoid Nutrient Broth #2.
  • test solution containing cither 0, 0.1, 0.5, 1, 5, 10, 50, 100, 250, or 500 ⁇ g/mL of the test compound was added.
  • the 0.7 mL mixture was vortexed and then pre ⁇ incubated while shaking for 20 minutes at 37°C.
  • 2 mL of molten top agar supplemented with histidine and biotin were added to the 0.7 mL mixture and immediately poured onto a minimal glucose agar plate (volume of base agar was 20 mL).
  • the top agar was allowed 30 minutes to solidify and then the plates were inverted and incubated for 44 hours at 37°C. After incubation the number of revertant colonies on each plate were counted.
  • the results are set forth in table form in U.S. Patent No. 5,399,719, from "Table 10(A)" through "Table 16(B)”.
  • Maron and Ames (1983) describe the conflicting views with regard to the statistical treatment of data generated from the test.
  • this example adopts the simple model of mutagenicity being characterized by a two-fold or greater increase in the number of revertants above background, as well as dose dependent mutagenic response to drug.
  • AMT showed frameshift mutagenicity at between 5 and 10 ⁇ g/plate in TA97a and TA98, at 5 ⁇ g/plate in TA1537 and at 1 ⁇ g/plate in TA1538.
  • AMT showed no significant base-substitution mutations.
  • Compound 1 the only mutagenic response detected was a weak frameshift mutagen in TA1538 at 5 ⁇ g/plate in the presence of S9.
  • Compound 1 also displayed mutation in the TA100 strain, but only in the absence of S9.
  • Compound 2 also showed weak frameshift mutagenicity in the presence of S9 in TA98 and TA1537.
  • Compounds 3 and 4 showed no mutagenicity.
  • Compound 6 had no base substitution mutagenicity, but showed a frameshift response in TA98 in the presence of S9 at concentrations of 250 ⁇ g/plate and above. It also showed a response at 50 ⁇ g/plate in TA1537 in the presence of S9.
  • Compound 18 showed only a weak response at high concentrations in the presence of S9 in strains TA 9o and TA 1537. The response was higher in the absence of S9, but still was significantly below that of AMT, which displayed mutagenicity at much lower concentrations (5 ⁇ g/plate).
  • Example 1 5 the compounds of the present invention exhibited the ability to inactivate pathogens in synthetic media.
  • FIG. 21A schematically shows the standard blood product separation approach used presently in blood banks.
  • Three bags are integrated by flexible tubing to create a blood transfer set (200) (e.g., commercially available from Baxter, Deerfield, 111.). After blood is drawn into the first bag (201), the entire set is processed by centrifugation (e.g., SorvallTM swing bucket centrifuge, Dupont), resulting in packed red cells and platelet rich plasma in the first bag (201).
  • centrifugation e.g., SorvallTM swing bucket centrifuge, Dupont
  • the plasma is expressed off of the first bag (201) (e.g., using a Fen wallTM device for plasma expression), through the tubing and into the second bag (202).
  • the first bag (201) is then detached and the two bag set is cenr ifuged to create platelet concentrate and platelet-poor plasma; the latter i-- expressed off of the second bag (202) into the third bag (203).
  • FIG. 21 B schematically shows an embodiment of the present invention by which synthetic media and photoactivation compound are introduced to platelet concentrate prepared as in FIG. 21A.
  • a two bag set (300) is sterile docked with the platelet concentrate bag (202) (indicated as "P.C”).
  • P.C platelet concentrate bag
  • Sterile docking is well-known to the art. See e.g., US Patent No. 4,412,835 to D.W.C. Spencer, hereby incorporated by reference. See also US Patents Nos. 4,157,723 and 4,265,280, hereby incorporated by reference. Sterile docking devices are commercially available (e.g., Terumo, Japan).
  • One of the bags (301) of the two bag set (300) contains a synthetic media formulation of the present invention (indicated as "STERILYTE").
  • the platelet concentrate is mixed with the synthetic media by transferring the platelet concentrate to the synthetic media bag (301) by expressing the platelet concentrate from the first blood bag into the second blood bag via a sterile connection means.
  • the photoactivation compound can be in the bag containing synthetic media (301), added at the point of manufacture.
  • the compound can be mixed with the blood at the point of collection, if the compound is added to the blood collection bag (FIG. 21A, 201) at the point of manufacture.
  • the compound may be either in dry form or in a solution compatible with the maintenance of blood.
  • FIG. 21 C schematically shows one embodiment of the decontamination approach of the present invention applied specifically to platelet concentrate diluted with synthetic media as in FIG. 21B.
  • platelets have been transferred to a synthetic media bag (301).
  • the photoactivation compound either has already been introduced in the blood collection bag (201) or is present in the synthetic media bag (301). Either the platelets are then expressed into the synthetic media bag via a sterile connection means (as shown) or the synthetic media is expressed into the platelet bag.
