WO1996008965A1 - Photodynamic inactivation of viral and bacterial blood contaminants with halogenated coumarin and furocoumarin sensitizers - Google Patents

Abstract

Viral, bacterial and parasitic contaminants in biological compositions are photodynamically inactivated by mixing halogenated coumarin and furocoumarin photosensitizers with the biological composition and irradiating the mixture. The figure depicts the proposed energy diagram of the instant photosensitizers.

Description

Photodyanmic Inactivation of Viral and Bacterial Blood Contaminants with Halogenated Coumarin and Furocoumarin Sensitizers

FIEL D OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

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. Of particular concern are 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). 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. In fact, some viruses are believed to be cell-associated, and are unlikely to be removed by extensive washing and centrifugal pelleting of the cells. Current theory suggests that safe levels require at least a 6 log (6 orders of magnitude) demonstrated reduction in infectious viral titer for cellular blood

components. This 6 log threshold may be greater for plasma protein components, especially the clotting factors (Factor VIII, Factor IX) that are administered throughout the life of some hemophilia patients. All blood collected in the United States is currently screened for six infectious agents: HIV-1, HIV-2, HTLV-1, Hepatitis B Virus, Hepatitis C Virus and Syphilis. Additionally, donors are screened for risk factors, and potential donors are eliminated that are considered at risk for HIV. Despite these measures, the risk of becoming infected by a potentially deadly virus or bacteria via the transfusion of blood or blood products remains serious. Screens for contaminants are by nature not foolproof. There is also the likely occurrence of new infectious agents that will enter the blood supply before their significance is known. For example, by the end of June 1992, the Center for Disease Control reports that 4,959 AIDS cases could be traced directly to the receipt of blood transfusions, blood components or tissue.

Viral inactivation by stringent sterilization is not acceptable since this method can also destroy the functional components of the blood, particularly the erythrocytes (red blood cells), thrombocytes (platelets) and the labile plasma proteins, such as clotting factor VIII. Viable red blood cells (RBC) can be characterized by one or more of the following:

capability of synthesizing ATP; cell morphology; P₅₀ values; filterability or deformability; oxyhemoglobin, methemoglobin and hemochrome values; MCV, MCH, and MCHC values; cell enzyme activity; and in vivo survival.

Thus, if virally inactivated cells are damaged to the extent that the cells are not capable of metabolizing or synthesizing ATP, or the cell circulation is compromised, then their utility in transfusion medicine is compromised.

Wet steam sterilization also destroys function blood components, in particular, blood cells and plasma proteins. Dry heat sterilization, like wet steam, is harmful to blood cells and blood proteins at the levels needed to reduce viral infectivity. The use of stabilizing agents, for example, carbohydrates, does not provide sufficient protection to the delicate blood cells and proteins from the general effects of exposure to high temperature and pressure.

Methods that are currently employed with purified plasma protein fractions, often followed by lyophilization of the protein preparation, include treatment with organic solvents and heat, or alternatively, extraction with detergents to disrupt the lipid coat of membrane enveloped viruses. Lyophilization, freeze-drying, alone has proven insufficient to either inactivate viruses or render blood proteins sufficiently stable to the effects of heat sterilization. The organic solvent or detergent method employed with purified blood proteins cannot be used with blood cells as these chemicals destroy the lipid membrane that surrounds the cells.

Another viral inactivation approach for plasma proteins; first demonstrated in 1958, involves the use of the chemical compound beta- propiolactone with ultraviolet (UV) irradiation. This method has not found acceptance in the United States due to concern over the toxicity of beta- propiolactone in the amounts necessary to achieve some demonstrable viral inactivation and to unacceptable levels of damage to the proteins caused by the chemical agents. Concern has been raised over the explosive potential for beta-propiolactone as well.

