EP0830057A1 - Procede d'inactivation de contaminants viraux et bacteriens dans le sang - Google Patents

Procede d'inactivation de contaminants viraux et bacteriens dans le sang

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Publication number
EP0830057A1
EP0830057A1 EP96916996A EP96916996A EP0830057A1 EP 0830057 A1 EP0830057 A1 EP 0830057A1 EP 96916996 A EP96916996 A EP 96916996A EP 96916996 A EP96916996 A EP 96916996A EP 0830057 A1 EP0830057 A1 EP 0830057A1
Authority
EP
European Patent Office
Prior art keywords
photosensitizer
blood
viral
cysteine
biological
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Withdrawn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
EP96916996A
Other languages
German (de)
English (en)
Other versions
EP0830057A4 (fr
Inventor
Samuel O. Sowemimo-Coker
Nagender Yerram
Raymond P. Goodrich, Jr.
Matthew Platz
Sang Chul Park
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Baxter International Inc
Original Assignee
Baxter International Inc
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Filing date
Publication date
Priority claimed from US08/474,459 external-priority patent/US5798238A/en
Priority claimed from US08/480,271 external-priority patent/US5955256A/en
Application filed by Baxter International Inc filed Critical Baxter International Inc
Publication of EP0830057A1 publication Critical patent/EP0830057A1/fr
Publication of EP0830057A4 publication Critical patent/EP0830057A4/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Withdrawn legal-status Critical Current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
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
    • A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
    • A61L2/0082—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using chemical substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/02—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using physical phenomena
    • A61L2/08—Radiation

Definitions

  • This invention relates to the general field of 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 persons, ex vivo media used in the preparation of anti-viral vaccines, and cell culture media such as fetal bovine serum, bovine serum or products derived 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 products derived from these sources.
  • the blood-borne viruses that cause hepatitis especially hepatitis A, hepatitis B, and hepatitis C
  • acquired immune deficiency syndrome AIDS
  • 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 unlikely to be removed by extensive washing and centrifugal pelleting of the cells.
  • Viral inactivation by stringent sterilization is not acceptable since this could also destroy the functional components of the blood, particularly the erythrocytes (red blood cells) and thrombocytes (platelets) and the labile plasma proteins, such as clotting factor VIII.
  • Viable RBC's can be characterized by one or more of the following: capability of synthesizing ATP; cell morphology; P 50 values; filterability or deformability; oxyhemoglobin, methemoglobin and hemochrome values; MCV, MCH, and MCHC values; cell enzyme activity; and in vivo survival.
  • capability of synthesizing ATP can be characterized by one or more of the following: capability of synthesizing ATP; cell morphology; P 50 values; filterability or deformability; oxyhemoglobin, methemoglobin and hemochrome values; MCV, MCH, and MCHC values; cell enzyme activity; and in vivo survival.
  • Viral inactivation by stringent steam sterilization is not acceptable since this also destroys the functional components of the blood, particularly the 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 mfectivity.
  • Use of stabilizing agents such as 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 extraction with detergents to disrupt the lipid coat of membrane enveloped viruses. Lyophilization (freeze-drying) alone has not proven sufficient to inactivate viruses, or to 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 has involved the use of a 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 used to achieve some demonstrable viral inactivation and also due to unacceptable levels of damage to the proteins caused by the chemical agents. Concern has also been raised over the explosive potential for beta-propiolactone as well.
  • UV ultraviolet
  • Non B hepatitis (hepatitis C), and ranges from 1:60,000 to 1:225,000 for HIV, depending on geographic location.
  • hepatitis C hepatitis C
  • Contamination problems also exist for blood plasma protein fractions, such as plasma fractions containing immune globulins and clotting factors.
  • plasma protein fractions such as plasma fractions containing immune globulins and clotting factors.
  • new cases of non A, non B hepatitis and hepatitis A have occurred in hemophilia patients receiving protein fractions containing Factor VIII which have been treated for viral inactivation according to approved methods. Therefore, there is a need for improved viral inactivation treatment of blood protein fractions.