  • the bag containing the mixture of platelet concentrate and synthetic media (301), which has UV light transmission properties and other characteristics suited for the present invention, is then placed in a device (such as that described in Example 1, above) and illuminated.
  • the decontaminated platelets are transferred from the synthetic media bag (301) into the storage bag (302) of the two bag set (300).
  • the storage bag can be a commercially available storage bag (e.g., CLX bag from Cutter).
  • FIG. 21 D schematically shows an embodiment of the decontamination approach of the present invention, which includes a capture device to remove photoinactivation compound from the treated material after phototreatment.
  • the present invention contemplates several adsorptive materials which may be used in a capture device to remove photoinactivation compounds, of which the following is a non-exclusive list: Amberlite XAD-4 (available from Rohm and Haas Ltd., Croydon, Surrey, UK) ("Resin hemoperfusion for Acute Drug Intoxication," Arch Intern Med 136:263 (1976)); Amberlite XAD-7 (“Albumin-Coated Amberlite XAD-7 Resin for Hemoperfusion in Acute Liver Failure," Artificial Organs, 3:20 (1979); Amberlite 200, Amberlite DP-1, Amberlite XAD-2, Amberlite XAD-16; activated charcoals, ("Charcoal haemoperfusion in Drug Intoxication," British J.
  • the present invention contemplates an absorptive material operating in conjunction with a filtering means to remove compounds.
  • This example involves an assessment of the impact of the compounds and methods of the present invention on platelet function.
  • Four indicators of platelet viability and function were employed: 1 ) GMP- 140 expression; 2) maintenance of pH; 3) platelet aggregation, and 4) platelet count.
  • the compounds tested in this experiment were Compound 2 ( 36 ⁇ L of 10 mM stock added to 4 ml PC), Compound 6 (173.5 ⁇ l of 9.8 mM stock added to 16.8 ml PC), Compound 17 (2.0 ml of ImM stock added to 18 ml PC) and Compound 18 (.842 ml of 2.0 mM stock to 16 ml PC).
  • the samples were pipetted gently up and down to mix. Then aliquots (either 3 ml or 8 ml) of each sample was transferred to two sterile TeflonTM Medi-bagsTM (American Fluoroseal Co., Silver Springs, MD) (presently owned by The West Company, Lionville, PA).
  • Samples were treated in one of two different sized bags, having either 3 ml or 8 ml capacity.
  • the bags both have approximately the same surface area to volume ratios, and previous experiments have shown that the two bags exhibit approximately equivalent properties during irradiation of samples. (Data not shown).
  • two control samples without compound were prepared by again removing aliquots of platelet concentrate (17 ml 'i using an 8 ml bag, 4 ml if using a 3 ml bag) from the same one of the first set of 50 ml centrifuge tubes from which the compound sample was drawn, and dividing into Medibags, as before.
  • GMP140 alpha granule membrane glycoprotein
  • p-selectin an alpha granule membrane glycoprotein called p-selectin
  • GMP140 a small aliquot of platelet rich plasma is placed in HEPES buffer containing a GMP140-binding antibody or an isotype control mouse IgG.
  • CD62 is a commercially available monoclonal antibody which binds to GMP140 (available from Sanbio, Uden, the Netherlands; Caltag Labs, So. San Francisco, CA, and Becton Dickinson, Mountain View, CA).
  • Goat F(ab') 2 Anti-Mouse IgG conjugated to FITC (Caltag Laboratories, So. San Francisco, CA) is added to the tube in saturating amounts and allowed to incubate at room temperature (RT) for 15 minutes.
  • the cells are diluted in 1%> paraformaldehyde in phosphate buffered saline and analyzed on a FACSCANTM (Becton Dickinson, Mountain View, CA).
  • the positive control is made by adding Phorbol Myristate Acetate (PMA) to the test system at a final concentration of 2 x IO "7 M.
  • PMA Phorbol Myristate Acetate
  • CD62 was employed to measure the impact, if any, of irradiation in the presence of several compounds of the present invention on platelet activation.
  • the antibody was mixed with HEPES buffer (10 ⁇ L antibody [0.1 mg/ml] : 2.49mL buffer) and stored in 50 ⁇ L aliquots at -40°C prior to use.
  • a positive control consisted of: 10 ⁇ L CD62, 8 ⁇ L PMA and 2.482 mL Hepes buffer.
  • a mouse IgGl control (0.05 mg/ml) (Becton Dickinson, Mountain View, CA #9040) 5X concentrated was also employed.
  • the antibody was diluted in HEPES buffer (20 ⁇ L antibody : 2.48 ml buffer) and stored at -40°C.
  • Phorbol Myristate Acetate (PMA) (Sigma, St. Louis, MO) was stored at -40°C. At time of use, this was dissolved in DMSO (working concentration was 10 ⁇ g/mL).
  • DMSO working concentration was 10 ⁇ g/mL.