There is significant interest in an effective viral inactivation treatment for human blood components, that will not damage the valuable blood cells or proteins. The treatment must be nontoxic and selective for viruses, while allowing the intermingled blood cells or proteins to survive unharmed. Thus, an immediate need exists to develop protocols for the inactivation of viruses that can be present in the human red blood cell supply. For example, only recently has a test been developed for Hepatitis C, but such screening methods, while reducing the incidence of viral transmission, do not make the blood supply completely safe or virus free. Current statistics indicate that the transfusion risk per unit of transfused blood is as high as 1 :3,000 for Hepatitis C, and depending on geographic location, ranges from 1 :60,000 to 1 :225,000 for HIV. Clearly, it is desirable to deyelop a method which inactivates or indiscriminately removes virus from blood.

Contamination problems also exist for blood plasma protein fractions, plasma fractions containing immune globulins and clotting factors.. For example, 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. Hence, there is a need for improved viral inactivation treatment of blood protein fractions.

In addition to the common viruses that are included in the category of enveloped viruses, it is also highly desirable to provide a viral

inactivation protocol that is effective for non-enveloped viruses. The 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.

Current procedures for inactivating viruses from plasma protein derivatives that have been incorporated into manufacturing processes are: 1) dry heat-heating in freeze-dried state; 2) heating in solution- pasteurization, wet heat (60°C, 10 hours); 3) heating in suspension-n- heptane; 4) vapor heat-freeze-dried state; 5) solvent detergent --tri(n-butyl) phosphate/cholate, Tween 80, Triton X-100; and 6) low pH-e.g. pH 4.25

(M.M.Mozen, "Viral Inactivation of Plasma Derivatives", in The Role of Virus-Inactivated Plasma in Clinical Medicine, Bethesda, MD, National Institutes of Health, March 23, 1994). None of these protocols is effective for the inactivation of B19. See, Luban, supra at 823 ("[Recent studies support] the concept that neither solvent/detergent treatment, heat treatment, nor the combination is sufficient to prevent transmission of B19").

The ability to inactivate bacterial contaminants from blood and blood products may be as critical as reducing viral contaminants. Between 1986 and 1991, the Food and Drug Administration reported that 15.9% of all transfusion related fatalities were associated with the transfusion of bacterially contaminated blood components. Most of these fatalities were due to the transfusion of bacterially contaminated platelets.

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 Ultraviolet A) and more recently various forms of lymphoma.

Psoralen binds to nucleic acid double helices by intercalation between base pairs; adenine, guanine, cytosine and thymine (DNA) or uracil (RNA). Upon absorption of UVA photons the psoralen excited state has been shown to react with a thymine or uracil double bond and covalently attach to both strands of a nucleic acid helix.

The crosslinking reaction is specific for a thymine (DNA) or uracil (RNA) base and proceeds only if the psoralen is intercalated in a site containing thymine or uracil. The initial photoadduct absorbs a second UVA photon and reacts with a second thymine or uracil on the opposing strand of the double helix to crosslink the two strands of the double helix.

Lethal damage to a cell or virus occurs when a psoralen intercalated into a nucleic acid duplex in sites containing two thymines (or uracils) on opposing strands sequentially absorb 2 UVA photons. This is .an inefficient process because two low probability events are required; the localization of the psoralen into sites with two thymines (or uracils) present and its sequential absorption of 2 UVA photons.

United States Patent 4,748,120 , to Wiesehahn ,is an example of the use of certain substituted psoralens by a photochemical decontamination process for the treatment of blood or blood products. The psoralens described for use in the process do not include halogenated psoralens, or psoralens with non-hydrogen binding ionic substituents. Using traditional psoralens, for example, S-methoxypsoralen (8-MOP), 4'-aminomethyl- 4,5',8-trimethylpsoralen (AMT) and 4^,-hydroxymethy-4,5',8- trimethylpsoralen (HMT), it is imperative that certain substances be added into the blood product solution in conjunction with UV irradiation in order to scavenge singlet oxygen and other highly reactive oxygen species formed by irradiation of the psoralen. Without the addition of reactive oxygen species scavengers, cellular components and protein components in the blood product are seriously damaged upon irradiation. (See also, United States Patent 5,176,921.) It is clear, therefore, that irradiation of traditional psoralens in aqueous solution creates a competition between the inefficient photocrosslinking reaction and the generation of highly reactive oxygen species. It is also possible that much of the viral deactivation seen using these photosensitizers actually results from the action of the reactive oxygen species against the viral contaminants rather than the inefficient

photocrosslinking mechanism.