  • 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 Ultra Violet A) and more recently various forms of lymphoma. Psoralen will bind 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 RNA base and will proceed only if the psoralen is intercalated in a site containing thymine or uracil.
  • the initial photoadduct can absorb a second UVA photon and react 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 number 4,748,120 of Wiesehan 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.
  • some of the best compounds for use in photo-assisted viral inactivation have a psoralen or coumarin backbone. These compounds are nucleic acid intercalators, and thus assure that the photosensitizer associates with the nucleic acid of viral or bacterial contaminants in biological solutions.
  • FIG. 1 depicts the possible structure of several ring-opened species that may result from the inactivation of a coumarin photosensitizer. Similar types of ring-opened species can be envisioned as arising from the inactivation of psoralen backbone photosensitizers.
  • the formation of non-productive reaction products upon irradiation leads to two concerns. The first concern is related to the toxicity of byproducts formed, and the second concern is the further reactivity of such byproducts. Additional reactions to the byproducts can either be photo assisted or simply due to the highly reactive nature of the compounds.
  • the 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 lipid bilayers, which are common to viruses, erythrocytes, and platelets.
  • lipid bilayers which are common to viruses, erythrocytes, and platelets.
  • neutral psoralens such as 8-MOP (8-methoxypsoralen) are uncharged and thus also have a high affinity for the interior of lipid bilayers and cell membranes.
  • AMT 4'-aminomethyl-4,5',8- trimethylpsoralen
  • AMT contains an acidic hydrogen which can bind to the phospholipid head group by hydrogen bonding, shown below.
  • AMT is believed to be an unacceptable photosensitizer because it will indiscriminately sensitize damage to viral membranes and to the membranes of erythrocytes and platelets.
  • Studies of the affects of cationic sidechains on furocoumarins as photosensitizers are reviewed in Psoralen DNA Photobiology, Vol. I, ed. F. Gasparro, CRC Press, Inc., Boca Raton, Fla., Chapter 2. The following points can be gleaned from this review:
  • United States patent number 5,216,176 of Heindel describes a large number of psoralens and coumarins that have some effectiveness as photoactivated inhibitors of epidermal growth factor. Included among the vast functionalities that could be included in the psoralen or coumarin backbone were halogens and amines. The inventors did not recognize the significance of either functionality or the benefits of a photosensitizer including both functionalities.
  • United States patent applications serial nos. 08/165,305 and 08/091 ,674 are commonly assigned with the present application, and are parent applications to this application. These applications 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.
  • the 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 al. Proc. Natl. Acad. Sci. USA, £1:5552-56 (1994).
  • 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 ultra-violet radiation or ionizing radiation, such as X-rays, which can penetrate the sample containing the contamination.
  • the present invention is also applicable to inactivation of blood- borne bacterial contaminants, and to blood-borne parasitic contaminants, since 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, particularly red blood cells and platelets lack their own genomic DNA RNA, the use of nucleic acid- binding photosensitizers is especially useful for the problem of treating blood contaminants.
  • the present invention may also be applied to viral inactivation of tissues and organs used for transplantation, and used in topical creams or ointments for treatment of skin disorders or for topical decontamination.
  • the present invention may also be used in the manufacture of viral vaccines for human or veterinary use, particularly to produce live, nonviable or attenuated viral 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 or superficial skin cancers.
  • the present invention includes the utilization of a class of compounds that have a selective affinity to nucleic acid.
  • the class of compounds also contains a halogen substituent and a water solubilization moiety, such as, 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 comprise the genetic material of viruses.
  • the bound compound can be activated by exposure to radiation, such as ultraviolet radiation (UV light 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.
  • radiation such as ultraviolet radiation (UV light of a defined wavelength)
  • ionizing radiation such as x-rays
  • 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 are characterized generally as follows: a) they are intercalators, and they are 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 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 photo inactivation of blood and blood products contaminated with Human Immunodeficiency Virus- 1
  • HIV-1 Sindbis virus
  • Cytomegalovirus 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) to confer new photoactive properties to the intercalator.