  • 1% Paraformaldehyde (PFA) (Sigma, St. Louis, MO) was prepared by adding 10 grams paraformaldehyde to 1 liter PBS. This wa» heated to 70°C, whereupon 3 M N ⁇ OH was added dropwise until the solution was clear. The solution was cooled and the pH was adjusted to 7.4 with 1 N HCl. This was filtered and stored.
  • Processing each of the samples of platelet concentrate after treatment involved adding 5 ⁇ l of platelet concentrate, diluted 1:3 in Hepes buffer, to each microcentrifuge tube containing the antibody CD62, and appropriate reagents and mixing very gently by vortex. The samples were then incubated for 15 minutes at room temperature.
  • FIGS 22C, 23C, 24C, and 25C The results are shown in FIGS 22C, 23C, 24C, and 25C.
  • FIGS 22D, 23D, 24D and 25D are bar graphs showing pH results for a dark control, a light control and an experimental light plus compound. These graphs indicate that the pH of platelet concentrate samples after illumination in the presence of any one of the compounds remains above a pH of 6.5. Thus platelets remain at a pH acceptable for stored platelets following photoinactivating treatment using compounds of the present invention. 3) Aggregation
  • Platelet aggregation is measured by the change in optical transmission that a platelet sample exhibits upon stimulation of aggregation. Platelet aggregation was measured using a Whole Blood Aggregometer (Chrono-Log Corp., Havertown, PA, model 560VS). The number of platelets in each sample was controlled to be constant for every measurement. A Model F800 Sysmex cell counter (Toa Medical Electronics, Kobe, Japan) was used to measure platelet count in the platelet samples and autologous plasma was used to adjust platelet counts to 300,000/mL of platelet concentrator.
  • the 100 % aggregation line is the level at which the recorder was set to zero.
  • the 0% aggregation line is where the platelets transmitted before the ADP and collagen were added.
  • the aggregation value for the sample is determined by taking the maximum aggregation value as a percent of the total range.
  • Three of the four compounds tested showed very little or no difference in aggregation levels between the samples treated with compound and the untreated samples which were stored for 5 days.
  • Compound 2 exhibited a small reduction in aggregation, of approximately 8% from the day 1 control.
  • the aggregation for the sample treated with compound and UV was the same as that for ti>. ⁇ UV only sample. This is supporting evidence that the decontamination compounds tested do not have a significant effect on platelet aggregation when used in the methods of the present invention.
  • a Sysmex cell counter was used to measure platelet count in the platelet samples. Samples were diluted 1 : 3 in blood bank saline.
  • FIGS. 22A, 23A, 24A, and 25A The results of the platelet count measurements appear in FIGS. 22A, 23A, 24A, and 25A.
  • the samples show little or no drop in platelet count between the Day 5 control and the Day 5 treated sample.
  • samples treated with Compounds 6, 17 and 18 all display a higher platelet count than samples treated with light alone.
  • samples treated with Compound 6 had counts equivalent to the 5 day control, but samples treated with only ultraviolet light showed approximately a 33% reduction in platelet count.
  • treatment with compounds of the present invention compatible with the maintenance of platelet count, but it actually appears to prevent the drop in count caused by ultraviolet light exposure.
  • a preferred compound for decontaminating blood subsequently used in vivo should not be mutagenic to the recipient of the blood.
  • some compounds were screened to determine their genotoxicity level in comparison to aminomethyltrimethylpsoralen.
  • the in vivo clastogenicity of some compounds of the present invention was measured by looking for micronucleus formation in mouse reticulocytes.
  • Mammalian . ell cultures are valuable tools for assessing the clastogenic potential of chemicals.
  • cells are exposed to chemicals with and /or without rat S-9 metabolic activation system (S-9) and are later examined for either cell survival (for a genotoxicity screen) or for changes in chromosome structure (for a chromosome aberration assay).
  • S-9 metabolic activation system S-9
  • CHO cells Chinese hamster ovary (CHO; ATCC CCL 61 CHO-K1 , proline- requiring) cells were used for the in vitro genotoxicity and chromosomal aberration tests.
  • the cells were grown in an atmosphere of 5% C0 2 at approximately 37° C in McCoy's 5a medium with 15% fetal bovine serum (FBS), 2 mM L- glutamine, and 1% penicillin-streptomycin solution to maintain exponential growth This medium was also used during exposure of the cells to the test compound when no S-9 was used.
  • FBS fetal bovine serum
  • penicillin-streptomycin solution was also used during exposure of the cells to the test compound when no S-9 was used.
  • Cell cultures were maintained and cell exposures were performed in T-75 or T-25 flask
  • the compound was added in complete McCoy's 5a medium. After the compound was added, cells were grown in the dark at approximately 37° C for approximately 3 hours. The medium containing the test compound was then aspirated, the cells were washed three times with phosphate-buffered saline (PBS) at approximately 37° C, and fresh complete McCoy's 5a medium was added.
