WO1996008965A1 - Procede d'inactivation photodynamique de contaminants du sang de nature virale et bacterienne a l'aide de sensibilisants a la coumarine ou la furocoumarine - Google Patents

Procede d'inactivation photodynamique de contaminants du sang de nature virale et bacterienne a l'aide de sensibilisants a la coumarine ou la furocoumarine Download PDF

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Publication number
WO1996008965A1
WO1996008965A1 PCT/US1995/012069 US9512069W WO9608965A1 WO 1996008965 A1 WO1996008965 A1 WO 1996008965A1 US 9512069 W US9512069 W US 9512069W WO 9608965 A1 WO9608965 A1 WO 9608965A1
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Prior art keywords
photosensitizer
viral
biological solution
blood
virus
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PCT/US1995/012069
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English (en)
Inventor
Sang Chul Park
Raymond P. Goodrich, Jr.
Nagender Yerram
Samuel O. Sowemino-Coker
Matthew S. Platz
Brian Aquila
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Baxter International, Inc.
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Priority claimed from US08/311,125 external-priority patent/US5516629A/en
Priority claimed from US08/343,680 external-priority patent/US6251644B1/en
Application filed by Baxter International, Inc. filed Critical Baxter International, Inc.
Priority to JP8511090A priority Critical patent/JPH10506391A/ja
Priority to EP95933899A priority patent/EP0782388A4/fr
Priority to AU36385/95A priority patent/AU691672B2/en
Publication of WO1996008965A1 publication Critical patent/WO1996008965A1/fr
Priority to NO971350A priority patent/NO971350L/no

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/10—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person
    • A61K41/17—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person by ultraviolet [UV] or infrared [IR] light, X-rays or gamma rays

Definitions

  • This invention relates to the general field of chemistry, and more specifically, the inactivation of viral and bacterial contamination of blood and blood products including compositions comprising peripheral blood cells (red blood cells, platelets, leukocytes, stem cells, etc.), plasma protein fractions (albumin, clotting factors, etc.) from collected whole blood, the blood of virally infected subjects, ex vivo media used in the preparation of anti-viral vaccines, and cell culture media such as fetal bovine serum, bovine serum or derivatives from these sources.
  • peripheral blood cells red blood cells, platelets, leukocytes, stem cells, etc.
  • plasma protein fractions albumin, clotting factors, etc.
  • cell culture media such as fetal bovine serum, bovine serum or derivatives from these sources.
  • This 6 log threshold may be greater for plasma protein components, especially the clotting factors (Factor VIII, Factor IX) that are administered throughout the life of some hemophilia patients.
  • All blood collected in the United States is currently screened for six infectious agents: HIV-1, HIV-2, HTLV-1, Hepatitis B Virus, Hepatitis C Virus and Syphilis. Additionally, donors are screened for risk factors, and potential donors are eliminated that are considered at risk for HIV. Despite these measures, the risk of becoming infected by a potentially deadly virus or bacteria via the transfusion of blood or blood products remains serious. Screens for contaminants are by nature not foolproof. There is also the likely occurrence of new infectious agents that will enter the blood supply before their significance is known. For example, by the end of June 1992, the Center for Disease Control reports that 4,959 AIDS cases could be traced directly to the receipt of blood transfusions, blood components or tissue.
  • Viral inactivation by stringent sterilization is not acceptable since this method can also destroy the functional components of the blood, particularly the erythrocytes (red blood cells), thrombocytes (platelets) and the labile plasma proteins, such as clotting factor VIII.
  • Viable red blood cells can be characterized by one or more of the following:
  • Wet steam sterilization also destroys function blood components, in particular, blood cells and plasma proteins. Dry heat sterilization, like wet steam, is harmful to blood cells and blood proteins at the levels needed to reduce viral infectivity.
  • stabilizing agents for example, carbohydrates, does not provide sufficient protection to the delicate blood cells and proteins from the general effects of exposure to high temperature and pressure.
  • Methods that are currently employed with purified plasma protein fractions, often followed by lyophilization of the protein preparation include treatment with organic solvents and heat, or alternatively, extraction with detergents to disrupt the lipid coat of membrane enveloped viruses. Lyophilization, freeze-drying, alone has proven insufficient to either inactivate viruses or render blood proteins sufficiently stable to the effects of heat sterilization.
  • the organic solvent or detergent method employed with purified blood proteins cannot be used with blood cells as these chemicals destroy the lipid membrane that surrounds the cells.
  • beta-propiolactone Another viral inactivation approach for plasma proteins; first demonstrated in 1958, involves the use of the chemical compound beta- propiolactone with ultraviolet (UV) irradiation. This method has not found acceptance in the United States due to concern over the toxicity of beta- propiolactone in the amounts necessary to achieve some demonstrable viral inactivation and to unacceptable levels of damage to the proteins caused by the chemical agents. Concern has been raised over the explosive potential for beta-propiolactone as well.
  • Contamination problems also exist for blood plasma protein fractions, plasma fractions containing immune globulins and clotting factors.
  • new cases of Hepatitis A and Hepatitis C have occurred in hemophilia patients receiving protein fractions containing Factor VIII which have been treated for viral inactivation according to approved methods.
  • Factor VIII Factor VIII which have been treated for viral inactivation according to approved methods.
  • non-enveloped viruses include Hepatitis A and human Parvovirus B19.
  • Non- enveloped viruses do not possess lipid coats but compensate by the presence of highly impenetrable protein capsids.
  • Human parvovirus B19 is a heat-stable single-stranded DNA virus of the genus Parvovirus. B19 is the only human parvovirus that produces clinical illness. In children and young adults, B19 causes erythema infectiosum, or fifth disease, a common childhood exanthema. However, in pregnant women, patients with disorders involving increased red blood cell production and those with either acquired or congenital immunodeficiency B19 can be life-threatening. The disease manifestations in these individuals include, respectively, hydrops fetalis, acute aplastic and hypoplastic anemia, and chronic anemia. See, Luban(1994) Transfusion 34:821.
  • Psoralens are naturally occurring compounds which have been used therapeutically for millennia in Asia and Africa. The action of psoralens and light has been used to treat vitiligo and psoriasis (PUVA therapy;
  • Psoralen binds to nucleic acid double helices by intercalation between base pairs; adenine, guanine, cytosine and thymine (DNA) or uracil (RNA). Upon absorption of UVA photons the psoralen excited state has been shown to react with a thymine or uracil double bond and covalently attach to both strands of a nucleic acid helix.
  • the crosslinking reaction is specific for a thymine (DNA) or uracil (RNA) base and proceeds only if the psoralen is intercalated in a site containing thymine or uracil.
  • the initial photoadduct absorbs a second UVA photon and reacts with a second thymine or uracil on the opposing strand of the double helix to crosslink the two strands of the double helix.
  • Lethal damage to a cell or virus occurs when a psoralen intercalated into a nucleic acid duplex in sites containing two thymines (or uracils) on opposing strands sequentially absorb 2 UVA photons. This is .an inefficient process because two low probability events are required; the localization of the psoralen into sites with two thymines (or uracils) present and its sequential absorption of 2 UVA photons.
  • United States Patent 4,748,120 to Wiesehahn , is an example of the use of certain substituted psoralens by a photochemical decontamination process for the treatment of blood or blood products.
  • the psoralens described for use in the process do not include halogenated psoralens, or psoralens with non-hydrogen binding ionic substituents.
  • photosensitizers that have been employed are typically dyes. Examples include dihematoporphyrin ether (DHE), Merocyanine 540 (MC540) and methylene blue.
  • DHE dihematoporphyrin ether
  • MC540 Merocyanine 540
  • methylene blue methylene blue
  • an effective radiation photosensitizer must bind specifically to nucleic acids and must not accumulate in significant amounts in the lipid bilayers that are common to viruses, erythrocytes, and platelets.
  • neutral psoralens such as 8-MOP are uncharged and thus also have a high affinity for the interior of lipid bilayers and cell membranes.
  • Positively charged psoralens for example, AMT, do not bind to the interior of phospholipid bilayer membranes because of the presence of the charge.
  • AMT contains an acidic hydrogen which binds to the phospholipid head group by hydrogen bonding, shown below.
  • AMT is an unacceptable photosensitizer because it indiscriminately sensitizes and damages viral membranes and the membranes of erythrocytes and platelets.
  • 08/091,674 commonly assigned with the present application, and parent applications to this application, disclose the use of a novel class of psoralen photosensitizers that are superior for use with irradiation to inactivate viral and bacterial contaminants in blood and blood products.