Attempts to inactivate viral contaminants using photosensitizers and light have also been developed using some non-psoralen photosensitizers. The photosensitizers that have been employed are typically dyes. Examples include dihematoporphyrin ether (DHE), Merocyanine 540 (MC540) and methylene blue.

In any event, 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. Although evidence shows that psoralens bind to nucleic acids by

intercalation, neutral psoralens such as 8-MOP are uncharged and thus also have a high affinity for the interior of lipid bilayers and cell membranes.

The binding of 8-MOP to cell membranes, shown above, is acceptable only if the psoralen bound to the lipid is photochemically inert. However, Midden (W.R. Midden, Psoralen DNA photobiology, Vol II (ed. F.P. Gaspalloco) CRC press, pp. 1. (1988)) has presented evidence that psoralens photoreact with unsaturated lipids and photoreact with molecular oxygen to produce active oxygen species such as superoxide and singlet oxygen that cause lethal damage to membranes. Thus, 8-MOP is an unacceptable photosensitizer because it sensitizes indiscriminate damage to both cells and viruses.

Positively charged psoralens, for example, AMT, do not bind to the interior of phospholipid bilayer membranes because of the presence of the charge. However, AMT contains an acidic hydrogen which binds to the phospholipid head group by hydrogen bonding, shown below.

Thus, AMT is an unacceptable photosensitizer because it indiscriminately sensitizes and damages viral membranes and 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.

Gaspano, CRC Press, Inc., Boca Raton, Fla., Chapter 2. The following points can be gleaned from this review:

1) the intent of this line of research is to improve the poor water solubility of the basic psoralen nucleus;

2) none of the psoralens described are halogenated as are the photosensitizers of the present invention;

3) later conducted studies show that a cationic group on a large linker, when added to the 5 or 8 position on the psoralen ring, gives the psoralen nucleus improved binding with native DNA relative to

corresponding 5-MOP and 8-MOP analogues;

4) sidechain substitution at the 5 position is less desirable than substitution at the 8 position; and

5) a study of 5-aminomethyl derivatives of 8-MOP shows that most of the amino compounds have a much lower ability to both photobind and form crosslinks to DNA, as compared to 8-MOP. These reports actually suggest that the primary amino functionality is the preferred ionic species for both photobinding and crosslinking.

United States Patent 5,216,176, to 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 do not recognize the significance of either the functionality or the benefits of a photosensitizer including both functionalities.

United States patent application serial nos. 08/165,305 and

08/091,674, commonly assigned with the present application, and parent applications to this application, disclose the use of a novel class of psoralen photosensitizers that are superior for use with irradiation to inactivate viral and bacterial contaminants in blood and blood products. Said psoralens are characterized by the presence of a halogen substituent and a non-hydrogen binding ionic substituent to the basic psoralen side chain. See also,

Goodrich et αl.(1994) Proc. Natl. Acad. Sci. USA 91:5552.

SUMMARY OF THE INVENTION

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. In preferred embodiments, 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.

In one embodiment of the present invention, 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 Herpes Simplex Virus Type 1 (HSV- 1), using the photosensitizers of the present invention.

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. In the present invention, essentially any intercalating molecule (psoralens, coumarins, or other polycyclic ring structures) 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.

In one embodiment, 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. In addition, as shown in Figures 1 and 2, 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.

In a second embodiment, 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. However, as shown in the present invention (Figure 2), halogenation of this class of intercalating molecules confers the ability to undergo the electron transfer mechanism, thereby imparting new properties to the molecule. Without intending to limit the present invention, 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.

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. For example, known classes of compounds that may be improved by the present invention include, porphyrins, phthalocyanines, quinones, hypericin, and organic dye molecules such as coumarins, for example, merocyanine dyes, methylene blue and eosin dyes.