  • any intercalating molecule psoralens, coumarins, or other polycyclic ring structures
  • 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 due to a novel electron transfer pathway created by the breaking of the carbon-halogen bond upon input of appropriate radiation energy.
  • the mechanism for this alternate chemical reaction requires a single photon of light 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 breakage of the carbon-halogen atom). 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 desired for viral inactivation.
  • 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 these principles can be extended to any intercalating molecule, to confer new photoactive properties. Due to the flexibility in adding halogen substituents or non- hydrogen bonding ionic substituents to virtually any cyclic or polycyclic ring structure, the inventors envision that new and useful molecules 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 many organic dye molecules (such as coumarins) including merocyanine dyes, methylene blue, eosin dyes, and others.
  • organic dyes such as 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 in such an application than the unmodified dye.
  • the photosensitizers of the present invention are irradiated in the presence of a "blocking" agent.
  • the blocking agent is a chemical that is capable of reducing deleterious side reactions such as cell surface modifications or protein modifications. Of particular concern are the blocking of deleterious side reactions that are a result of irradiation- induced reactions to the photosensitizers that do not result in viral or bacterial inactivation. More specifically, are reactions that occur to psoralen and coumarin backbone photosensitizers upon irradiation; presumably these reactions lead to ring-opened species that are highly reactive.
  • a variety of blocking agents have been found to be suitable for this purpose, some of which are traditional antioxidants, and some of which are not.
  • the blocking agent is cysteine and in the most preferred embodiment the blocking agent is N-acetyl-L-cysteine.
  • the present invention includes a method for inactivating viral and bacterial contaminants from a biological solution comprising mixing said biological solution with a photosensitizer and a blocking agent and irradiating said mixture under conditions whereby substantially all of said contaminants are inactivated and the physiological activity of said biological solution is substantially unimpaired.
  • the photosensitizer upon irradiation participates in deleterious side reactions when not in the presence of said blocking agent that are not exclusively or predominantly the result of reactive oxygen species, and the mode of action of the blocking agent is not predominantly in the quenching of reactive oxygen species.
  • the present invention also includes an additional novel family of photosensitizers.
  • the defining characteristic of their new family of photosensitizers is a chemical backbone of quinoline or quinolone. It has been shown that such photosensitizers, modified in a variety of different ways, leads to the viral inactivation of biological solutions with reduced damage to the physiological functions of the biological solution.
  • the present invention includes, therefore, a method for inactivating viral and bacterial contaminants from a biological solution comprising mixing said biological solution with a photosensitizer, wherein said photosensitizer is a quinoline or quinolone, and irradiating said mixture under conditions whereby substantially all of said contaminants are inactivated and the physiological activity of said biological solution is substantially unimpaired.
  • Figure 1 depicts the potential photo products resulting from the irradiation of Photosensitizer A.
  • Figure 2 shows the results of Capture-P assay for Photosensitizers A and B in the presence and absence of blocking agent cysteine.
  • Figure 3 depicts the aggregation response to ristocetin of control (a), Photosensitizer B (b), and Photosensitizer B and blocking agent cysteine (c).
  • Figure 4 is a plot showing the adhesion of platelets to protein coated glass slides following irradiation. The graph plots percentage platelet adherent versus flow rate for control (-D-), Photosensitizer B (- ⁇ -), and cysteine (-
  • Figure 5 is a plot showing the kinetics of inactivation of Sindbis virus using 218 ⁇ M Photosensitizer A with (- ⁇ -) and without (- ⁇ -) 10 mM N- acetyl-L-cysteine in human plasma. The graph plots virus titer versus UVA fluence.
  • Figure 6 is a plot showing the inactivation of Sindbis virus using 55 ⁇ M (- ⁇ *-), 109 ⁇ M (- ⁇ -) and 218 ⁇ M (- ⁇ -) of Photosensitizer A with 10 mM N- acetyl-L-cysteine in plasma.
  • the graph plots virus titer versus UVA fluence.