  • PBS phosphate-buffered saline
  • the positive control was methylmethane sulfonate.
  • the solvent control was dimethylsulfoxide (DMSO) diluted in culture medium. For assays using metabolic activation (see below) the activation mixture was also added to the solvent control. The cultures were then incubated for an additional time of approximately 12 hours before they were harvested. Colchicine (final concentration, 0.4 ⁇ g/ml) was adde ⁇ approximately 2.5 hours prior to the harvest.
  • the cells were harvested. Cells were removed from the surface of the flasks using a cell scraper. The resulting cell suspension was centrifuged, the supernatant, aspirated, and 4 ml of a hypotonic solution of 0.075 M KCl added to the cells for 15 minutes at approximately 37°C. The cells were then centrifuged, the supernatant aspirated, and the cells suspended in a fixative of methanol: acetic acid (3:1). After three changes of fixative, air-dried slides were prepared using cells from all flasks.
  • the cell density and metaphase quality on the initial slide from each flask was monitored using a phase-contrast microscope; at least two slides of appropriate cell density were prepared from each flask. The slides were stained in 3% Giemsa for 20 min, rinsed in deionized water, and passed through xylene. Coverslips were mounted with Permount. Slides are then examined to determine what concentration of each test compound represented a toxic dose.
  • Saline solutions were prepared for Compounds 2, 6, 17 and 18 at various concentrations. Male Balb/c mice were then injected with 0.1 ml of a compound solution via the tail vein. At least 3 mice were injected per dose level. Saline only was used as a negative control. For a positive control, cyclophosphamide (cycloPP) was administered at a dose of 30 mg/kg. In the experimental group, the injections were repeated once per day for four days. In the positive control group, the sample was administered only once, on day three. On day 5, several microliters of blood were withdrawn from each subject and smeared on a glass slide. Cells were fixed in absolute methanol and stored in a slide rack.
  • cyclophosphamide cyclophosphamide
  • reticulocytes were stained with acridine orange and visualized under a fluorescence microscope by counting: (i) the number of reticulocytes per 5000 erythrocytes; and (ii) the number of micronucleated reticulocytes per 1000 reticulocytes.
  • Reticulocytes were distinguished by their red fluorescence due to the presence of RNA.
  • Micronuclei were distinguished by their green fluorescence due to the presence of DNA.
  • the percentage of reticulocytes was then calculated.
  • a decrease in the frequency of erythrocytes, represented by an increase in the percentage of reticulocytes, is an indication of bone marrow toxicity.
  • the percentage of reticulocytes with micronuclei was also calculated. An increase in %PCE with MN is a measure of clastogenicity.
  • EXAMP ⁇ E 13 the inactivation of cell-free HIV virus, using compounds and methods of the present invention, is shown. This example shows inactivation of cell-associated HIV also using compounds of the present invention.
  • H9 cells chronically infected with HIV ⁇ iB were used. (H9/HTLV-III- B NIH 1983 Cat.#400). Cultures of these cells were maintained in high glucose Dulbecco Modified Eagle Medium supplemented with 2 mM L- glutamine, 200 u/mL penicillin, 200 ⁇ g/ml streptomycin, and 9% fetal bovine serum (Intergen Company, Purchase, N.Y.) For maintenance, the culture was split once a week, to a density of 3 x IO 5 to 4 x IO 5 cells /ml and about four days after splitting, 3.3% sodium bicarbonate was added as needed. For the inactivation procedure, the cells were used three days after they were split.
  • PC human platelet concentrate
  • the samples were serially diluted directly in 96-well plates (Corning Glass Works, Corning, N.Y.). The plates were mixed on an oscillatory shaker for 30 seconds and incubated at 37°C in a 5% C0 2 atmosphere for 1 to 18 hours.
  • MT-2 cells 0.025 mL [clone alpha-4, available (catalog number 237) from the National Institutes of Health AIDS Research and Reference Reagent Program, Rockville, Md.] were added to each well to give a concentration of 80,000 cells per well.
  • the plates were incubated for 5 days at 37°C in 5% C0 2 and stained by the addition of 0.05 mL of 50 ⁇ g/mL propidium iodide (Sigma Chemical Co.) in phosphate- buffered saline (pH 7.4) to each well. After 24 to 48 hours, the red fluorescence-stained microplaques were visualized by placing the plates on an 8,000 ⁇ W/cm 2 304 nm UV light box (Fotodyne, Inc., New Berlin, Wis.). The plaques were counted at a magnification of x20 to x25 through a stereomicroscope.
  • EXAMPLE 22 This example involves an assessment of new synthetic media formulations as measured by the following in vitro platelet function assays: 1) maintenance of pH; 2) platelet aggregation ("Agg") and 3) GMP140 expression. The assays for each of these tests have been described above. TABLE 13
  • PRP human platelet rich plasma
  • the unit was allowed to rest for 1 hour, after which it was gently kneaded to resuspend the platelets.