Without intending to limit the present invention, the inventors anticipate that new classes of compounds prepared according to the principles of this invention will find application in numerous fields, in addition to the filed of decontamination of blood and blood products. The new chemical reaction properties imparted by halogenation and the selective binding properties imparted by the use of non-hydrogen bonding ionic substituents, may be grafted onto known classes of molecules to impart advantageous chemical reaction and targeting properties to these molecules. Psoralens for example, such as 8-methyoxypsoralen (8-MOP) have been used in therapeutic photophoresis to treat cutaneous T Cell Lymphoma, Scleroderma, and other cancers and skin disorders. The modified psoralen derivatives of the present invention, or other classes of compounds modified according to the present invention, may prove more efficacious in

therapeutic photophoresis applications.

As a second example, 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.

Without intending to limit the present invention, 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. Examples of photosensitizers that absorb in the visible light region include hypericin, pthalocyanines, porphyrins, and organic dyes such as methylene blue. See, for example, International Patent Application WO/94 14956, wherein hypericin is activated via a chemiluminescent reaction between luciferin and luciferase.

Other fields of application wherein the present invention may find application include the preparation of non-infectious viral vaccines, therapeutic treatment of immune system disorders by photophoresis, elimination of viable nucleated cells such as leukocytes via the cytotoxicity of nucleic acid binding photosensitizers and possible treatment for certain accessible cancers and tumors exploiting the cytotoxic effects of nucleic acid binding photosensitizers.

The inventors further anticipate that the problem of singlet oxygen production by UV irradiation of traditional psoralen molecules can also be reduced by incorporating a quenching sidechain moiety onto the psoralen nucleus. An example of such a compound is shown below.

In this compound 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).

While not intending to be bound by theory, in principle the quenching pyridyl group, or a comparable functional 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. In the presence of the pyridium ion 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. Although, in principle, 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.

Reduction of singlet oxygen production minimizes damage to lipid membranes or proteins. Attachment of a quenching group directly onto the psoralen nucleus provides proximity to the excited psoralen, and obviates the need for addition of exogenous quenching agents, such as oxygen scavengers, reducing agents, or sugars, into the medium. Without limiting the scope of the present invention, the inventors anticipate that quenching sidechains that comprise both a non-hydrogen bonding ionic feature and a triplet quenching feature will be useful for selective viral inactivation of complex biological systems such as blood, blood plasma, or isolated blood cell fractions.

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. In the preferred mode of this embodiment, 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. According to a preferred embodiment of the invention, 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.

Other features and advantages of the present invention will become apparent form the following detailed description, taken in conjunction with the accompanying figures, that illustrate by way of example, the principles of the instant invention. BRIEF DESCRIPTION OF THE DRAWINGS

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

Virus- (HIV-1) using long wavelength ultraviolet radiation (UVA) in the presence of different concentrations of photosensitizer B. Viral reduction, log 10, is plotted versus UVA fluence, Joules/cm².

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 Herpes Simplex Virus Type 1 (HSV-1) in the presence of photosensitizer B and UVA. Viral inactivation is plotted versus UVA fluence.

FIGURE 9 depicts the synthetic scheme for the synthesis of photosensitizer A.

FIGURE 10 depicts the absorption spectrum of photosensitizer A. FIGURE 11 depicts the fluorescence spectrum of photosensitizer A.

FIGURE 12 depicts the inactivation of Sindbis Virus with photosensitizers B, A, AX, CX, D, DX and E.

FIGURE 13 depicts the synthetic scheme for the synthesis of photosensitizer D. DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.

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.

According to the present invention, 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.

Assays of viral infectivity demonstrate the effectiveness of the compounds to inactivate the viruses as compared to radiation treatment alone.

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.

The following definitions will be helpful in understanding the specification and claims. The definitions provided herein should be borne in mind when these terms are used in the following examples and

throughout the instant application.

One of the most critical elements of the present invention 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 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.

In one embodiment of the invention, 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.

Those skilled in the art are familiar with a relatively large number of intercalators and are generally able to predict the types of chemical species that are able to function as intercalators based on the chemical structure of the backbone of the chemical species. Psoralens and coumarins, the preferred basic structure for the intercalators of the present invention, are just two examples of chemical backbone structures capable of nucleic acid intercalation.