  • Said psoralens are characterized by the presence of a halogen substituent and a non-hydrogen binding ionic substituent to the basic psoralen side chain. See also,
  • the present invention provides a method for the inactivation of viral and bacterial contaminants present in blood and blood protein fractions.
  • the present invention involves utilization of photosensitizers which bind selectively to a viral nucleic acid, coat protein or membrane envelope.
  • the photosensitizer is also a moiety which can be activated upon exposure to radiation, which may be in the form of ultraviolet radiation or ionizing radiation, such as X-rays, that penetrate the contaminated sample.
  • the present invention is also applicable to the inactivation of blood- borne bacterial contaminants and blood-borne parasitic contaminants because such infectious organisms rely on nucleic acids for their growth and propagation. Since purified blood plasma protein fractions are substantially free of human nucleic acids, and mature human peripheral blood cells, in particular, red blood cells and platelets, lack their own genomic DNA/RNA, nucleic acid-binding photosensitizers are especially useful for treating the problem of blood contaminants.
  • the present invention may also be applied to viral inactivation of tissues and organs used for transplantation, to topical creams or ointments for treatment of skin disorders and for topical decontamination.
  • the present invention may also be used in the manufacture of virally-based vaccines for human or veterinary use, in particular, to produce live, nonviable or attenuated virus vaccines.
  • the present invention may also be used in the treatment of certain proliferative cancers, especially solid localized tumors accessible via a fiber optic light device and superficial skin cancers.
  • the present invention utilizes a class of compounds that have a selective affinity to nucleic acids.
  • the class of compounds also contains a halogen substituent and a water soluble moiety, for example, a quaternary ammonium ion or phosphonium ion. These materials comprise a relatively low toxicity class of compounds, which can selectively bind to the nucleic acid (single-stranded DNA, double-stranded DNA, or RNA) that comprises the genetic material of viruses.
  • the bound compound can be activated by exposure to radiation, such as ultraviolet radiation of a defined wavelength or ionizing radiation such as x-rays, after which the activated compound damages the bound viral nucleic acid or viral membranes rendering the virus sterile and non-infectious.
  • Activation of the selectively bound chemical photosensitizer focuses the photochemistry and radiation chemistry to the viral nucleic acid or viral membranes and limits exposure to nearby cellular components or plasma proteins.
  • the preferred class of photosensitizers for use with the present invention is characterized, generally, as follows: a) intercalators comprised of either b) at least one halogen substituent or c) at least one non-hydrogen bonding ionic substituent.
  • the photosensitizers comprise at least one halogen substituent and at least one non-hydrogen bonding ionic substituent.
  • Particularly preferred photosensitizers are psoralens and coumarins comprising at least one halogen substituent and at least one non-hydrogen bonding ionic substituent.
  • the preferred photosensitizers are intercalators that fluoresce and that are comprised of either a) at least one halogen substituent or b) at least one non-hydrogen bonding ionic substituent.
  • the preferred photosensitizers according to this embodiment are the substituted coumarins having the structure as shown below.
  • the photosensitizers disclosed herein are suited for the inactivation of a variety of viral and bacterial contaminants associated with blood and blood products.
  • the present invention specifically includes the
  • HIV-1 Human Immunodeficiency Virus- 1
  • Sindbis Virus Cytomegalovirus.
  • VSV Vesicular Stomatitis Virus
  • HSV- 1 Herpes Simplex Virus Type 1
  • the present invention also demonstrates the flexibility of adding one or more halogen atoms to any cyclic ring structure capable of intercalation between the stacked nucleotide bases in a nucleic acid (either DNA or
  • RNA in order to confer new photoactive properties to the intercalator.
  • intercalating molecule psoralens, coumarins, or other polycyclic ring structures
  • halogenation or addition of non-hydrogen bonding ionic substituents can be selectively modified by halogenation or addition of non-hydrogen bonding ionic substituents to impart advantages in its reaction photochemistry and its competitive binding affinity for nucleic acids over cell membranes or charged proteins.
  • halogenation of psoralen enables the molecule, once intercalated within the nucleic acid, to undergo a strand cleavage reaction upon light activation that non-halogenated psoralens cannot initiate.
  • the nucleic acid strand cleavage is attributable to a novel electron transfer pathway (see Figure 1) created by the breaking of the carbon-halogen bond upon the application of the appropriate radiation energy.
  • the mechanism for this alternative chemical reaction requires a single UV photon and is more efficient than the crosslinking reaction that normally occurs with non- halogenated psoralens.
  • the electron transfer reaction involves transfer from a donor (usually a guanine base when the intercalator is inserted in nucleic acid) and an acceptor (the carbon radical created by the broken carbon-halogen bond). Since the donor and acceptor species must be in close physical proximity for the transfer reaction to proceed, most damage is limited to the nucleic acid, as is desired in viral inactivation.
  • a donor usually a guanine base when the intercalator is inserted in nucleic acid
  • an acceptor the carbon radical created by the broken carbon-halogen bond
  • halogenation of a coumarin imparts totally new photoactive properties useful for viral inactivation.
  • Coumarins unlike psoralens, do not have an inherent ability to crosslink nucleic acid strands upon exposure to radiation, and hence have not heretofore found application as photosensitizers.
  • halogenation of this class of intercalating molecules confers the ability to undergo the electron transfer mechanism, thereby imparting new properties to the molecule.
  • the inventors believe that the example of coumarin halogenation demonstrates that the principles disclosed herein can be extended to any intercalating molecule to confer new photoactive properties.
  • halogen substituents or non-hydrogen bonding ionic substituents can be created by adapting the present invention to many known classes of ring compounds, whether those compounds comprise intercalating agents or not.
  • known classes of compounds that may be improved by the present invention include, porphyrins, phthalocyanines, quinones, hypericin, and organic dye molecules such as coumarins, for example, merocyanine dyes, methylene blue and eosin dyes.
  • organic dyes for example, methylene blue which is not considered a nucleic acid intercalating compound, have been used for viral inactivation treatments of blood plasma with questionable success. It is contemplated that such organic dyes, modified according to the present invention, may prove more efficacious than the unmodified dye in such an application.
  • the inventors further anticipate that the fluorescent coumarin photosensitizers described herein may also be used in combination with known photosensitizing molecules that absorb in the visible light wavelength region.
  • Figure 11 shows the fluorescence emission spectrum of one such coumarin molecule
  • Photosensitizer A having an emission peak at 414 nm in the visible light spectrum.
  • the emission spectrum of Photosensitizer A extends beyond 500 nm, which can overlap the absorbance range of certain visible light activated molecules. It is therefore anticipated that a combination of a visible fluorescing photosensitizer with one or more photosensitizers that absorb in the visible light region may be utilized for enhanced virucidal or cytotoxic effect.
  • photosensitizers that absorb in the visible light region include hypericin, pthalocyanines, porphyrins, and organic dyes such as methylene blue. See, for example, International Patent Application WO/94 14956, wherein hypericin is activated via a chemiluminescent reaction between luciferin and luciferase.
  • nucleic acid binding photosensitizers include the preparation of non-infectious viral vaccines, therapeutic treatment of immune system disorders by photophoresis, elimination of viable nucleated cells such as leukocytes via the cytotoxicity of nucleic acid binding photosensitizers and possible treatment for certain accessible cancers and tumors exploiting the cytotoxic effects of nucleic acid binding photosensitizers.
  • the inventors further anticipate that the problem of singlet oxygen production by UV irradiation of traditional psoralen molecules can also be reduced by incorporating a quenching sidechain moiety onto the psoralen nucleus.
  • An example of such a compound is shown below.
  • the non-hydrogen bonding ionic substituent of the present invention further comprises a quaternary ammonium pyridyl group.
  • This quaternary ammonium pyridyl group acts as a quencher of the UV excited triplet state of the psoralen molecule (see Figure 1).
  • the quenching pyridyl group deactivates the triplet state of any psoralen or intercalator, thereby preventing formation of undesired singlet oxygen.
  • the pyridyl group quenches the excited triplet state by promoting electron transfer.
  • the halo-intercalator serves as the donor, and carbon-centered radicals are not formed.
  • the electron is transferred from the halo-intercalator to the pyridium ion and back. This reversible electron transfer shorts out the triplet state before it can react to make singlet oxygen.
  • the pyridium ions quench the excited singlet state of the halo intercalator, the lifetime of the singlet state is so short that little quenching actually occurs.