  • Figure 7 is a plot showing the inactivation of Sindbis virus using
  • Figure 8 shows HPLC chromatograms of a solution containing Photosensitizer A following irradiation alone (Fig. 8 A) and in the presence of 10 mM N-acetyl-L-cysteine (Fig. 8B).
  • Figure 9 is a plot showing the kinetics of inactivation of Sindbis virus using 300 ⁇ g/mL of Photosensitizer QA (- ⁇ -), QB (-*-) and CARBOSTYRIL 124 (- ⁇ -) in human plasma. The graph plots virus reduction versus UVA fluence.
  • Figure 10 is a plot showing the kinetics of inactivation of Sindbis virus using 300 ⁇ g/mL of bromoquinoline (- ⁇ -), bromoquinoline and 10 mM N- acetyl-L-cysteine (- ⁇ -), CARBOSTYRIL 124 (-0-), and CARBOSTYRIL 124 and
  • Figure 11 shows HPLC chromatograms of a solution containing Photosensitizer QA before (Fig. 11 A) and after 20 J/cm 2 UVA irradiation (Fig. 11B).
  • Figure 12 is a plot showing the kinetics of inactivation of Sindbis virus using 16.7 ⁇ g/ml CARBOSTYRIL 124 with 10 mM N-acetyl-L-cysteine in plasma. The graph plots virus reduction versus UVA fluence.
  • Figure 13 is a plot showing the kinetics of inactivation of Sindbis virus using 16.7 ⁇ g/ml CARBOSTYRIL 124 (- ⁇ -) and 300 ⁇ g/ml (- ⁇ -) with 10 mM N-acetyl-L-cysteine in plasma.
  • the graph plots virus reduction versus UVA fluence.
  • Figure 14 is a plot showing the kinetics of inactivation of Sindbis virus using Photosensitizer A (- ⁇ -) or CARBOSTYRIL 124 (- ⁇ -) with 10 mM N- acetyl-L-cysteine in plasma. The graph plots virus reduction versus UVA fluence.
  • 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 to decontaminate liquid compositions, such as blood, blood components, reconstituted lyophilized cells, and the like, using UV irradiation.
  • a radiation sensitizing chemical compound is added to a liquid suspension of infectious viruses and/or bacteria and/or parasites, and the mixture is exposed to UV light or ionizing radiation.
  • Assays of viral infectivity demonstrate the effectiveness of the compounds to inactivate the viruses, compared to radiation treatment alone.
  • the present invention includes a method for reducing viral, bacterial and other parasitic contamination from a biological solution.
  • Biological solutions include, but are not limited to, solutions comprising blood, a blood component, cell culture or a component of a cell culture.
  • the method comprises mixing the composition in a liquid state with a photochemical photosensitizer capable of binding to the viral, bacterial or parasitic contamination.
  • the photochemical 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.
  • one of the most critical elements is the use of a novel class of photosensitizer.
  • a photosensitizer is defined for the purposes of this application as a chemical compound that has a light-absorbing chromophore that absorbs radiation in the ultraviolet or visible spectrum, and that is capable of inactivating viral or bacterial 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.
  • Photodetitizers included in 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 which are two of the preferred basic structures for the intercalators of the present invention, are just two examples of chemical backbone structures capable of nucleic acid intercalation.
  • Certain 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.
  • Certain 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
  • Compound A is a coumarin based photosensitizer
  • compound B is a psoralen or furocoumarin based photosensitizer. Additional preferred embodiments of the present invention include the following coumarin based photosensitizers:
  • compound A Upon irradiation with UV light, 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 acts to reduce the production of highly reactive oxygen species in blood and blood components.
  • 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 halo-C bond homolysis and the generation of a coumarin radical that can attack the nucleic acid backbone.
  • Bromopsoralens, and Photosensitizer B specifically, do not form free radicals upon irradiation in solution.
  • a donor is required to activate
  • Photosensitizer B Using fluorescence spectroscopy it has been shown that amino acids are not suitable donors to activate Photosensitizer B. Thus 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, are capable of fluorescence; and either 1) are halogenated; or 2) have the psoralen backbone structure.