Preferred photosensitizers of the present invention comprise at least one halogen substituent. The halogens include F, Cl, Br and I. In the preferred embodiments of the present invention, 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.

In the preferred embodiments of the 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(CH₂)₃-.

The most preferred non-hydrogen bonding ionic functionalities are quaternary ammonium functionalities, more specifically, trialkyl quaternary ammonium, and even more specifically, -O(CH₂)₃ N•(CH₂CH₃)₃. Two preferred photosensitizers of the present invention are the following:

Compound A is a coumarin based photosensitizer, and compound B is a psoralen or furocoumarin based photosensitizer.

Additional preferred embodiments of the present invention include the following coumarin based photosensitizers:

The synthesis of photosensitizer A is described in Example 9 below, according to the scheme shown in Figure 7. The synthesis of photosensitizer D is described in Example 14 below, according to the scheme shown in Figure 13.

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. According to the proposed mechanism -- which is speculative and not intended to limit the scope of the invention -- 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. 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.

It is, therefore, one preferred embodiment of the method of the present invention to use a photosensitizer that is capable of fluorescence.

Coumarins and furocoumarins that fluoresce are known to those skilled in the art and the screening of photosensitizers to determine fluorescent properties is easily determined. properties is easily determined.

Photosensitizers that are capable of fluorescence appear to be superior to non-fluorescent varieties. For 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. Thus, in this embodiment of the invention the preferred photosensitizers are intercalators capable of fluorescence and either 1) are halogenated or 2) have the psoralen backbone structure.

According to an additional embodiment of the present invention, 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.

An example of a photosensitizer of this embodiment of the invention is as follows:

The non-hydrogen bonding ionic substituent comprises a quaternary ammonium pyridyl group. Such a compound can be easily prepared by one skilled in the art without undue experimentation. The quaternary

ammonium pyridyl group 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.

The 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. 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. In addition, 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):

wherein μ is an integer from 1 to 6; X is an anionic counterion; Z is N or P; R₁, R₂, R₃, R₄, R₅ and R₆ are independently halo; H; linear or branched alkyl of 1-10 carbon atoms; linear or branched alkoxy of 1-10 carbon atoms; (CH₂)-_mO (CH₂)_pZ•R',R",R'" or -O(CH₂)_nZ•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 R₁, R₂, R₃, R₄, R₅ or R₆ is

(CH₂)_mO(CH₂)_pZ•R',R",R'" or -O(CH₂)_nZ•R',R",R"'. Particularly preferred are compounds wherein R₄ is -O(CH₂)_nN•R',R",R"', especially wherein R', R" and R'" are ethyl and n=3. Preferably, R₆, R₅, R₂ and R₁ are hydrogen and R₃ is H or halo, preferably bromo. An additional preferred class of photosensitizers is selected from the group consisting of the formula (II).

wherein μ is an integer from 1 to 6; X is an anionic counterion; Z is N or P; R₁, R₂, R₃, R₄, R₅, and R₆ are independently halo; H; linear or branched alkyl of 1-10 carbon atoms; linear or branched alkoxy of 1-10 carbon atoms; (CH₂)-_mO (CH₂)_pZ•R'.R",R'" or -O(CH₂)_nZ•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 R₁, R₂, R₃, R₄, R₅ or R₆ is

(CH₂)_mO(CH₂)_pZ•R',R",R'" or -O(CH₂)_nZ•R',R",R"'. Particularly preferred are compounds wherein R₄ is -O(CH₂)_nN•R',R",R"', especially wherein R', R" and R'" are ethyl and n=3. Preferably, R₃, R₅, R₂ and R₁ are hydrogen and R₃ is H or halo, preferably bromo.

In general, the above compounds are made by halogenating psoralens and isolating the appropriately substituted isomers. For compounds wherein 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.

As described above, the most preferred photosensitizers of the present invention are comprised of ionic functionalities that are non- hydrogen bonding. However, included within the scope of this invention are 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.