  • the present invention includes methods for the viral inactivation of non-enveloped viruses such as Hepatitis A and Human Parvovirus B19.
  • the method generally includes the irradiation of blood and blood components in the presence of photosensitizers under operating conditions that "loosen” or increase the permeability of the viral protein capsid.
  • non-enveloped viruses found as contaminants in plasmid protein compositions are inactivated by irradiation of said compositions containing one of the photosensitizers of the present invention.
  • the operating conditions for the irradiation are selected so as to increase the permeability of the capsid.
  • Operating conditions that may be adjusted in order to increase access to the nucleic acid core of the non- enveloped virus include reduced ionic strength, solvent detergent concentration, pH, chaotrophic agents, reducing agents, freeze-thaw cycles and elevated temperature.
  • a photosensitizer is added to the blood product solution under operating conditions which increase the permeability of non-enveloped viruses contaminating said solution.
  • the solution is then inactivated under conditions where substantially all of the non-enveloped viruses in the solution are inactivated without substantially impairing the biological functions of the components of the solution being treated.
  • FIGURE 1 depicts the proposed energy diagram of photosensitizer A of the present invention.
  • FIGURE 2 depicts the proposed reaction mechanism for the inactivation of nucleic acid upon irradiation of photosensitizer A.
  • FIGURE 3 depicts the inactivation of Human Immunodeficiency
  • HIV-1 Virus- (HIV-1) using long wavelength ultraviolet radiation (UVA) in the presence of different concentrations of photosensitizer B. Viral reduction, log 10, is plotted versus UVA fluence, Joules/cm 2 .
  • FIGURE 4 depicts the same data as Figure 3 as described above, where viral reduction is plotted versus concentration of photosensitizer B.
  • FIGURE 5 depicts the inactivation of Sindbis Virus
  • photosensitizer A and photosensitizer B are photosensitizers A and photosensitizer B. Virus inactivation is shown versus concentration of photosensitizer.
  • FIGURE 6 depicts the inactivation of Cytomegalovirus using photosensitizer B and UVA. Viral inactivation is plotted versus UVA fluence.
  • FIGURE 7 depicts the inactivation of Vesicular Stomatitis Virus
  • VSV in platelet concentrate using photosensitizer B and UVA. Viral inactivation is plotted versus UVA fluence.
  • FIGURE 8 depicts the inactivation of Herpes Simplex Virus Type 1 (HSV-1) in the presence of photosensitizer B and UVA. Viral inactivation is plotted versus UVA fluence.
  • HSV-1 Herpes Simplex Virus Type 1
  • FIGURE 9 depicts the synthetic scheme for the synthesis of photosensitizer A.
  • FIGURE 10 depicts the absorption spectrum of photosensitizer A.
  • FIGURE 11 depicts the fluorescence spectrum of photosensitizer A.
  • FIGURE 12 depicts the inactivation of Sindbis Virus with photosensitizers B, A, AX, CX, D, DX and E.
  • FIGURE 13 depicts the synthetic scheme for the synthesis of photosensitizer D. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention is directed to methods for reducing viral, bacterial and other parasitic contamination in blood, blood components, cell cultures or cell culture components by irradiation in the presence of a chemical photosensitizer.
  • Photosensitizers are disclosed which are particularly useful in the decontamination of liquid or frozen-state liquid compositions, for example, blood, blood components, reconstituted lyophilized cells and the like, using UV radiation.
  • a radiation sensitizing chemical compound is added to a suspension of blood, blood components, cell cultures or cell culture components contaminated with virus and/or bacteria and/or parasites, and the mixture is exposed to UV or ionizing radiation.
  • the present invention includes a method for reducing viral, bacterial and other parasitic contamination from a biological sample, for example, a solution.
  • Biological solutions include, but are not limited to, solutions comprising blood, blood components, cell culture or components of a cell culture.
  • the method comprises mixing the composition in a liquid state with a chemical photosensitizer capable of binding to the viral, bacterial or parasitic contamination.
  • the chemical photosensitizer is capable of being activated by irradiation under conditions of sufficient wavelength, intensity and period of exposure to inactivate the contaminant, while at the same time the conditions for irradiation are insufficient to produce reactive oxygen species in the composition at levels which substantially impair the physiological activity of the treated composition.
  • the composition containing the photosensitizer is then irradiated under conditions where the concentration of biologically active contaminant is reduced and the physiological activity of the composition is substantially unimpaired.
  • a photosensitizer is defined for the purposes of this application as a chemical compound that has a light- absorbing chromophore that absorbs radiation between 780 and 200 nm, and is capable of inactivating viral, bacterial or parasitic contaminants in blood or blood products.
  • the photosensitizers of the present invention are characterized by their ability to bind to the nucleic acid components of the viral or bacterial contaminants that are to be inactivated.
  • the blood and blood product compositions that are to be treated according to the method of this invention all contain at least some cellular components or complex proteins.
  • the photosensitizers of this invention are characterized as comprising a lipophilic moiety, a hydrophilic moiety and a photoreactive moiety.
  • the photosensitizers of this invention are preferably nucleic acid intercalators that are comprised of either 1) at least one halogen atom; and b) at least one non-hydrogen bonding ionic moiety.
  • Intercalators are defined broadly herein as any chemical compound that has a specific affinity to double or single stranded nucleic acid. More specifically, intercalators are chemicals -- not including nucleic acids, proteins or peptides -- that locate themselves between neighboring base pairs in nucleic acids. Intercalators are generally characterized by the presence of a relatively planar rigid, multi-cyclic pi-conjugated chemical backbone.
  • intercalators Those skilled in the art are familiar with a relatively large number of intercalators and are generally able to predict the types of chemical species that are able to function as intercalators based on the chemical structure of the backbone of the chemical species.
  • Psoralens and coumarins the preferred basic structure for the intercalators of the present invention, are just two examples of chemical backbone structures capable of nucleic acid intercalation.
  • Preferred photosensitizers of the present invention comprise at least one halogen substituent.
  • the halogens include F, Cl, Br and I.
  • the photosensitizer contains at least one bromine or chlorine atom.
  • Preferred photosensitizers of the present invention comprise at least one non-hydrogen bonding ionic substituent.
  • Chemical functionalities that are ionic and non-hydrogen bonding include quaternary ammonium functionalities and phosphonium functionalities.
  • a variety of additional functionalities that are both ionic and non-hydrogen bonding are familiar to those skilled in the art, and equally applicable for use with this invention.
  • the non-hydrogen bonding ionic substituent is linked to the backbone of the chemical intercalator via a spacer unit.
  • the spacer can be selected from any of a number of chemical subunits known to those skilled in art, but in the preferred embodiments is composed of a saturated linear alkoxy group. In the most preferred embodiment the spacer element is -O(CH 2 ) 3 -.
  • the most preferred non-hydrogen bonding ionic functionalities are quaternary ammonium functionalities, more specifically, trialkyl quaternary ammonium, and even more specifically, -O(CH 2 ) 3 N•(CH 2 CH 3 ) 3 .
  • Two preferred photosensitizers of the present invention are the following:
  • Compound A is a coumarin based photosensitizer
  • compound B is a psoralen or furocoumarin based photosensitizer.
  • compound A Upon UV irradiation, compound A has been shown to be effective at viral inactivation while compound B has been shown to be effective at viral and bacterial inactivation.
  • Compounds A, D and E also fluoresce upon UV irradiation. It is theorized by the present inventors that the fluorescence pathway for the dispersion of energy from the excited state of irradiated compounds A, D and E, as depicted in Figure 1, acts to reduce the production of highly reactive oxygen species in blood and blood
  • the proposed reaction mechanism for the inactivation of viral contaminants using compound A and UV radiation is shown in Figure 2.
  • the photoreaction is initiated by an electron transfer from a guanine residue to the photosensitizer in its executed singlet state. Electron transfer is followed by Br-C bond homolysis and the generation of a coumarin radical that can attack the nucleic acid backbone.
  • Bromopsoralens specifically photosensitizer B, do not form free radicals upon irradiation in solution.
  • a donor is required to activate photosensitizer B.
  • fluorescence spectroscopy it has been shown that amino acids are not suitable donors to activate photosensitizer B.
  • any of these photosensitizers bound or associated with proteins should not generate radicals capable of damaging proteins.
  • Photosensitizers that are capable of fluorescence appear to be superior to non-fluorescent varieties.
  • a photosensitizer to be useful, there must be a mechanism for viral and bacterial inactivation.
  • Non- halogenated psoralens may still function as useful photosensitizers if they are properly situated in the solution to be treated. Such compounds can inactivate viruses via the traditional photocrosslinking mechanism.
