EP0782388A1 - 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

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Publication number
EP0782388A1
EP0782388A1 EP95933899A EP95933899A EP0782388A1 EP 0782388 A1 EP0782388 A1 EP 0782388A1 EP 95933899 A EP95933899 A EP 95933899A EP 95933899 A EP95933899 A EP 95933899A EP 0782388 A1 EP0782388 A1 EP 0782388A1
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EP
European Patent Office
Prior art keywords
photosensitizer
viral
biological solution
blood
virus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95933899A
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German (de)
English (en)
Other versions
EP0782388A4 (fr
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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Baxter International Inc
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Publication date
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
Publication of EP0782388A1 publication Critical patent/EP0782388A1/fr
Publication of EP0782388A4 publication Critical patent/EP0782388A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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.
  • a major concern in the transfusion of donated, stored whole human blood or the various blood cell or protein fractions isolated therefrom is the possibility of viral or bacterial contamination.
  • the blood-borne viruses that cause Hepatitis (especially Hepatitis A, Hepatitis B, and Non-A, Non-B Hepatitis (Hepatitis C)) and Acquired Immune Deficiency Syndrome (AIDS).
  • Hepatitis especially Hepatitis A, Hepatitis B, and Non-A, Non-B Hepatitis (Hepatitis C)
  • AIDS Acquired Immune Deficiency Syndrome
  • While any number of cell washing protocols may reduce the viral contamination load for samples of blood cells by physical elution of the much smaller virus particles, such washing alone is insufficient to reduce viral contamination to safe levels.
  • some viruses are believed to be cell-associated, and are unlikely to be removed by extensive washing and centrifugal pelleting of the cells.
  • 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: capability of synthesizing ATP; cell mo ⁇ hology; P 50 values; filterability or deformability; oxyhemoglobin, methemoglobin and hemochrome values; MCV, MCH, and MCHC values; cell enzyme activity; and in vivo survival.
  • 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.
  • 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 abso ⁇ tion 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 abso ⁇ tion 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.
  • psoralens for example. S-methoxypsoralen (8-MOP), 4'-aminomethyl- 4,5',8-trimethylpsoralen (AMT) and 4 , -hydroxymethy-4,5',8- trimethylpsoralen (HMT)
  • the photosensitizers that have been employed are typically dyes. Examples include dihematopo ⁇ hyrin ether (DHE), Merocyanine 540 (MC540) and methylene blue.
  • DHE dihematopo ⁇ hyrin 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.
  • 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, Goodrich et ⁇ /.(1994) Proc. Natl. Acad. Sci. USA 91:5552.
  • 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 photoinactivation of blood and blood products contaminated with Human Immunodeficiency Virus- 1 (HIV-1), Sindbis Virus. Cytomegalovirus. Vesicular Stomatitis Virus (VSV), and He ⁇ es Simplex Virus Type 1 (HSV- 1), using the photosensitizers of the present invention.
  • HIV-1 Human Immunodeficiency Virus- 1
  • Sindbis Virus Cytomegalovirus.
  • VSV Vesicular Stomatitis Virus
  • HSV- 1 He ⁇ es 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, po ⁇ hyrins, phthalocyanines, quinones, hypericin, and organic dye molecules such as coumarins, for example, merocyanine dyes, methylene blue and eosin dyes.
  • modified psoralen derivatives of the present invention may prove more efficacious in therapeutic photophoresis applications.
  • 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, po ⁇ hyrins, 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 inco ⁇ orating 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 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 with photosensitizer 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 He ⁇ es Simplex Virus Type 1 (HSV-1) in the presence of photosensitizer B and UVA. Viral inactivation is plotted versus UVA fluence.
  • HSV-1 He ⁇ es Simplex Virus Type 1
  • FIGURE 9 depicts the synthetic scheme for the synthesis of photosensitizer A.
  • FIGURE 10 depicts the abso ⁇ tion 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 DESCRffTTON OF THE TNVENTTON
  • 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 pu ⁇ oses 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.
  • Psoralens and coumarins 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 -0(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 components.
  • 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.
  • a quaternary 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,, R 2 , R 3 , R 4 , R 5 and R 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 ) ⁇ Z"R',R",R'” wherein n, m and p are independently integers from 1 to 10 and R', R", and R'" are 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,, R 2 , R 3 , R 4 , R 5 or Rg is (CH 2 ) m O(CH
  • ⁇ is an integer from 1 to 6;
  • X is an anionic counterion;
  • Z is N or P;
  • Rstrich R 2 , R 3 , R 4 , R 5 , and R 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 ⁇ p Z'R'.R" ⁇ '" or -0(CH 2 ) n Z # R',R",R"' wherein n, m andp are independently integers from 1 to 10 and R', R", and R'" are 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, R 2 , R 3 , R 4 , R 5 or R is (CH 2 ) m O(CH : ) p Z , R',R%R'" or -0(CH 2 ) n Z*R',R",R"'.