  • quaternary ammonium or phosphonium substituted halo- intercalators described herein do not accumulate in the interior of lipid bilayers
  • One preferred class of photosensitizers is selected from the group consisting of compounds of the formula (I):
  • u 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 ) n Z ffi 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 ⁇ is
  • Rg, R 5 , R 2 and R 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).
  • u is an integer from 1 to 6;
  • X is an anionic counterion;
  • Z is N or P;
  • RNase 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 ffl 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 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 perpetrat R 2 , R 3 , R 4 , R 5 or ⁇ is (CH 2 )
  • R 3 , R 5 , R 2 and R are hydrogen and R 3 is H or halo, preferably bromo.
  • the above compounds may be 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.
  • Quaternary ammonium sensitizers osp on um sens tzers 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. Proc. Nat'l. Acad. Sci. USA, £1:5552-56 (1994). 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.
  • An additional novel class of photosensitizers useful in the present invention have either quinoline or quinolone backbone structures. It has surprisingly been shown that such compounds are capable of inactivating virus in biological solutions upon irradiation. This is true even for compounds that do not contain a halogen. In addition, it is not known that quinolines or quinolones will undergo crosslinking reactions with nucleic acids such as psoralens. Thus, the exact mechanism for light induced viral inactivation by such photosensitizers is not known.
  • R, - R ]3 are independently any chemical substituent, including but not limited to, halo, H, linear or branched or substituted alkyl of 1-15 carbon atoms, linear or branched or substituted alkoxy of 1-15 carbon atoms, any alkenyl (C r C 15 ), alkynyl (C--C- I5 ) aryl, amino, thio, thioester, metal, silicon based substituent.
  • Certain preferred photosensitizers in this class included the following:
  • CARBOSTYRIL 124 (7-amino-4-methyl-2 (1 H)-quinolinone
  • the experimental data below involves a study of viral inactivation of Sindbis virus using several quinolones (Photosensitizers QA and QB and CARBOSTYRIL 124; see Figure 9).
  • the CARBOSTYRIL 124 compound was most effective against Sindbis virus seeded into human plasma and irradiated with UVA light.
  • CARBOSTYRIL 124 either with or without N-acetyl-L-cysteine is more effective against Sindbis virus than the 3 -bromoquinoline, which appears to be relatively ineffective against Sindbis.
  • Table 3 shows that
  • CARBOSTYRIL 124 treated platelets exhibit some loss of function, particularly the aggregation response at 300 ⁇ g/mL CARBOSTYRIL 124 and 30 J/cm 2 light energy. Even at lower CARBOSTYRIL 124 concentration levels cells exhibit some loss of function.
  • Table 5 shows platelet properties after CARBOSTYRIL 124 treatment with and without N-acetyl-L-cysteine.
  • FIGS. 12 and 13 illustrate that CARBOSTYRIL 124 with N-acetyl- L-cysteine inactivates Sindbis virus, by 4-fold, at a concentration of 16.7 ⁇ g/ml in plasma; more effectively inactivates Sindbis virus, by 6-fold, at a concentration of 300 ⁇ g/ml.
  • Figure 14 shows that the former concentration of CARBOSTYRIL 124 with N-acetyl-L-cysteine is more effective than
  • Phage T 4 is a model for Parvo virus and Hepatitis A found in blood sample.
  • the DNA of this virus is surrounded by capsid proteins which do not allow any sensitizer to get inside and inactivate the viral DNA.
  • the goal is to inactivate the viral DNA without destroying the capsid proteins of the virus.
  • Phage T 4 as a model for viruses found in the blood, it is intended to find a sensitizer that is able to destroy the T 4 virus without destroying the capsid proteins.
  • CARBOSTYRIL 124 compound is water soluble although it carries no formal charge. This is due to resonance structures that can form a zwitterionic species.
  • the 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 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/or 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 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 can lead to DNA cleavage by known mechanisms.
  • RNA ribose
  • DNA deoxyribose
  • 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, which 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.