  • Other photosensitizers, such as those having the coumarin backbone structure must be halogenated in order to accomplish significant viral or bacterial inactivation.
  • the preferred photosensitizers are intercalators capable of fluorescence and either 1) are halogenated or 2) have the psoralen backbone structure.
  • the photosensitizer of the invention comprises a quenching sidechain moiety attached to the intercalating backbone.
  • Figure 1 provides a diagrammatic energy diagram for certain halogenated photosensitizers that are capable of fluorescence. According to the theory expressed herein, the ability to fluoresce provides a rapid means for the excited singlet state species to revert to ground energy state that competes with intersystem crossing to the triplet excited state. For photosensitizers that do not fluoresce in particular, the presence of a quenching moiety attached to the intercalator can also serve the same function.
  • the non-hydrogen bonding ionic substituent comprises a quaternary ammonium pyridyl group. Such a compound can be easily prepared by one skilled in the art without undue experimentation.
  • the quaternary ammonium pyridyl group Such a compound can be easily prepared by one skilled in the art without undue experimentation.
  • ammonium pyridyl group can serve as a quencher of the UV excited triplet state of the psoralen compound. While not intending to be bound by theory, it is proposed that the quenching group will deactivate the triplet state of any intercalator, thereby preventing formation of undesired singlet oxygen. The reduction of singlet oxygen production as such minimizes damage to lipid membranes or proteins.
  • the proximity of the quenching moiety to the intercalator should make quenching highly preferred to any reaction with oxygen in solution, and should also obviate the need for the addition of exogenous quenching agents (such as oxygen scavengers, reducing agents or sugars) into the medium.
  • the quenching moiety may be attached to the backbone of the photosensitizer at any position, and can consist of any chemical functionality known to those skilled in the art to function as an excited state quenching agent.
  • quaternary ammonium or phosphonium substituted halo- intercalators described herein do not accumulate in the interior of lipid bilayers (membranes) found in blood and blood products because of the presence of the charge, nor will they bind to the phospholipid head groups of the membrane because they lack acidic hydrogen for hydrogen bonding.
  • Prior art psoralens for example, 8-MOP and AMT, must often be used in combination with a quencher (e.g. mannitol, dithiothreitol, vitamin E, etc.) to protect, repair or otherwise offset the deleterious effects of the photosensitizer and light on cell membranes, and to quench the production of free oxygen radicals in solution that cause indiscriminate damage.
  • a quencher e.g. mannitol, dithiothreitol, vitamin E, etc.
  • the photosensitizers described herein do not accumulate in viral membranes and as a consequence do not require the presence of a quencher additive to the blood product.
  • the photosensitizers described herein containing halogen generate a minimal amount of free radicals in solution, thereby avoiding the need for quenchers.
  • One preferred class of photosensitizers is selected from the group consisting of compounds of the formula (I):
  • ⁇ is an integer from 1 to 6;
  • X is an anionic counterion;
  • Z is N or P;
  • R 1 , R 2 , R 3 , R 4 , R 5 and R 6 are independently halo; H; linear or branched alkyl of 1-10 carbon atoms; linear or branched alkoxy of 1-10 carbon atoms; (CH 2 )- m O (CH 2 ) p Z•R',R",R'" or -O(CH 2 ) n Z•R',R",R'" wherein n, m and p are independently integers from 1 to 10 and R', R", and R'" are
  • R 1 , R 2 , R 3 , R 4 , R 5 or R 6 independently H or linear or branched alkyl of 1 to 10 carbon atoms with the proviso that on each Z atom, not more than two of R', R", or R'" may be H; and at least on one of R 1 , R 2 , R 3 , R 4 , R 5 or R 6 is
  • R 6 , R 5 , R 2 and R 1 are hydrogen and R 3 is H or halo, preferably bromo.
  • An additional preferred class of photosensitizers is selected from the group consisting of the formula (II).
  • ⁇ is an integer from 1 to 6;
  • X is an anionic counterion;
  • Z is N or P;
  • R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently halo; H; linear or branched alkyl of 1-10 carbon atoms; linear or branched alkoxy of 1-10 carbon atoms; (CH 2 )- m O (CH 2 ) p Z•R'.R",R'" or -O(CH 2 ) n Z•R',R",R"' wherein n, m and p are independently integers from 1 to 10 and R', R", and R'" are
  • R', R", or R' independently H or linear or branched alkyl of 1 to 10 carbon atoms with the proviso that on each Z atom, not more than two of R', R", or R'" may be
  • R 1 , R 2 , R 3 , R 4 , R 5 or R 6 is
  • R 3 , R 5 , R 2 and R 1 are hydrogen and R 3 is H or halo, preferably bromo.
  • the above compounds are made by halogenating psoralens and isolating the appropriately substituted isomers.
  • the ring substituent is a quaternary ammonium alkoxy or phosphonium alkoxy group
  • that group may be made from the corresponding hydroxy- substituted psoralens, as exemplified by the following scheme.
  • the most preferred photosensitizers of the present invention are comprised of ionic functionalities that are non- hydrogen bonding.
  • photosensitizers comprised of amine functionalities having one and in some cases two amine hydrogens. These compounds, of course, are capable of forming hydrogen bonds. It has been shown that there is a direct correlation between the number of hydrogens available on the amine and the cellular destruction caused by a class of psoralen compounds. Goodrich, et al (1994) Proc. Nat'l. Acad. Sci. USA, 91:5552. Thus, photosensitizers containing amine functionalities having two hydrogens are less preferred than those having one hydrogen, which are in turn less preferred than those having no hydrogen attached to the amine.
  • sensitizing compounds for viral inactivation preferably, do not contain substituents which possess free hydrogen groups capable of exhibiting hydrogen bonding to the cell membrane.
  • sensitizing compounds for viral inactivation preferably, do not contain substituents which possess free hydrogen groups capable of exhibiting hydrogen bonding to the cell membrane.
  • the present invention can be used to selectively bind a chemical photosensitizer to blood-transmitted viruses, bacteria, or parasites.
  • monoclonal or polyclonal antibodies directed against specific viral antigens, either coat proteins or envelope proteins, may be covalently coupled with a
  • cell compositions also comprise a variety of proteins
  • the method of decontamination of cells described herein is also applicable to protein fractions, particularly blood plasma protein fractions, including, but not limited to, fractions containing clotting factors (such as Factor VIII and Factor IX), serum albumin and immune globulins.
  • the viral and bacterial inactivation may be accomplished by treating a protein fraction with a photosensitizer as described herein.
  • the halogenated psoralens and coumarins according to the present invention are improved and more efficient photosensitizers because they require only a single UVA photon for activation.
  • the ability of the halogen photosensitizer to react with any base pair imposes no limitation for the site of intercalation.
  • absorption of a UVA photon by a bromocoumarin in the presence of guanine (or any nucleotide base) leads to electron transfer and the formation of bound radicals and ultimately nucleic acid cleavage and viral or cell death. This cleavage mechanism is more efficient than the conventional crosslinking reaction of non-halogenated psoralens.
  • the coumarin radical ( Figure 2) can inflict damage on the nucleic acid double helix to which it is bonded by abstraction of a ribose (RNA) or deoxyribose (DNA) sugar carbon hydrogen bond. This leads to DNA cleavage by known mechanisms.
  • the guanine radical cation shown as an example is also known to react with molecular oxygen, initiating a series of reactions which cleave DNA.
  • the byproduct of the bound radical photochemistry is debrominated coumarin 4 that is incapable of forming crosslinks to DNA, unlike psoralens.
  • a preferred class of photosensitizers comprise nucleic acid intercalators which may be added to plasma or plasma fractions followed by UV radiation to reduce the viral contamination therein.
  • nucleic acid intercalators which may be added to plasma or plasma fractions followed by UV radiation to reduce the viral contamination therein.
  • the reduction of viral contamination can be unexpectedly reduced by utilizing halogenated intercalators.
  • the bromopsoralens are about 200,000 times more effective in reducing viral activity when compared to use of their non-brominated counterparts.
  • the brominated intercalators are an improvement over the known psoralens and other substituted psoralens when used as photosensitizers because only one photon of light is required to activate the brominated photosensitizer whereas two photons are required to activate a non- brominated photosensitizer.
  • a brominated intercalator is effective in virtually every intercalative site, whereas a non-brominated
  • photosensitizer is effective only in intercalation sites containing a uracil or thymine on different strands of the DNA or RNA.