  • R 3 , R 5 , R : and R 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 photosensitizer compound.
  • 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.
  • abso ⁇ tion 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.
  • 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 counte ⁇ arts.
  • 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.
  • Some of the conditions that have been manipulated in order to loosen the capsid of non-enveloped viruses include: ionic strength of the solution, pH, solvent detergent treatments, chaotrophic agents such as urea, reducing agents, chelating agents, freeze-thaw cycles, and temperature.
  • ionic strength of the solution pH
  • solvent detergent treatments chaotrophic agents such as urea, reducing agents, chelating agents, freeze-thaw cycles, and temperature.
  • Example 15 adjustment of the pH, ionic strength and freeze-thaw cycles on a plasma solution containing Porcine Parvovirus yielded dramatic improvements upon irradiation in the presence of one of the photosensitizers of the present invention. It is, therefore, an embodiment of this invention to reduce non- enveloped viral contamination in a biological solution.
  • 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 inco ⁇ orating 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 inco ⁇ oration 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 or EGTA, sodium pyrophosphate or any polyanion in different combinations.
  • This embodiment of the invention includes the use of methods for the inactivation of non-enveloped viruses using the osmotic shock process for selective inco ⁇ oration 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 He ⁇ es Simplex Virus Type I.
  • the results of the inactivation using photosensitizer B are depicted in Figure 8. Because 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 concentration of 4.36 ⁇ g/mL per test plate do not cause any mutagenic effects in Salmonella strains TA98, TA100, TA1535, TA1537 and TA1538.
  • the maximum residual concentrations of photosensitizer B under use conditions correspond to about 3.4 times the expected concentration of photosensitizer B per therapeutic dose of platelet concentrates of 1.28 ⁇ g/mL.
  • photosensitizer B is non-mutagenic when photolyzed in platelet concentrates such that the initial concentration is reduced by at least 60% under use conditions (>25 J/cm 2 UVA and 12.8 ⁇ g/mL photosensitizer B plate).
  • the mutagenicity results for photosensitizer A show that for both irradiated and non-irradiated solutions there is no significant increase in reversion rate with any of the five test strains in either the absence or presence of S-9 activation.
  • Example 7 describes the mouse fibroblast protocol used to determine the cytotoxicity of the photosensitizers of the present invention. The results of these tests for photosensitizer B at 72 hr are depicted in Table 2.
  • 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.
  • Example 10 describes the protocol employed for analyzing the specificities that a variety of photosensitizers have for nucleic acids.
  • the photosensitizers of the present invention have been examined with regard to their effects on the constituents of platelet concentrates under conditions that are sufficient for obtaining complete contaminant inactivation.
  • the general procedure for conducting these experiments is disclosed in Example 11 below.
  • 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 photosensitizer A. 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;
  • virucidal efficacy of brominated psoralen is substantially higher than that of 8-MOP or AMT with respect to inactivation of non-enveloped bacteriophages such as lambda and R- 17.
  • 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.
  • 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 following examples serve to explain and illustrate the present invention. Said examples are not to be construed as limiting of the invention in anyway. Various modification are possible within the scope of the invention as described and claimed herein.
  • Example 1 Inactivation of HIV-1 Virus in Platelet Concentrate
  • 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 performed in order to verify the elimination of HIV- 1 from platelet concentrates.
  • 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 Sample Set 1
  • PHASE II 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
  • Light intensity (including distance of sample from the light source)
  • Step 1 Place a transparent sample platform equidistance between the top and bottom UVA lamps.
  • 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: Prepare solution A by dissolving photosensitizer B in
  • Solution C 10 mg/mL Solution D: 20 mg/mL Solution E: 30 mg/mL
  • Step 8 Platelet Concentrate Preparation for UVA Irradiation: Pool four units of ABO compatible platelet concentrates together in a standard platelet collection bag to obtain a final volume of about 182 mL of platelet rich plasma (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.
  • Step 8 Preparation of Platelet Concentrates with HIV-1 for UVA Irradiation
  • 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 51 immunoassay for the detection of p24 antigen of HTV in plasma, serum or tissue culture media.
  • This assay uses a murine monoclonal antibody (anti- HTV 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 HTV which react with conjugated streptavidin horseradish peroxidase, and develop color from the reaction of the peroxidase with hydrogen peroxide in the presence of tetramethylbenzidine substrate.