Therefore, according to this invention, sensitizing compounds for viral inactivation, preferably, do not contain substituents which possess free hydrogen groups capable of exhibiting hydrogen bonding to the cell membrane.

From the foregoing description, it will be realized that the present Therefore, according to this invention, sensitizing compounds for viral inactivation, preferably, do not contain substituents which possess free hydrogen groups capable of exhibiting hydrogen bonding to the cell membrane.

From the foregoing description, it will be realized that the present invention can be used to selectively bind a chemical photosensitizer to blood-transmitted viruses, bacteria, or parasites. Also monoclonal or polyclonal antibodies directed against specific viral antigens, either coat proteins or envelope proteins, may be covalently coupled with a

photosensitizer compound.

Since 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.

Although described in connection with viruses, it will be understood that the methods of the present invention are generally also useful to inactivate any biological contaminant found in stored blood or blood products, including bacteria and blood-transmitted parasites.

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. As shown in Figure 2, absorption of a UVA photon by a bromocoumarin in the presence of guanine (or any nucleotide base) leads to electron transfer and the formation of bound radicals and ultimately nucleic acid cleavage and viral or cell death. This cleavage mechanism is more efficient than the conventional crosslinking reaction of non-halogenated psoralens.

The coumarin radical (Figure 2) can inflict damage on the nucleic acid double helix to which it is bonded by abstraction of a ribose (RNA) or deoxyribose (DNA) sugar carbon hydrogen bond. This leads to DNA cleavage by known mechanisms. The guanine radical cation shown as an example is also known to react with molecular oxygen, initiating a series of reactions which cleave DNA. The byproduct of the bound radical photochemistry is debrominated coumarin 4 that is incapable of forming crosslinks to DNA, unlike psoralens.

A preferred class of photosensitizers comprise nucleic acid intercalators which may be added to plasma or plasma fractions followed by UV radiation to reduce the viral contamination therein. According to the present invention, the reduction of viral contamination can be unexpectedly reduced by utilizing halogenated intercalators. For example, it was observed that 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. Secondly, 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.

Patents 4,889,129 and 4,878,891 and 4,613,322.

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.

According to one embodiment of the present invention, 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. Although not limited by theory, 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.

Past attempts to inactivate non-enveloped viruses by irradiation in the presence of photosensitizers have been almost totally unsuccessful. It is presumed that the reason for this failure is the inability of the

photosensitizers to gain access to the genetic material of the virus.

Moreover, unrelated efforts have been made to determine conditions under which the capsid surrounding non-enveloped viruses are loosened. Despite such efforts, none of the commercially employed techniques for treating compositions for viral inactivation are effective against non-enveloped viruses. 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. In one preferred embodiment of the present invention, described in detail in

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.

Although brominated psoralens with quaternary ammonium side chains have been tried to inactivate non-enveloped viruses, results obtained to date show that these compounds are relatively ineffective against non- enveloped viruses. The psoralen compounds tested are quite hydrophilic due to the presence of the furan ring. However, experiments performed using DNA intercalators that are relatively less hydrophilic, the level of inactivation of Porcine Parvovirus -- a non-enveloped single stranded DNA virus -- increased. For example, with photosensitizer B, a brominated psoralen photosensitizer, 0.26 log reduction was obtained in Porcine

Parvovirus with an initial titer of 6 logs. On the other hand, with a brominated coumarin such as photosensitizer A, which does not have the hydrophilic furan ring, 0.56 log reduction was obtained (see Table 17). These data suggest that DNA intercalators with reduced hydrophilicity may be more effective in penetrating the tight protein capsid of non-enveloped viruses such as Porcine Parvovirus.

In an alternate version of this embodiment, osmotic shock is used to loosen the protein capsid. When a cell or virus is suspended in a low ionic strength hypotonic solution, the cell will be subjected to an osmotic shock resulting in volume expansion. In some viruses, hypotonicity may lead to rupture of the protein capsid with discharge of their nucleic acid contents. The present invention includes a method for incorporating photosensitizers into non-enveloped virus in which a short but intense period of osmotic stress will cause the virus to become transiently permeable and allows partial incorporation of photosensitizers with low molecular weights.