  • the brominated intercalators are also an improvement over the known coumarins, which unlike the known psoralens have no crosslinking ability, and therefore, have generally not been used previously as photosensitizers for viral inactivation or as light activated drugs in therapeutic photophoresis procedures for certain cancer treatments and immune disorders.
  • Brominated or halogenated intercalators are particularly useful for inactivation in hydrated systems such as plasma, immune sera, tissue culture media containing animal serum or serum components, for example, fetal calf serum, or recombinant products isolated from tissue culture media.
  • the present invention may be applied to treatment of liquid blood in ex vivo irradiation, such as by methods and apparatus described in U.S.
  • the photosensitizers disclosed herein may also be utilized in vivo, delivered in liposomes (artificial cells) or drug-loaded natural cells. After introduction of the liposome or drug-loaded cell, the patient may be treated by radiation to activate the photosensitizer.
  • the present invention is applicable to contaminants which comprise single or double-stranded nucleic acid chains, including RNA and DNA, viruses, bacteria and parasitic contamination.
  • certain biological solutions that are contaminated with non-enveloped viruses are treated in order to inactivate all viral contaminants in the solution, including non-enveloped viruses.
  • the treatment required for inactivating non- enveloped viruses includes the manipulation of operating conditions in order to loosen or increase the permeability of the capsid surrounding the genetic core of the virus.
  • the inventors hereto speculate that the adjustment of operating conditions to increase the permeability of the capsid allows the photosensitizers of the present invention access to the genetic material of the virus, thereby allowing viral inactivation to occur -- by harming the genetic material of the virus -- upon irradiation.
  • Example 15 adjustment of the pH, ionic strength and freeze-thaw cycles on a plasma solution containing Porcine Parvovirus yielded dramatic
  • This method encompasses the adjustment of the operating conditions of the solution so as to loosen the capsid of the virus either prior or subsequent to the addition of a photosensitizer of the present invention into the solution.
  • the solution is then irradiated under conditions to inactivate the non-enveloped viruses.
  • osmotic shock is used to loosen the protein capsid.
  • a cell or virus When a cell or virus is suspended in a low ionic strength hypotonic solution, the cell will be subjected to an osmotic shock resulting in volume expansion. In some viruses, hypotonicity may lead to rupture of the protein capsid with discharge of their nucleic acid contents.
  • the present invention includes a method for incorporating photosensitizers into non-enveloped virus in which a short but intense period of osmotic stress will cause the virus to become transiently permeable and allows partial incorporation of photosensitizers with low molecular weights.
  • the virus In the osmotic shock procedure, the virus is first incubated for a short time with dimethylsulfoxide (DMSO) or another chemical such as polyol (i.e., glycerol), or organic solvents in addition to DMSO (i.e., ethanol) that permeate to viral capsid.
  • DMSO dimethylsulfoxide
  • the virus is rapidly diluted in a solution containing photosensitizers.
  • the abrupt change in extracellular DMSO concentration induced by rapid dilution creates an osmotic gradient that spontaneously decays as DMSO reaches a new equilibrium.
  • the composition of the diluent has a profound effect on the viral capsid during osmotic shock.
  • the diluent may contain photosensitizer, inositol
  • hexaphosphate IHP
  • EDTA EDTA
  • EGTA sodium pyrophosphate
  • any polyanion in different combinations.
  • Using distilled water alone without DMSO to induce osmotic shock in Parvovirus it is possible to increase the inactivation of the virus from 0.62 to 2.46 logs. Data to this effect were obtained when the osmolality of plasma was reduced by 50%. However, further reduction in osmolality of the medium to either 30, 60, 90 or 120 mOsmol (i.e. 1, 20, 30 and 40% dilution of the native plasma with water) significantly increases the inactivation of Parvovirus from 2.46 logs to as high at 4 logs.
  • This embodiment of the invention includes the use of methods for the inactivation of non-enveloped viruses using the osmotic shock process for selective incorporation of photosensitizer into the virus.
  • this invention also includes the use of small molecular weight nucleic acid intercalators that are more effective at penetrating the protein capsid of viruses that have been subjected to osmotic shock, either alone or in combination with a freeze-thaw cycle, metal chelators, and polyanions or other operating conditions that help loosen the capsid.
  • this invention covers the following photosensitizer compounds (or derivatives thereof) that offer potentially desirable intercalation properties and that are less hydrophilic than psoralen-based photosensitizers:
  • the present invention includes the inactivation of specific viral species that are found as contaminants in blood and blood products.
  • Example 1 describes in great detail the experimental protocol for the inactivation of HIV-1 virus in platelet concentrate.
  • the results obtained from this series of experiments validate the ability of the photosensitizers of the present invention to inactivate HIV-1 virus in a blood product.
  • the results of this study are summarized in Table 1.
  • Reductions in viral titer were obtained by subtracting the viral titer of treated samples from control samples.
  • Figures 3 and 4 show a graphic representation of the results of the study.
  • Figure 3 shows the viral reduction versus light intensity for a number of different concentrations of photosensitizer B
  • Figure 4 shows viral reduction versus concentration of photosensitizer B.
  • Example 1 The procedure described in detail in Example 1 for the inactivation of the HIV-1 virus in platelets is typical of the type of experimental protocol utilized to examine the inactivation of a variety of viral species.
  • Example 2 describes the general protocol used to demonstrate the inactivation of Sindbis Virus in human plasma. The results of the inactivation using photosensitizer A and photosensitizer B are depicted in Figure 5.
  • Example 3 describes the general protocol used to demonstrate the inactivation of Cytomegalovirus in human platelet concentrates. The results of the inactivation using photosensitizer B are depicted in Figure 6.
  • Example 4 describes the general protocol used to demonstrate the inactivation of Vesicular Stomatitis Virus in human platelet concentrates.
  • Example 5 describes the general protocol used to demonstrate the inactivation of Herpes Simplex Virus Type I.
  • the results of the inactivation using photosensitizer B are depicted in Figure 8.
  • the photosensitizers of the present invention are to be used to inactivate blood and blood products for use in the transfusion into human patients, it is imperative that they be safe for transfusion following irradiation.
  • Example 6 describes the mutagenicity protocol used to verify the safeness of the photosensitizers of the present invention.
  • Example 6 is specific for photosensitizer B, before and after irradiation, under conditions suitable for the inactivation of viral and bacterial components in blood and blood products.
  • the results of the mutagenicity tests for photosensitizer B demonstrate that a mixture of photosensitizer B photolysis products and a maximum residual photosensitizer B
  • photosensitizer B is non-mutagenic when photolyzed in platelet
  • Example 8 describes the Chinese hamster ovary hybridoma cell and AE-L cell protocol used to determine the cytotoxicity of the photosensitizers of the present invention.
  • the results of these tests for photosensitizer B are depicted in Tables 3 and 4.
  • Compound A 3-bromo-7-( ⁇ -triethylammonium propyloxy) coumarin bromide, is one of the most preferred photosensitizers of the present invention.
  • the synthesis of Compound A is given in Example 9.
  • photosensitizers is the extent to which the photosensitizer tends to associate with nucleic acids rather than to cellular membrane components or proteins in blood or blood products.
  • Example 10 describes the protocol employed for analyzing the specificities that a variety of photosensitizers have for nucleic acids.
  • Table 6 presents a summary of the in vitro platelet properties after photoactivation in the presence of 300 ⁇ g/mL of photosensitizer B, with and without bicarbonate. Bicarbonate is added to offset the effects on the pH of the solution resulting from irradiation.
  • Table 7 presents a summary of the phoresed platelet in vitro properties following photoinactivation in the presence 300 ⁇ g/mL of photosensitizer B.
  • Table 8 summarizes the platelet in vitro properties following photoinactivation in the presence of
  • the pH does not substantially change with the use of photosensitizer A.
  • photoinactivated platelet concentrates using photosensitizer B (60 ⁇ M photosensitizer concentration and 4.5 J/cm 2 ) maintain normal properties following post-treatment storage for 5 days in a standard platelet incubator at 22 ⁇ 2°C;
  • Example 13 describes the results of a comparison study of the ability of a variety of photosensitizers of the present invention to inactivate Sindbis Virus in human plasma.
  • the compounds tested in this series of experiments were photosensitizers A, B, D and E and non-halogenated forms of A, C and D. These results of these experiments are depicted graphically in Figure 12. The results show that under the same conditions: 1) the coumarin-based photosensitizers A, C and D are superior to the psoralen- based photosensitizer B; 2) the non-halogenated coumarin-based
  • photosensitizers are not suitable for photoactivated inactivation of virus; and 3) the methylated coumarins, photosensitizers D and E, appear to be the most efficient photosensitizers for viral inactivation.