  • the intensity of the color developed is directly proportional to the amount of HTV 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 TdD 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.
  • 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 values are then converted to TCID 50 /ml using a sample inoculum volume of 0.1 ml.
  • the p24 assay is the Coulter HIV p24 Ag Assay which is an enzyme Negative Control Article: RPMI 1640 Medium
  • Test System MT-4 cells (L013-T) Source: National Institute of Health,
  • 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
  • Cytomegalovirus (CMV) in human platelet concentrate is conducted under ambient oxygen tension using a photosensitizer and long wavelength ultraviolet radiation (UVA) at 22 ⁇
  • 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 He ⁇ es Simplex Virus type 1
  • UVA long wavelength ultraviolet light
  • kinetics studies are conducted in order to determine the optimal conditions for inactivation of HSV-1 in calf serum.
  • Three logs of HSV- 1 virus are added to 100 mL of calf serum.
  • the contaminated sera are incubated at ambient non-UVA laboratory light for 10 ⁇ 5 minutes. Following incubation, the sera are exposed to UVA at various fluences (4.20-8.40 J/cm 2 ).
  • Inactivation of HSV virus is evaluated by an infectivity assay.
  • Inactivation of 3 logs of HSV- 1 using photosensitizer B is obtained at a UVA fluence of 12.6 J/cm 2 .
  • 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 pKMlOl 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 concentrates is equivalent to a standard single therapeutic dose (1TD). Calculation of the theoretical concentration of photosensitizer B is based on the following deductions assuming homogenous distribution of the drug in a 70 kg normal individual:
  • Example 7 Measurement of Photosensitizer Cytotoxicity Using Mouse Fibroblasts Historically, in vitro mammalian cell culture studies have been used to evaluate the cytotoxicity of biomaterials and complex chemical compounds.
  • Mouse fibroblasts (L-929) are grown to confluency in 25 cm 2 culture flasks using sterile minimum essential medium (MEM) supplemented with 5% fetal calf serum and nontoxic concentrations of penicillin, streptomycin and amphotericin B. Confluent monolayers of L-
  • 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
  • N A uniform confluent monolayer containing primarily elongated cells with discrete intracytoplasmic granules present at 24 hours. At 48 and 72 hours, there should be an increasing number of rounded cells as cell population increases and crowding begins. Little or no vacuolization, crenation or swelling should be present.
  • Toxic (T) Greater than 50% of all cells have been lysed. Extensive vacuolization, swelling, or crenation are usually present in the cells remaining on the flask surface.
  • Chinese Hamster Ovary (CHO ⁇ Hvbridoma Cells and AE- L Cells 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 pretreatment of the sera with irradiated and non-irradiated photosensitizer B.
  • the solution is then filtered and the solids washed 3 times with 1 : 1 ethyl acetate and hexane and then dried for 2 hours.
  • 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.
  • the resulting mixture is then filtered, the solids washed three times with 1 : 1 ethyl acetate and hexane and then dried for 2-5 hours.
  • the product is checked by TLC (ethyl acetate:hexane (4:6)).
  • the crude product (13-16 g) is dissolved in 100-170 mL of dichloromethane and purified by flash chromatography (100-150 g SiO 2 (70 - 230 mesh), 35 mm O.D. column, approximately 60 cm in length), using dichloromethane as the eluting solvent. Fractions are collected in a 50-250 mL beakers and monitored by TLC (developing solvent: ethyl acetate:hexane (4:6)). The fractions containing product are combined, concentrated by rotary evaporator and dried.
  • photosensitizer A fluoresces when treated with UV radiation.
  • the abso ⁇ tion spectrum of photosensitizer A in water is shown in Figure 10.
  • the fluorescence spectrum of photosensitizer A is shown in Figure 11.
  • Dialysis experiments are carried out using a custom-made polystyrene dialysis chamber.
  • 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.
  • Polyadynelic acid (Poly A; Sigma), Calf thymus DNA (DNA; Sigma) and bovine serum albumin (BSA) solutions are prepared in PBS (4.0 mg/mL).
  • the dialysis cells containing solutions are allowed to equilibrate with constant agitation for a period of 24 hours at room temperature.
  • the solutions are removed from individual chambers and absorbance is determined at 350 nm using a spectrophotometer.
  • 5% Titron X-100 (Sigma) is used to clarify the solutions prior to absorbance reading.
  • Quantitative determination of photosensitizer in plasma and platelets is carried out by high performance liquid chromatography (HPLC) equipped with a C 18 reverse phase column.
  • 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 t ⁇ . 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 D and E 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.
  • Virus PPV 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
  • Random donor platelet concentrates were pooled and subsequently divided into control and treated units.
  • Photoinactivation was in CI.X containers containing 50 mL plalolet concentrate antl to mL sensitizer solution in 0 9% saline (D, I 8 mg/ml. ) 2 mL ot 57o soduim bicaibnoale solution (20 mM final concentration in platelet concentrates) was added following photoinactivation treatment
  • Pheresed platelet concentiales were divided into control and treated units.
  • Photoinactivation was carried out in PL 732 containers containing 50 mL platelet concentrates and 10 mL of sensitizer solution in 0.9% saline (B 1.8 mg/mL).