  • the reconstituted unit was assayed for pH and other tests the next day, with the following results:
  • bacterial inactivation efficiency of several compounds of the present invention was evaluated by examining the ability of the compounds to inactivate a variety of bacteria.
  • twelve phylogenetically distinct and clinically pathogenic strains of bacteria were studied. Table 18 contains a list of these bacteria. All bacteria were obtained from the Department of Public Health Services in Berkeley, CA. Inactivation assays were performed as follows.
  • E. cloacae E. coli. K pneumoniae. L. monocvtogenes. P. aeruginosa.
  • aeruginosa recently outdated platelet concentrate units from a local Blood Bank were stored in Helmer platelet incubator and shaker at 22°C until use. Two units (each comprising 4 pooled platelet concentrates) of approximately 50ml each were pooled using a Haemonetics SCD 312 sterile docker (Terumo; Braintree, MA) in order to have sufficient volume for each experiment. The platelet concentrate was divided evenly in 50 ml conical centrifuge tubes (Starstedt). Tubes were spun at 3800 rpm for 6 minutes with brake 6 on a Sorvall RC3B Centrifuge (DuPont Company; Wilmington, DE).
  • a synthetic media comprising: 116 mM NaCl, 10 mM Na3 citrate, 30 mM Na acetate, having pH adjusted to 7.2 with HCl
  • Platelets were resuspended via serial pipette (Falcon, Becton Dickinson; Rutherford, NJ). For the rest of the bacteria, apheresis units were used. These also contained the same synthetic media, however, S. aureus had additional phosphate (26 mM) and S. Group A, L. monocytogenes had additional phosphate (26 mM) and mannitol (20 mM).
  • Ciba-Corning 238 pH/Blood Gas Analyzer (Ciba-Corning; Medfield, MA) and ranged from 6.57 to 7.08 pH. Platelet count was performed by diluting platelet concentrate 1:3 in S/P Certified Blood Bank Saline (Baxter), diluting that sample using a Sysmex
  • Treated platelet concentrate (1ml) was incubated in 9 ml of sterile LB in a 50 ml Starstedt tube. The lid was loosely attached with tape. Culture was shaken overnight at 37°C and was then streaked on sterile LB plates using a sterile loop. C. minutissimum, L. monocytogenes and S. Group A were plated on Soy Agar with 5% Sheep blood plates These plates were incubated overnight at 37°C. Complete kill was determined by clean plates after the overnight incubation. The sampling procedure was repeated at the 3 joule point for gram negative bacteria samples.
  • Colonies were counted and the numbers obtained were multiplied by the appropriate dilution factor to get cfu/ml. Titers for each treatment were averaged and the logio bacteria was calculated. Logio kill was calculated by subtracting the treated bacteria logio from the untreated bacteria loglO- For samples that showed no colonies, 1 ml samples were then plated, incubated and counted. If these plates showed no colonies, the kill was considered to be complete.
  • the life cycle of HIV includes entry into a host cell, reverse transcription of the viral RNA into DNA and integration of the viral DNA into the host genome.
  • This integrated viral DNA is called proviral DNA.
  • the proviral DNA can be replicated, transcribed and translated into viral protein.
  • the viral RNA is packaged into particles that bud out of the infected cell.
  • Viable virus budding from infected cells is termed cell-associated virus.
  • Intact viable virus particles after budding from the infected cell are called cell-free virus. Both cell-free and cell- associated virus have been demonstrated to be sensitive to Compound 2 treatment in platelet concentrates.
  • Proviral DNA can also be maintained in the absence of transcription and translation resulting in a latent infection. Since it is possible for latently infected lymphocytes to contaminate platelet concentrates, experiments were designed to measure inactivation of provirus by Compound 2 plus UVA light.
  • the OM-10.1 cell line is a clone of HL-60, a promyelocyte cell line, that harbors a single copy per cell of proviral DNA of the IIIB strain of HIV-l.
  • the provirus is an obligate intermediate in the replication cycle of HIV.
  • the provirus present in the OM-10.1 is in a state of "absolute" latency as defined by the absence of proviral transcription. Butera ST, et al.
  • OM-10.1 Human Immunodeficiency virus type 1 RNA expression by four chronically infected cell lines indicated multiple mechanisms of latency" Journal of Virology 68: 2726-30. There is, however, a subpopulation of individual cells within a culture that do produce HIV at a low constitutive level. Upon stimulation by the cytokine TNF- ⁇ , OM-10.1 cells begin to produce infectious HIV. Viral expression, once initiated, produces large amounts of infectious progeny and viral p24 protein. At the same time expression of CD4, the cellular receptor for HIV, is down regulated. Butera, Journal of Virology 65:4645 (1991). OM10.1 is used here as a sensitive model system with a broad dynamic range for the study of the sensitivity of proviral HIV to Compound 2 treatment.