  • Example 14 describes the synthesis of photosensitizer D.
  • the procedure follows the synthetic scheme depicted in Figure 13. Following this general procedure, believed to be novel, one skilled in the art may also synthesize photosensitizer E and other photosensitizers of the present invention. (See, e.g., Sethna (1945) Chem. Rev. 36:10 ; Sethna et al. (1953) Organic Reactions 7:1 ).
  • Example 15 describes the results of a series of experiments showing the effectiveness of the present invention in inactivating the non-enveloped
  • Porcine Parvovirus in plasma Manipulation of the operating conditions -- particularly ionic strength, pH and freeze/thaw cycles -- make it possible to significantly inactivate Porcine Parvovirus with photosensitizer A and irradiation.
  • the experimental design for the viral validation studies involves the addition of photosensitizer B to platelet concentrates in standard platelet collection bags and subsequent activation of the photosensitizer by ultraviolet irradiation at 320-400 nm.
  • the following studies were:
  • Photosensitizer Toxicity Test This study establishes the degree of toxicity of the photosensitizer to the indicator cell lines used in the assay and rules out any interference, by the photosensitizer, with the ability of the chosen viruses to infect the indicator cell lines. Photosensitizer Toxicity to Viral Indicator Cells
  • Photosensitizer Dose Response This study determines the optimum concentration of photosensitizer for complete inactivation of HIV-1.
  • Kinetics of Inactivation This study establishes the optimal exposure time for effective inactivation of HIV-1.
  • UVA UVA
  • HIV-1 HIV-1
  • Light intensity (including distance of sample from the light source)
  • UVA Reactor Orbital shaker
  • Ambient means ambient laboratory light (Non-UVA light Source).
  • Step 1 Place a transparent sample platform equidistance
  • Step 2 Outline a square on the sample platform of the reactor.
  • Step 3 Switch on the top-bank of UVA light and turn on the fan for maintenance of ambient temperature during photolysis.
  • Step 4 Place a light intensity meter at both the four corners of the square and the center. Record the light intensity meter readings at these locations for the top bank of lights.
  • Step 5 Repeat step 4 for the bottom bank of lights.
  • Step 6 If the light intensity is different for the various locations, redefine the "square" such that light intensity is the same at all the different sections of the square.
  • Step 7 Preparation of Stock Solution Photosensitizer B:
  • Step 8 Platelet Concentrate Preparation for UVA Irradiation:
  • Platelet suspension F Place 50 mL of platelet concentrates into a standard platelet collection bag to be used in Phase 1A (platelet suspension G). Set aside the remaining 132 mL of platelet concentrate for Phase II.
  • Step 9 For samples 1-12, place 7.0 mL aliquots of suspension
  • Step 10 Pipette 71 ⁇ l of working solutions C and E and add to platelet concentrates from step 8 and allow said samples to incubate with photosensitizer at 24 °C for 10 minutes in ambient light. Add 71 ⁇ l of phosphate buffered saline (PBS) to the control samples and incubate as described above.
  • PBS phosphate buffered saline
  • Step 11 Place 3.0 mL aliquots of treated and untreated samples from Step 10 in covered 35 mm petri dishes and irradiate samples according to the experimental conditions as outlined in Phase IA.
  • Step 12 After irradiation, pour platelet samples into 5 mL test tubes and test control and treated samples for (1) cellular toxicity for viral assay system; and (2) viral interference for assay system.
  • METHODS-PHASE II After irradiation, pour platelet samples into 5 mL test tubes and test control and treated samples for (1) cellular toxicity for viral assay system; and (2) viral interference for assay system.
  • Step 8 Preparation of Platelet Concentrates with HIV-1 for
  • Step 9 Prepare samples for viral elimination studies.
  • Step 10 Place 3.0 mL aliquots of treated and untreated samples from Step 4 into covered 35 mm petri dishes and irradiate samples according to the experimental conditions as outlined above.
  • Step 11 After irradiation, pour platelet samples into 5 mL test tubes and determine HIV-1 infectivity in control and treated samples.
  • HIV is generally titrated in vitro by an MT-4 syncytium assay.
  • MT-4 is a cell line developed specifically to facilitate the recognition of HIV infection. These cells adhere to and abundantly express the CD4 receptor used by HIV during the infection of a cell. When infected with HIV, cells develop easily-detectable multinucleated cells or syncytium forming units.
  • Twenty-four well cluster plates are seeded with MT-4 cells in a total immunoassay for the detection of p24 antigen of HIV in plasma, serum or tissue culture media.
  • This assay uses a murine monoclonal antibody (anti- HIV core antigen) coated onto microwell strips and binds the present antibody to the antibody-coated microwells.
  • the bound antigen is recognized by biotinylated antibodies to HIV which react with conjugated streptavidin horseradish peroxidase, and develop color from the reaction of the peroxidase with hydrogen peroxide in the presence of
  • the intensity of the color developed is directly proportional to the amount of HIV antigen present in the sample.
  • the p24 assay negative control is RPMI 1640 and the positive control is antigen reagent.
  • Culture fluid from each well is analyzed by the HIV p24 assay and the absorbance value is compared to the cut off value for a positive result.
  • the cut off value for a positive result is determined by adding the mean absorbance value of the ELISA negative control to a predetermined factor of 0.055.
  • the expected range of the cut off value is 0.055 to 0.155. If the absorbance value for the well exceeds the cut off value, then the well is considered positive for HIV p24 antigen.
  • the level of HIV p24 in each well is not quantitated.
  • the TCID S0 of the sample is determined from the sum of the percentage of wells positive for HIV p24 antigen at each dilution using the standard formula, as stated above.
  • the samples are spiked with Human Immunodeficiency Virus- 1.
  • the spiked samples are then carried through the inactivation process. All samples are tested undiluted or diluted in RPMI medium (negative control) at various dilutions. Retained samples are stored frozen at -60°C or below. Titration of Samples for the Presence of HIV- 1
  • Twenty-four well cluster plates are seeded with MT-4 cells in a total volume of 1.0 ml/well.
  • Ten fold serial dilutions are made in culture medium from the spiked sample or positive control,.
  • a 0.1 ml volume of each of the samples is tested.
  • Cultures are fed twice a week by removal of 1.0 ml of medium and addition of 1.0 ml of fresh medium.
  • On days 7, 14 and 28 the cultures are evaluated for cytopathic effects to determine the TCID 50
  • 1.0 ml of each culture is removed for analysis by HIV-1 p24 antigen capture ELISA.
  • the p24 assay is the Coulter HIV p24 Ag Assay which is an enzyme Negative Control Article: RPMI 1640 Medium
  • Cytotoxicity is observed with all undiluted samples, however, the cultures appear to recover from the effects by day 7. Cytotoxicity is observed with all the samples diluted 1 : 10 on day 3, however, the cultures recover by day 7. These effects are most likely due to the excessive amount of cellular material in the samples.
  • Results for samples taken at various points during the inactivation of HIV-1 study show no evidence of replication competent HIV-1: 34 A, 42 and 44.
  • One well of four inoculated with undiluted sample 34 and sample 32 is positive for CPE on day 28.
  • Two wells of four inoculated with undiluted sample 40 are positive for CPE on day 28. The remaining samples have significant levels of replicating HIV-1.
  • Example 2 Inactivation of Sindbis Virus in Plasma Solution
  • Human plasma is spiked with Sindbis Virus to a final concentration of > 7 log 10 plaque forming units (PFU)/mL.
  • Photosensitizer is then added to the virus spiked plasma at either 100 or 300 ⁇ g/mL final concentration.
  • V R Virus reduction
  • V s is the starting virus titer
  • V f is the virus titer after treatment.
  • CMV Cytomegalovirus
  • UVA long wavelength ultraviolet radiation
  • VSV Vesicular Stomatitis
  • UVA long wavelength ultraviolet light
  • Inactivation of VSV is then evaluated by an infectivity assay (plaque assay) using Vero cells. Inactivation of 6 logs of VSV using photosensitizer B is obtained at a minimum UVA fluence of 4.20 J/cm 2 .