  • the use of the brominated or halogenated intercalators is particularly useful for activation in hydrated systems such as plasma, immune sera, tissue culture media containing animal serum or serum components (such as 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. Patents
  • the photosensitizers also may be utilized in vivo and 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, and viruses, bacteria or other parasites comprising RNA and/or DNA.
  • the present invention includes the use of "blocking" agent along with the photosensitizer in the method of inactivating contaminants in biological solutions.
  • blocking agents prevent the photosensitizer from undergoing non ⁇ productive side reactions and side reactions that can lead to a reduction in the physiological properties of the components of the biological solution.
  • the blocking agents of the present invention act predominantly by preventing reactions of the photosensitizer that lead to the formation of reactive byproducts that are not associated with reactive oxygen species such as singlet oxygen.
  • Examples 1 , 2 and 3 and Figures 3 and 4 demonstrate that use of cysteine blocking agents improves the functional quality of treated platelets, as measured by three in vitro assays: IgG binding, aggregation induced by Ristocetin, and cell adhesion to an artificial substrate.
  • IgG binding is most important, since any alterations to the cell surface chemistry that induce nonspecific binding of plasma IgG cause rapid in vivo removal of the circulating blood cells by the immune system.
  • Table 1 summarizes the IgG binding data, using numerous agents in combination with Photosensitizer A and UVA radiation with human platelets.
  • the assay used was in a commercial test kit sold by Immucor Inc. for detection of IgG binding to platelets ("Capture-P Assay").
  • Capture-P Assay A photograph of one such developed assay is included in Figure 2.
  • some classical antioxidants such as ascorbate are effective in blocking IgG binding to the cells, but other quenchers such as mannitol and glucose are ineffective. Degassing is also effective, as are certain reducing agents (DTT) and food preservatives (BHT, BHA), which act as antioxidants.
  • DTT reducing agents
  • BHT food preservatives
  • the related N-acetyl-L-cysteine is the preferred compound of the invention, since it is as effective as cysteine or ascorbate in preventing IgG binding, but in addition is commercially available in pharmaceutical grade, and exhibits better storage properties in solution with halogenated sensitizers.
  • Figures 3 and 4 show the aggregation response data and the adhesion assay data, respectively. Irradiation of platelets in the presence of cysteine and Photosensitizer B results in an aggregation response profile that is closer to normal than irradiation with Photosensitizer B alone.
  • FIG. 5 shows a comparison between Photosensitizer A alone and Photosensitizer A + 10 mM N- acetyl-L-cysteine for inactivation of Sindbis virus, a mammalian enveloped RNA virus.
  • Figure 6 shows the dose dependence of viral kill with increasing concentrations of Photosensitizer A in the presence of 10 mM N-acetyl-L- cysteine.
  • Figure 7 shows a similar experiment using 25 mM N-acetyl-L-cysteine. In all cases the inclusion of the "blocking" agent did not affect the viral inactivation kinetics or dose dependence.
  • FIG 8 shows two HPLC traces of Photosensitizer A exposed to UVA light in saline solution.
  • Trace 1 shows the profile of Photosensitizer A irradiated by itself. Residual amounts of Photosensitizer A parent compound are seen as peak 4.36 (some 0.72% of the total material).
  • the main peak at 3.53 corresponds to the debrominated Photosensitizer A, accounting for some 61% of the material.
  • the remaining peaks, accounting for some 38% of the total material comprises various photolysis products of Photosensitizer A, which are believed to include a mixture of the possible ring-opened structures shown in Figure 1 (each ring-opened product can exist in a brominated or debrominated form).
  • HPLC data clearly show that one mechanism of action of the "blocking" agent may be to block formation of reactive ring-opened compounds, which can in turn react with cells via their aldehyde or carboxylic acid groups.
  • Photosensitizer B only more photolysis products are evident in the absence of a blocking agent since the psoralen nucleus can open at either the furan or pyrone rings.