  • OM-10.1 cells obtained from American Type Culture Collection, #CRL10850 were propagated in RPMI 1640 culture medium (RPMI) (available from Gibco, Inc., Gathersburg Maryland) with 10% heat-inactivated FBS and 2 mM L-glutamine, 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin. Cultures were maintained in a 37°C, 5% C ⁇ 2, humidified incubator. The subculturing schedule required a 1:10 split of each culture every 48-72 hours. It was important not to allow the cell culture to overgrow to greater than 1 x 10 ⁇ cells per mL. Overgrowth stimulated the expression of infectious HIV. Low passage stocks of cell suspension in FBS with 10% DMSO were archived in a vapor phase liquid nitrogen freezer.
  • a 48 hour old culture of OM-10.1 cells was counted (Neubauer type hemocytometer) to determine the concentration of cells in suspension. The concentration was then adjusted by pelleting the cells at 1600 RPM for 10 minutes in a Beckman Inst uments J-6B centrifuge with a JS-4.2 rotor (Palo Alto, California). The supernatant was removed and discarded. The cells were then resuspended in tissue culture medium (RPMI/10% FBS) at a final density of 2 x 10 ⁇ cells per mL. Five mL of this OM-10.1 cell suspension was then added to each well of a 6-well tissue culture plate (1 x 10 ⁇ cells in 5 mL per well).
  • tissue culture medium RPMI/10% FBS
  • the 6-well tissue culture plate was placed in the UVA light device described in Example 1 and illuminated with 1 J/cm 2 total illumination without reciprocal shaking.
  • Four control samples were also prepared, none of which received any Compound 2.
  • Two of these controls were also illuminated with 1 J/cm 2 UVA, while the other two were not illuminated.
  • titration of the production of infectious HIV was performed as described in Example 13. Samples of each culture were serially 10-fold diluted prior to assay.
  • Determination of cell density in a cell culture was performed as follows: cells were stained with ethidium bromide which is non-permeant to viable cells, and acridine orange which is permeant to viable cells. A Neubauer type hemacytometer was used to count individual cells for both number and viability using an inverse microscope equipped with a UV light source. Characterization of the OM-10.1 system
  • the kinetics of cell growth and viral expression were studied with and without TNF-a stimulation.
  • Two cultures of OM-10.1 were set up in parallel, one stimulated by TNF-a and the other an unstimulated control. The cultures were sampled at day 0, and daily thereafter until day 4. Samples were taken for titration of infectious virus in the HIV microplaque assay, for quantitation of p24 concentration, and for determination of cell culture density. Sampling of the stimulated culture on the fourth day was not done because of a technical error.
  • the growth kinetics experiment was carried out twice with very similar results (FIG. 26).
  • TNF-a stimulation clearly initiated the productive infection of the latently infected cell causing an overall decrease in cell number relative to control. This can occur by two mechanisms; an inhibition of cell growth because of metabolic demands of viral assembly, or cell death among some of the virus producing cells.
  • OM-10.1 cells were photochemically treated with 1 Joule/cm 2 UVA light and Compounds 2, 6, 17 or 18, each at concentrations of 0.1, 1.0, 10 and 100 ⁇ M. The cells were then isolated from the psoralen-containing medium and cultured in RPMI 1640 medium with 20 U/ml TNF-a. Samples were taken after 72 hours of stimulation and examined for total p24 (FIG. 27) and for infectious HIV titer (Table 20).
  • Controls were prepared with each psoralen derivative at a concentration of 100 ⁇ M to test for cytotoxicity in the absence of UVA illumination. All four compounds were cytotoxic at 100 ⁇ M to varying degrees. Compounds 6 and 17 in the absence of light completely inhibited the expression of any detectable infectious HIV. The samples treated with Compound 2 were still able to produce 2 to 3 logs (pfu/ml) of infectious virus compared to 4.5 logs in the analogous control sample.
  • the p24 concentration of the inactivated samples was measured following TNF-a stimulation for 72 hours (FIG. 27).
  • the 100 ⁇ M, UVA illuminated samples were not included in FIG. 27, as they were essentially identical to the 10 ⁇ M samples.
  • TNF-a stimulated controls had 550 ng/ml p24 and a titer of 4.5 logio pfu/ml, whereas the unstimulated controls had 235 ng/ml p24 and a titer of 4.2 logio pfu/ml. This is probably the result of sub optimal cell culture conditions which initiated proviral expression. Infectious titer was measured to correlate well with the p24 data (Table 20).
  • Compound 2 at a concentration of 0.01 ⁇ M allowed some breakthrough expression of p24 (9.7 ng/ml) and infectious virus (2.5 logio pfu/ml).
  • the comparable control culture had a p24 level of 185 ng/ml and an infectious titer of 4.8 logio pfu/ml. This represents close to a 95% reduction in p24 expression and more than a 200-fold reduction in infectious titer compared to the control sample.