  • HSV-1 Herpes Simplex Virus type 1
  • UVA long wavelength ultraviolet light
  • the Ames Mutagenicity test is based upon the use of five specially constructed strains of Salmonella typhimurium containing a specific mutation in the histidine operon. These genetically altered strains, TA98, TA100, TA1535, TA1537 and TA1538, cannot grow in the absence of histidine. When they are placed in a histidine-free medium, only those cells which mutate spontaneously back to their wild type state -- non-histidine- dependent by manufacturing their own histidine - are able to form colonies.
  • the spontaneous mutation rate, or reversion rate, for any one strain is relatively constant, but if a mutagen is added to the test system, the mutation rate is significantly increased.
  • Each test strain contains, in addition to a mutation in the histidine operon, two additional mutations that enhance sensitivity to some mutagens.
  • the rfa mutation results in a cell wall deficiency that increases the permeability of the cell to certain classes of chemicals, for example, those chemicals containing large ring systems that are otherwise excluded.
  • the second mutation is a deletion in the uvrB gene resulting in a deficient DNA excision-repair system.
  • Test strains TA98 and TA100 also contain the pKM101 plasmid that carries the R-factor. It has been suggested that the plasmid increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.
  • TA98, TA1537 and TA1538 revert from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutations.
  • TA100 reverts by both frameshift and base substitution mutations and TA1535 reverts only by substitution mutations.
  • the experiment is designed such that the concentrations of photosensitizer B on the agar plate is equivalent to the expected final dose in a recipient given 5 units of platelet concentrates. Note that 5 units of platelet
  • a Salmonella/mammalian microsome mutagenicity test is conducted to determine whether a plasma test solution of photosensitizer B in platelet concentrate causes mutagenic changes in histidine-dependent mutant strains of Salmonella typhimurium.
  • the Ames mutagenicity test system has been widely used as a rapid screening procedure for the determination of mutagenic and potential carcinogenic hazards of pure compounds, complex compounds and commercial products.
  • Mouse fibroblasts (L-929) are grown to confluency in 25 cm 2 culture flasks using sterile minimum essential medium (MEM)
  • 929 cells are exposed to extract dilutions of photosensitizer B.
  • a standard solution of photosensitizer B is prepared by dissolving 12 mg in 20 mL of MEM supplemented with 5% bovine serum and then incubated at 37 °C for 24 hours. Following incubation, different dilutions (1:2 to 1:16) of standard stock of photosensitizer B are prepared with fresh MEM. A 5 mL aliquot of the different dilutions of photosensitizer B is added to confluent monolayers of L-929 cells and then incubated at 37°C for 72 hours. A 5 mL MEM aliquot is added as a negative control.
  • CTE cytotoxic effects
  • Nontoxic (N) A uniform confluent monolayer containing
  • intracytoplasmic granules present at 24 hours. At 48 and 72 hours, there should be an increasing number of rounded cells as cell population
  • T Toxic
  • Chinese hamster ovary and AE-L cells are grown to confluency in 25 cm 2 culture flasks using sterile Eagles Minimum Essential Medium
  • EMEM EMEM supplemented with 2 mM L-glutamine, 1% proline and 5% calf serum treated with various concentrations of photosensitizer B (30-150 ⁇ g/mL) in the presence of UVA.
  • photosensitizer B 30-150 ⁇ g/mL
  • Nontoxic concentrations of penicillin, streptomycin and amphoteric B are also added to the culture medium to prevent bacterial growth.
  • Control samples contain non-treated calf serum. All samples are incubated at 37°C for 2 to 7 days. The number of viable cells are measured at the end of each incubation period. Results show that the growth and viability of the two cell types are not affected by
  • the solution is concentrated by rotary evaporator. 50 mL of a 1 : 1 mixture of ethyl acetate and hexane is added to the concentrate and the mixture is stirred for 30 minutes at room temperature. The solution is then filtered and the solids washed 3 times with 1 : 1 ethyl acetate and hexane and then dried for 2 hours. To obtain additional product, the filtrate is concentrated by rotary evaporator and 30 mL of a 1 :1 mixture of ethyl acetate and hexane is added to the concentrate.
  • photosensitizer A fluoresces when treated with UV radiation.
  • the absorption spectrum of photosensitizer A in water is shown in Figure 10.
  • the fluorescence spectrum of photosensitizer A is shown in Figure 11.
  • the unit consists of three chambers capable of holding a volume of 10 mL of solution. Each chamber is separated from the adjoining chamber by a dialysis membrane (MW cut off, 5000, Fisher).
  • the center chamber is loaded with 100 ⁇ M photosensitizer solution either in phosphate buffered saline (PBS) or plasma.
  • PBS phosphate buffered saline
  • the other two adjoining chambers are loaded with solutions containing the agents for which the binding is to be tested.
  • Liposomes are prepared by vortexing dioleyl phosphatidylserine (4.0 mg/mL, Avanti polar lipids) solution in PBS.
  • Platelet units are aseptically pooled and subsequently split into controls and treatments. Ten milliliters of photosensitizer solution in 0.9% saline is added to 50 mL platelet concentrates in CLX (Miles) containers to obtain the photosensitizer final preset concentration. After addition of the photosensitizer, the platelet units are incubated at room temperature while mixing on a shaker for 10 minutes. Platelet concentrate samples containing photosensitizers are UVA irradiated from top and bottom in a prototype
  • UVA reactor to deliver 25 J/cm 2 fluence.
  • samples are placed on a linear shaker.
  • UVA exposure the samples are stored in a platelet incubator with shaking for an additional 4 days.
  • 3 mL aliquots from each unit are collected and subsequently analyzed for platelet in vitro properties.
  • Platelet units are spiked with 6 logs of bacteriophage ⁇ 6. Equimolar concentrations (60 ⁇ M) of 8-MOP, AMT and brominated psoralen are added to the platelet concentrations and then incubated at 22 ⁇ 2 °C for 10 minutes. Treated samples are irradiated from top and bottom with a constant total UVA source intensity of 7 mW/cm 2 . During UVA exposure samples are continuously agitated to ensure adequate mixing. Virucidal properties are evaluated using a standard double agar plaque assay consisting of host bacteria Pseudomonas Syringae.
  • photosensitizer AX non-halogenated forms of photosensitizer A
  • photosensitizer CX photosensitizer CX
  • photosensitizer DX photosensitizer DX
  • the TC1D 50 assay is used to measure the affects of virus inactivation.
  • the photosensitizer is added to virus spiked plasma.
  • the virus employed is Sindbis and the plasma is spiked to a working titer of > 1 x 10 7 .
  • Each test unit is exposed to ultraviolet radiation at 320-400 nm (peak absorbance 365 nm) for 30 min. to achieve an irradiation of about 24 J/cm 2 .
  • Virus inactivation is quantitated by plaque assay.
  • a monolayer of indicator cells are grown on a solid support and exposed to sample materials to allow for virus attachment.
  • a foci of infection develops as a virus replicates and lyses and released virus diffuses to and infects neighboring cells, or virus infects neighboring cells via cell-cell fusion.
  • CPE develops after several days of incubation.
  • the virus titer in the sample is calculated from number of units exhibiting CPE. The results of this experiment are shown in Figure 12.
  • Human parvovirus B 19 is a major concern with respect to the safety of plasma derived products. This class of virus exhibits high resistance to chemical reagents such as alcohol, detergents and solvents that are currently employed for inactivating viruses in plasma.
  • Sample Set 1 Normal Plasma Samples at pH 7.0 - 7.5
  • Sample Set 2 Normal Plasma: Freeze - at -30°C and Thaw at
  • Sample Set 3 Normal Plasma Diluted 1:1 with Saline at pH 5.5-
  • UVA Light source
  • UVA transparent petri dishes are labeled along the sides with the sample numbers 1-13 (see section 3.2 and attachment 1 for the sample identification).
  • Two 50 mL sterile centrifuge tubes are labeled (Tube 1 - pH
  • the thawed plasma is centrifuged at 3000 g for 20 minutes. Using a
  • the pH of plasma in Tube 2 was adjusted to a value between 5.5 and 6.
  • samples 4, 6-13 are frozen at temperatures between -20°C and -40°C.
  • Sample 4 is thawed at 37°C (about 10-15 minutes at 37°C) and samples 6- 13 at temperatures between 2 and 8°C. Freeze-thaw cycles are repeated 10 times over 24 hour period. At completion of the freeze-thaw cycles, 6 mL of each sample were exposed to UVA at 30 J/cm 2 . At the end of the 60 minute incubation, 6 mL of the contents from tubes 1, 2, 3 and 5 are transferred to appropriately labeled petri dishes for photoinactivation treatment. The results of this experiment are shown in Table 17.