  • Immunoglobulin (IgG)-sensitized platelets are phagocytosed by the monocytes and macrophages of the reticuloendothelial system (RE). RE cells are located throughout the body but are concentrated in the spleen, liver, lungs and bone marrow. Platelets can be immune sensitized by several ways, e.g. platelet specific antigens, etc. Platelets can be IgG-sensitized by nonspecific binding of IgG to platelets which contributes to accelerated immune clearance. IgG binding to platelets can be easily detected by using the Immucor Capture-P assay
  • Platelet concentrates or platelet rich plasma were irradiated to deliver 10 to 30 J/cm 2 in the presence of sensitizers Photosensitizer B and Photosensitizer A (100 ⁇ g/mL or 300 ⁇ g/mL) and blocking agents under investigation (10 or 20 mM, see Tables 1 and 2).
  • the platelets were analyzed by the Capture-P assay.
  • the results are summarized in Table 1 (Photosensitizer B) and Table 2 (Photosensitizer A). Test results are interpreted as positive (+) for IgG binding (IgG sensitized) and negative (-) for non-binding (absence of IgG sensitization) according to the text kit manufacturer's described method.
  • EXAMPLE 2 RISTOCETIN AGGREGATION RESPONSE
  • Photoinactivation treatment in the presence of sensitizer Photosensitizer B reduces the aggregation response of platelets to Ristocetin agonist compared to control (Fig. 3). However, the presence of cysteine (Fig. 4) and the other agents listed in table 1 during the photoinactivation treatment reduces the platelet aggregation damage. Aggregation response was measured in an aggregometer using 2.5 mg/mL final concentration.
  • the adhesion property of photoinactivated platelets was measured using a micro flow system equipped with syringe pump and a microscope. Photoinactivated platelets were allowed to rest on glass flow cells in the presence of plasma. Following incubation at 37°C for 30 minutes different shear stress was applied by pumping plasma with different flow rates. Adherent cells were quantitated and compared with controls. The results are shown in Fig. 4. These results clearly demonstrate that the presence of Cysteine during photoinactivation substantially reduces adhesive properties of platelets (stickiness) compared to the sample without cysteine.
  • STERICON bags Two sets of three bags each were used. The first set contained 109, 218 and 327 ⁇ M Photosensitizer A and 25 mM N-acetyl-L-cysteine (NAC) and the second set contained 55, 109 and 218 ⁇ M Photosensitizer A and 10 mM NAC. Two other bags served as controls, each containing saline instead of the sensitizer. The bags were exposed to different levels of ultraviolet light A, except for the controls that where exposed to normal light. Samples were taken at 0, 5,
  • the results of this experiment indicate that virus inactivation is sensitizer and NAC concentration dependent (Fig. 5 and 6 and 7).
  • the viral inactivation capacity of 100 ⁇ g/mL of Photosensitizer A and 10 mM N-acetyl-L- cysteine was ⁇ 6 logs of Sindbis compared to 5 logs with 100 ⁇ g/mL and 25 mM N-acetyl-L-cysteine.
  • 50 ⁇ g/mL of Photosensitizer A achieved significant viral inactivation of 3 and 4 logs of virus with 25 mM and 10 mM N-acetyl-L-cysteine, respectively.
  • CARBOSTYRIL 124 (7-AMINO.4-METHYL.2- HYDROXY QUINOLINES AND ULTRAVIOLET LIGHT.
  • Bacteriophage T 4 a nonenveloped bacterial virus, was used to examine inactivation by CARBOSTYRIL 124 and ultraviolet light. T 4 is known to be quite resistant to inactivation and has been shown to be resistant to both CP- 38 and CP-45 (data not shown).
  • a lyophilized E. coli bacterial pellet was obtained from ATCC. Said pellet was suspended in 1.0 mL of LB medium by vortexing. A portion of the bacterial suspension was picked up with a sterilized inoculating loop and streaked on the lawn of an LB plate. The streaked plate was placed in an incubator for 24 hours at 37 °C in order to grow isolated bacterial colonies. A colony of bacteria was picked up with the sterile platinum loop and washed with 50 mL of LB medium and 0.5 mL of 20 % maltose solution in a screw cap flask.
  • the flask was then left on a roller drum at 120 rpm in an incubator at 37°C, with a loosened flask cap to ensure proper aeration to yield a saturated bacterial suspension.