  • Cytomegalovirus is a member of the Herpes family of viruses. CMV is prevalent in the United States and, as a result, within the blood supply. While the infection in a healthy individual is seldom symptomatic, patients who are immunosuppressed CMV-seronegative and become infected via transfusion of a CMV infected blood product can suffer serious complications or death. Quinley, E.D., "Immunohematology Principles and Practice," J.B. Lippincott Co., Philadelphia, PA (1993). In this example, methods of the present invention are tested for the ability to inactivate cytomegalovirus (CMV).
  • CMV Cytomegalovirus
  • a IO 7 pfu/ml stock of cell associated CMV was made by harvesting flasks of MRC-5 cells (obtained from American Type Culture Collection) on the eighth day of infection with CMV strain AD 169 (obtained from American Type Culture Collection.)
  • the infected cells were trypsinized and counted using a Neubauer type hemocytometer.
  • the infected cells were then centrifuged and resuspended in phosphate buffered saline (PBS) so that the final concentration was IO 7 cells/ml.
  • PBS phosphate buffered saline
  • MRC-5 cells were grown to confluence in a 24 well plate. Samples were quickly thawed and diluted 1:10 in Modified Eagles Medium with Earle's salts with Yeastolate (Gibco) and Bovine Serum Albumin (BSA) and allowed to clot at 37°C for 30 minutes before a second 1:10 diluting in the same medium. Each well was washed with diluent medium (to remove any inhibitory proteins) and then infected with 125 ⁇ l of diluted sample, and four wells are infected for each dilution.
  • diluent medium to remove any inhibitory proteins
  • Plaques have a markedly increased uptake of the neutral red stain as compared to the background lawn of cells. Plaques are visualized on a light box using a stereomicroscope. Cells that are questionable are confirmed using a light microscope, as are wells where no plaques are found.
  • HCV Hepatitis C virus
  • SDP single-donor platelet apheresis
  • the study was structured as four replicate inactivation experiments. In each of the four replicates the "Test” unit contained approximately 4 x 10 ⁇ platelets suspended in 35% autologous plasma and 65% synthetic medium plus phosphate, with a total volume of approximately 300 mL. In addition, each "Test” unit contained approximately 15 mg of Compound 2 and approximately 10 ⁇ pfu/mL of cell-free BVDV stock, which was prepared as follows.
  • One-day-old roller bottles of MDBK cells (approx. 70-80% confluent) were inoculated with approximately 5 mL of previously prepared BVDV stock. The roller bottles were then incubated for 1-2 hours at 37° C. After incubation, the inoculum was removed and fresh medium was added, and the roller bottles returned to the 37° C incubator. After approximately 72 hours, the new viral stock was harvested and centrifuged at 3000 rpm (approximately 2000 g) for 10' to remove cellular debris. The supernatant was either used immediately as BVDV inoculum, or stored frozen at or below -70° C until use.
  • the "Control” units were prepared from the same SDP or SDP pool as the "Test” unit.
  • the "Control” units differed only in that no Compound 2 was added to the “Control” units, the "Control” units were not illuminated and in two of the four replicates, and the "Control” units were prepared in TeflonTM "mini” bags containing approximately 30 mL instead of the approximately 300 mL in the "Test” unit. Mini bags were used in the Control units to conserve BVDV stock. Samples were withdrawn from the "Test” units after cumulative UVA exposures of 0 J/cm 2 , 0.5 J/cm 2 , 1 J/cm 2 , 2 J/cm 2 and 3 J/cm 2 and evaluated for virus infectivity.
  • the average titers at the subsequent sampling times were 10 -9 +/- 0.6 p f u /mL, l ⁇ 4-9 +/- 0.6 p f u /mL, 10 ⁇ -0 +/- 0.6 p f u / m L and l ⁇ 5.0 + /- 0.6 pfu/mL, respectively. Therefore there was no significant decrease in BVDV titer as a result of dilution into SDP during the course of the Compound 2 and UVA treatment.
  • the "Test" samples had an average initial titer of 10 ⁇ -8 +/- 0.7 pf u / m L at the first sampling time (prior to illumination).
  • the average titers at the subsequent sampling times were ⁇ 10 " l-5 pfu/mL, 10 " 0- 2 + / " 0- pfu/mL, ⁇ 10 " 0-3 pfu/mL and ⁇ 10 " 1- 7 pfu/mL, respectively.
  • the average decrease in BVDV titer was at least 10 4 -8 pfu/mL/10 "1 ' 7 pfu/mL which represents a 10 ⁇ -5 - fold reduction in BVDV infectivity.