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Abstract

Ont peut inactiver de manière photodynamique les contaminants de nature virale, bactérienne ou parasitaire présents dans des compositions biologiques en mélangeant auxdites compositions des photosensibilisants halogénés de coumarine et de furocoumarine, puis en irradiant le mélange. La figure 1 représente le schéma énergétique proposé des photosensibilisants à action instantanée.
PCT/US1995/012069 1994-09-22 1995-09-21 Procede d'inactivation photodynamique de contaminants du sang de nature virale et bacterienne a l'aide de sensibilisants a la coumarine ou la furocoumarine WO1996008965A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP8511090A JPH10506391A (ja) 1994-09-22 1995-09-21 ハロゲン化クマリンおよびフロクマリン増感剤を用いるウイルス性および細菌性血液混入物の光力学的不活化
EP95933899A EP0782388A4 (fr) 1994-09-22 1995-09-21 Procede d'inactivation photodynamique de contaminants du sang de nature virale et bacterienne a l'aide de sensibilisants a la coumarine ou la furocoumarine
AU36385/95A AU691672B2 (en) 1994-09-22 1995-09-21 Photodynamic inactivation of viral and bacterial blood contaminants with halogenated coumarin and furocoumarin sensitizers
NO971350A NO971350L (no) 1994-09-22 1997-03-21 Fotodynamisk inaktivering av virus- og bakterielle blod-kontaminanter med halogenerte kumarin- og furokumarin-sensibilisatorer

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US08/311,125 1994-09-22
US08/311,125 US5516629A (en) 1990-04-16 1994-09-22 Photoinactivation of viral and bacterial blood contaminants using halogenated coumarins
US08/343,680 1994-11-22
US08/343,680 US6251644B1 (en) 1990-04-16 1994-11-22 Method for inactivating non-enveloped viral contaminants with a photosensitizer by increasing viral permeability to the photosensitizer
US08/427,080 US5789601A (en) 1990-04-16 1995-04-21 Method of inactivation of viral and bacterial blood contaminants
US08/427,080 1995-04-21
US08/461,626 1995-06-05
US08/461,626 US5869701A (en) 1990-04-16 1995-06-05 Method of inactivation of viral and bacterial blood contaminants

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EP0796094A1 (fr) * 1994-02-04 1997-09-24 New York Blood Center, Inc. Utilisation de la vitamine e et de ses derives pour empecher les lesions de globules rouges sterilises au moyen de phthalocyamines et de lumiere
WO2000030682A1 (fr) * 1998-11-19 2000-06-02 Roecken Martin Preparation de khelline et son utilisation pour des traitements a action locale
WO2000034446A1 (fr) * 1998-12-04 2000-06-15 Csl Limited Inactivation de virus non enveloppes
WO2002002152A1 (fr) * 2000-07-04 2002-01-10 Blutspendedienst der Landesverbände des Deutschen Roten Kreuzes Niedersachsen, Sachsen-Anhalt, Thüringen, Oldenburg und Bremen gGmbH Traitement photodynamique et exposition aux rayons u.v.-b d'une suspension de thrombocytes
US8580192B2 (en) 2006-10-31 2013-11-12 Ethicon, Inc. Sterilization of polymeric materials
WO2016014854A1 (fr) 2014-07-23 2016-01-28 Cerus Corporation Procédés de préparation de produits contenant des plaquettes
WO2016057965A1 (fr) 2014-10-10 2016-04-14 Cerus Corporation Compositions et méthodes de traitement de la fièvre hémorragique virale
WO2016210374A1 (fr) 2015-06-26 2016-12-29 Cerus Corporation Compositions de cryoprécipités et leurs procédés de préparation
WO2017070619A1 (fr) 2015-10-23 2017-04-27 Cerus Corporation Compositions de plasma et leurs procédés d'utilisation
WO2017120545A2 (fr) 2016-01-07 2017-07-13 Cerus Corporation Systèmes et méthodes pour la préparation de plaquettes
WO2018161020A1 (fr) 2017-03-03 2018-09-07 Cerus Corporation Kits et méthodes de préparation de compositions de plaquettes inactivées par des agents pathogènes
WO2019060610A1 (fr) 2017-09-20 2019-03-28 Cerus Corporation Compositions et méthodes d'inactivation de pathogènes de plaquettes
WO2019133929A1 (fr) 2017-12-29 2019-07-04 Cerus Corporation Systèmes et procédés pour traiter de fluides biologiques
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CA2221733A1 (fr) * 1995-06-07 1996-12-19 Baxter International Inc. Procede d'inactivation de contaminants viraux et bacteriens dans le sang

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IL104530A0 (en) * 1992-01-27 1993-05-13 Cryopharm Corp Method of inactivation of viral and bacterial blood contaminants
CA2221733A1 (fr) * 1995-06-07 1996-12-19 Baxter International Inc. Procede d'inactivation de contaminants viraux et bacteriens dans le sang

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EP0796094A4 (fr) * 1994-02-04 2000-03-08 New York Blood Center Inc Utilisation de la vitamine e et de ses derives pour empecher les lesions de globules rouges sterilises au moyen de phthalocyamines et de lumiere
EP0796094A1 (fr) * 1994-02-04 1997-09-24 New York Blood Center, Inc. Utilisation de la vitamine e et de ses derives pour empecher les lesions de globules rouges sterilises au moyen de phthalocyamines et de lumiere
WO2000030682A1 (fr) * 1998-11-19 2000-06-02 Roecken Martin Preparation de khelline et son utilisation pour des traitements a action locale
WO2000034446A1 (fr) * 1998-12-04 2000-06-15 Csl Limited Inactivation de virus non enveloppes
WO2002002152A1 (fr) * 2000-07-04 2002-01-10 Blutspendedienst der Landesverbände des Deutschen Roten Kreuzes Niedersachsen, Sachsen-Anhalt, Thüringen, Oldenburg und Bremen gGmbH Traitement photodynamique et exposition aux rayons u.v.-b d'une suspension de thrombocytes
US8580192B2 (en) 2006-10-31 2013-11-12 Ethicon, Inc. Sterilization of polymeric materials
US8585965B2 (en) 2006-10-31 2013-11-19 Ethicon, Inc. Sterilization of polymeric materials
US10842818B2 (en) 2014-07-23 2020-11-24 Cerus Corporation Methods for preparing platelet products
WO2016014854A1 (fr) 2014-07-23 2016-01-28 Cerus Corporation Procédés de préparation de produits contenant des plaquettes
WO2016057965A1 (fr) 2014-10-10 2016-04-14 Cerus Corporation Compositions et méthodes de traitement de la fièvre hémorragique virale
WO2016210374A1 (fr) 2015-06-26 2016-12-29 Cerus Corporation Compositions de cryoprécipités et leurs procédés de préparation
US11096963B2 (en) 2015-06-26 2021-08-24 Cerus Corporation Cryoprecipitate compositions and methods of preparation thereof
WO2017070619A1 (fr) 2015-10-23 2017-04-27 Cerus Corporation Compositions de plasma et leurs procédés d'utilisation
US10799533B2 (en) 2015-10-23 2020-10-13 Cerus Corporation Plasma compositions and methods of use thereof
WO2017120545A2 (fr) 2016-01-07 2017-07-13 Cerus Corporation Systèmes et méthodes pour la préparation de plaquettes
WO2018161020A1 (fr) 2017-03-03 2018-09-07 Cerus Corporation Kits et méthodes de préparation de compositions de plaquettes inactivées par des agents pathogènes
US11235090B2 (en) 2017-03-03 2022-02-01 Cerus Corporation Kits and methods for preparing pathogen-inactivated platelet compositions
WO2019060610A1 (fr) 2017-09-20 2019-03-28 Cerus Corporation Compositions et méthodes d'inactivation de pathogènes de plaquettes
WO2019133929A1 (fr) 2017-12-29 2019-07-04 Cerus Corporation Systèmes et procédés pour traiter de fluides biologiques
US11554185B2 (en) 2017-12-29 2023-01-17 Cerus Corporation Systems and methods for treating biological fluids
US11883544B2 (en) 2019-06-28 2024-01-30 Cerus Corporation System and methods for implementing a biological fluid treatment device

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NO971350L (no) 1997-05-22
AU3638595A (en) 1996-04-09
CA2199372A1 (fr) 1996-03-28
EP0782388A4 (fr) 2000-03-08
EP0782388A1 (fr) 1997-07-09
AU691672B2 (en) 1998-05-21
NO971350D0 (no) 1997-03-21

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