  • the suspension was transferred into a sterile tube and centrifuged at 5000 rpm for 10 minutes, the supernatant subsequently discarded.
  • the resulting bacterial pellet was resuspended in about 50 mL of 0.1 M magnesium sulfate solution and stored in a refrigerator.
  • the lyophilized T 4 pellet obtained form ATCC was reconstituted with 0.3 mL of LB medium by vortexing in a small sterile vial. 100 ⁇ L of the resulting viral solution was removed for a 10-fold serial dilution. To effect the serial dilution, 900 ⁇ L of SM medium were introduced into 6 sterile culture tubes. 100 ⁇ L of viral suspension was transferred from the original viral solution into the first culture tube. The tube was then vortexed and 100 ⁇ L of suspension from the first culture tube was transferred to the second culture tube. This process being repeated until the six serial dilutions into the six culture tubes were achieved.
  • each of the six prepared viral dilutions were placed into a second set of six sterile culture tubes.
  • 100 ⁇ L of previously prepared bacterial suspension was added.
  • the six tubes were then incubated at 37 °C for about 30 minutes to allow the T 4 virus to infect the E. coli bacteria.
  • the viral stock solution for the viral inactivation experiment was prepared by isolating the plates of a very large number of clear plaques. To each of them was added 3 mL of SM medium. The plates were then left in the refrigerator for 7 hours. Next, the plates were scraped off and the supernatant was placed in a centrifuge tube and centrifuged at 5000 rpm for 15 minutes. The viral supernatant was then separated and stored with a drop of distilled chloroform at 4°C.
  • T 4 virus was exposed to 100 ⁇ g/mL of sensitizer for 24 hours, after which it was exposed to UV light for 10 minutes, estimated to be 3-4 J/cm 2 .
  • the effects on the viral titer after exposure are shown in Table 4.
  • the experimental data show that starting with a virus titer of 1.5 x 109 plaque-forming units (pfu) and adding CARBOSTYRIL 124 dissolved in either phosphate-buffered saline (PBS) or 7% bovine serum albumin in PBS, resulted in residual virus titers of 2.0 x 106 pfu or 1.0 x 105 pfu, respectively.
  • PBS phosphate-buffered saline
  • bovine serum albumin 7% bovine serum albumin
  • NAC 10 mM N- Acetyl Cysteine

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Abstract

Des contaminants viraux et bactériens présents dans des solutions biologiques sont inactivés en mélangeant ladite solution à un photosensibilisateur faisant partie d'une nouvelle classe de photosensibilisateurs, et en irradiant le mélange obtenu. Des composés de quinoline et de quinolone sont utiles en tant que photosensibilisateurs dans ce procédé. Avec certains photosensibilisateurs, un agent bloquant peut également être utilisé.
EP96916996A 1995-06-07 1996-06-03 Procede d'inactivation de contaminants viraux et bacteriens dans le sang Withdrawn EP0830057A4 (fr)

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US08/474,459 US5798238A (en) 1990-04-16 1995-06-07 Method of inactivation of viral and bacterial blood contaminants with quinolines as photosensitizer
US474459 1995-06-07
US08/480,271 US5955256A (en) 1990-04-16 1995-06-07 Method of inactivation of viral and bacterial blood contaminants
US480271 1995-06-07
PCT/US1996/008421 WO1996039816A1 (fr) 1995-06-07 1996-06-03 Procede d'inactivation de contaminants viraux et bacteriens dans le sang

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CA2199372A1 (fr) * 1994-09-22 1996-03-28 Raymond P. Goodrich, Jr. Procede d'inactivation photodynamique de contaminants du sang de nature virale et bacterienne a l'aide de sensibilisants a la coumarine ou la furocoumarine
DE69826521T2 (de) 1998-01-06 2005-09-29 Cerus Corp., Concord Verfahren zum quenchen von pathogen-inaktivatoren in biologischen materialien
US6268120B1 (en) * 1999-10-19 2001-07-31 Gambro, Inc. Isoalloxazine derivatives to neutralize biological contaminants
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