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
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  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
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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.
EP95933899A 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 Withdrawn EP0782388A4 (fr)

Applications Claiming Priority (9)

Application Number Priority Date Filing Date Title
US343680 1982-01-28
US311125 1989-02-15
US08/311,125 US5516629A (en) 1990-04-16 1994-09-22 Photoinactivation of viral and bacterial blood contaminants using halogenated coumarins
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
US427080 1995-04-21
US08/427,080 US5789601A (en) 1990-04-16 1995-04-21 Method of inactivation of viral and bacterial blood contaminants
US461626 1995-06-05
US08/461,626 US5869701A (en) 1990-04-16 1995-06-05 Method of inactivation of viral and bacterial blood contaminants
PCT/US1995/012069 WO1996008965A1 (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

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EP0782388A1 true EP0782388A1 (fr) 1997-07-09
EP0782388A4 EP0782388A4 (fr) 2000-03-08

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EP (1) EP0782388A4 (fr)
AU (1) AU691672B2 (fr)
CA (1) CA2199372A1 (fr)
NO (1) NO971350L (fr)
WO (1) WO1996008965A1 (fr)

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DE19853425A1 (de) * 1998-11-19 2000-05-25 Martin Roecken Khellin-Zubereitung und deren Verwendung zur topischen Therapie der Psoriasis, ihrer Subtypen, Sonderformen, sowie zur topischen Therapie von Ekzemen
AUPP751298A0 (en) * 1998-12-04 1999-01-07 Csl Limited Inactivation of non-enveloped viruses
DE10031851B4 (de) * 2000-07-04 2005-10-13 Blutspendedienst der Landesverbände des Deutschen Roten Kreuzes Niedersachsen, Sachsen-Anhalt, Thüringen, Oldenburg und Bremen gGmbH Photodynamische Behandlung und UV-B-Bestrahlung einer Thrombozyten-Suspension
US8580192B2 (en) 2006-10-31 2013-11-12 Ethicon, Inc. Sterilization of polymeric materials
EA201790265A1 (ru) 2014-07-23 2017-07-31 Сирус Корпорейшн Способы получения тромбоцит-содержащих продуктов
EP3204426B1 (fr) 2014-10-10 2020-11-25 Cerus Corporation Compositions pour l'utilisation dans le traitement de la fièvre hémorragique virale
CA2990864A1 (fr) 2015-06-26 2016-12-29 Cerus Corporation Compositions de cryoprecipites et leurs procedes de preparation
WO2017070619A1 (fr) 2015-10-23 2017-04-27 Cerus Corporation Compositions de plasma et leurs procédés d'utilisation
IL260393B2 (en) 2016-01-07 2024-06-01 Cerus Corp Systems and methods for preparing platelets
EP3589297A1 (fr) 2017-03-03 2020-01-08 Cerus Corporation Kits et méthodes de préparation de compositions de plaquettes inactivées par des agents pathogènes
AU2018338097B2 (en) 2017-09-20 2023-07-13 Cerus Corporation Compositions and methods for pathogen inactivation of platelets
CN111770789B (zh) 2017-12-29 2023-05-05 塞鲁斯公司 用于处理生物流体的系统和方法
MX2021015653A (es) 2019-06-22 2022-04-11 Cerus Corp Sistemas de tratamiento de fluidos biologicos.
EP3991179A1 (fr) 2019-06-28 2022-05-04 Cerus Corporation Système et procédés permettant de mettre en oeuvre un dispositif de traitement de fluide biologique

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EP0830057A1 (fr) * 1995-06-07 1998-03-25 Baxter International Inc. Procede d'inactivation de contaminants viraux et bacteriens dans le sang

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EP0782388A4 (fr) 2000-03-08
AU3638595A (en) 1996-04-09
AU691672B2 (en) 1998-05-21
WO1996008965A1 (fr) 1996-03-28
CA2199372A1 (fr) 1996-03-28
NO971350D0 (no) 1997-03-21
NO971350L (no) 1997-05-22

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