  • Reagents were prepared for the experiment as follows. First, 3 mM Compound 2 /10 mM sodium ascorbate was prepared by combining 100 ⁇ l 15.1 mM Compound 2; 5 ⁇ l 1 M sodium ascorbate; and 395 ⁇ l dH2 ⁇ . Then 2.5 mg/ml Psoralen/ 25 mM sodium ascorbate was prepared by combining 250 ⁇ l 10 mg/ml Psoralen; 25 ⁇ l 1 M sodium ascorbate; and 725 ⁇ l DMSO. (The present invention contemplates that the use of ascorbate during inactivation aids in the preservation of blood product function during photochemical inactivation.
  • ascorbate While ascorbate is not believed to be necessary for the methods of the present invention, previous experiments to evaluate the use of ascorbate for the preservation of blood product function during photochemical inactivation have suggested improvements attributable to the presence of ascorbate (data not shown). Without restricting the present invention to any particular mode of operation, it is hypothesized that ascorbate acts as a quencher, binding molecular oxygen from the treated sample so that oxygen radicals which may cause damage to the blood product do not have an opportunity to react with the blood product, thereby protecting the function of the blood product).
  • Compound 2 /normal plasma and Psoralen /normal plasma samples - 20 ⁇ l was removed from each well into an eppendorf tube for HPLC analysis (60 ⁇ l).
  • Compound 2 /B19(+) sample - 10 ⁇ l was removed for assay into 90 ⁇ l IMDM/2% FBS (IO "1 0 ). The dilution series was repeated (10 ⁇ l + 90 ⁇ l) out to IO "3 - 0 . A second 10 ⁇ l was removed into 90 ⁇ l of PBSC cells for undiluted sample. Psoralen /B19(+) sample - 10 ⁇ l was removed for assay into 90 ⁇ l IMDM/2% FBS (10"1 - Q ). The dilution series was repeated (10 ⁇ l + 90 ⁇ l) out to 10 " -0. A second 10 ⁇ l was removed into 90 ⁇ l of PBSCs for undiluted sample.
  • Psoralen /B19(+) sample - 10 ⁇ l was removed for assay into 90 ⁇ l IMDM/2% FBS (10 " l-0). The dilution series was repeated (10 ⁇ l + 90 ⁇ l) out to 10 " -0. A second 10 ⁇ l was removed into 90 ⁇ l of PBSC cells for undiluted sample. The last 10 ⁇ l was removed into 40 ⁇ l IMDM/2% FBS for PCR analysis and samples were frozen. Compound 2 /normal plasma and Psoralen /normal plasma samples - 20 ⁇ l was removed from each well and placed in an eppendorf tube for HPLC analysis (60 ⁇ l total).
  • the infectious assay was performed in peripheral blood stem cells.
  • One ampule of cryopreserved PBSCs was thawed rapidly in 37°C water bath, and the contents were transferred into a 50 ml conical centrifuge tube. While vortexing the cell suspension gently -48 ml ice cold IX D-PBS was added slowly. The mixture was centrifuged at 300 RCF for 5 min and the supernatant was discarded. The pellet was resuspended with 2 ml cold D-PBS and triturated vigorously. The volume was brought to 50 ml with more D-PBS, the mixture was centrifuged at 300 RCF for 5 min, and the supernatant was discarded. This was repeated once.
  • the pellet was then resuspended with 2 ml ice cold IMDM/ 2% FBS by triturating vigorously. A count of 100 ⁇ l was taken using trypan blue as a vital stain. The volume of the cell suspension was adjusted to give ⁇ 1 X IO 7 cells/ml. A 1:10 dilution of this cell suspension was prepared for each sample to be tested (infected) to yield ⁇ 1 X 10 6 cells /ml. Aliquots of 270 ⁇ l of this suspension were prepared for each dilution to be tested and 90 ⁇ l for the undiluted samples. The infected cultures were then incubated for 2 hours at 4°C.
  • FIGS. 29 and 30 The results appear in FIGS. 29 and 30.
  • the assay measures the number of cells that manage to divide and give rise to colony forming units- erythroid (CFU-E). Positive controls show that no BFU-E are present in the B19 infected samples that remain untreated. In the highest dilution samples (IO -3 ) even the controls show BFU-E at around the average number. It is presumed that at this concentration either no virus was present in the samples or the virus present was not sufficient to cause infection.
  • the results show that both psoralen and Compound 2 have inactivation activity, which is displayed by the contrast between surviving BFU-E in the photochemically treated infective samples and the untreated infective samples.

Abstract

L'invention concerne des procédés permettant d'inactiver des virus pathogènes à l'aide de psoralènes nouveaux et de psoralènes connus, qui ne compromettent pas la fonction des produits sanguins destinés à la transfusion. Spécifiquement, la présente application décrit des procédés utilisant des aminopsoralènes et la lumière U.V. pour inactiver le parvovirus B19 humain dans les produits sanguins.
PCT/US1996/009837 1995-12-14 1996-06-06 Inactivation des virus non enveloppes WO1997021346A1 (fr)

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