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. Next, 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. This transient osmotic gradient causes the cells to swell and become permeable for a short period of time to photosensitizers (the smaller the sensitizers the easier the movement through the viral capsid). The composition of the diluent has a profound effect on the viral capsid during osmotic shock. In one preferred embodiment, the diluent may contain photosensitizer, inositol

hexaphosphate (IHP), EDTA or EGTA, sodium pyrophosphate or any polyanion in different combinations. Using distilled water alone without DMSO to induce osmotic shock in Parvovirus, it is possible to increase the inactivation of the virus from 0.62 to 2.46 logs. Data to this effect were obtained when the osmolality of plasma was reduced by 50%. However, further reduction in osmolality of the medium to either 30, 60, 90 or 120 mOsmol (i.e. 1, 20, 30 and 40% dilution of the native plasma with water) significantly increases the inactivation of Parvovirus from 2.46 logs to as high at 4 logs.

This embodiment of the invention includes the use of methods for the inactivation of non-enveloped viruses using the osmotic shock process for selective incorporation of photosensitizer into the virus. In addition, 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. Furthermore, 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:

1. Cinnamic acid

2. Caffeic acid

3. Ferulic acid 4. Coumarins

5. Gallic acid

6. Polyacetylenes

7. Thiophenes

8. Alpha terthienyl

The present invention includes the inactivation of specific viral species that are found as contaminants in blood and blood products.

Example 1 below 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, and Figure 4 shows viral reduction versus concentration of photosensitizer B.

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 below 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 below 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 below describes the general protocol used to demonstrate the inactivation of Vesicular Stomatitis Virus in human platelet concentrates. The results of the inactivation using photosensitizer B are depicted in Figure 7. Example 5 below describes the general protocol used to demonstrate the inactivation of Herpes Simplex Virus Type I. The results of the inactivation using photosensitizer B are depicted in Figure 8.

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 below 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 μg/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 (25 J/cm² of UVA) correspond to about 3.4 times the expected concentration of photosensitizer B per therapeutic dose of platelet concentrates of 1.28 μg/mL. The results, thus, demonstrate that

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/cm² UVA and 12.8 μg/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-(γ-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.

The reaction scheme for the synthesis is shown in Figure 9.

One of the best measures of the effectiveness of potential

photosensitizers is the extent to which the photosensitizer tends to associate with nucleic acids rather than to cellular membrane components or proteins in blood or blood products. Example 10 describes the protocol employed for analyzing the specificities that a variety of photosensitizers have for nucleic acids.

Independent of the mechanism of photosensitized inactivation of viral and bacterial contaminants in blood or blood products, it is generally clear that the greater the preference the photosensitizer has to the nucleic acid components of the contaminants -- as opposed to cellular membranes or proteins in solution -- the better the performance of the photosensitizer. The quality of a photosensitizer being determined by the rate of efficiency of contaminant inactivation, absolute contaminant inactivation and impairment of the physiological activity of the treated composition. Of course, these factors are all interrelated. The results of specificity experiments comparing the photosensitizers of the present invention with prior art photosensitizers reveal the superior properties of the novel photosensitizers disclosed herein. These results are shown in Table 5.

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 μg/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 μg/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.

Additional experiments were conducted in order to compare the photosensitizers of the present invention with two prior art photosensitizers, 8-MOP and AMT. The protocol for this evaluation of photosensitizers irradiated in human platelet concentrate is described in Example 12. The results of this comparison can be summarized as follows:

1. complete inactivation of bacteriophage Φ6(≥6 logs of viral reduction) is obtained with photosensitizer B without alteration in platelet in vitro properties (HSR, morphology, aggregation response to collagen) under normal oxygen content at UVA fluence of 7.6 J/cm²;

2. equimolar concentrations of AMT and 8-MOP required 45 and 68 J/cm² of UVA energy, respectively, to obtain greater than 4 logs of viral inactivation and are associated with major alterations in platelet in vitro properties;

3. photoinactivated platelet concentrates using photosensitizer B (60 μM photosensitizer concentration and 4.5 J/cm²) maintain normal properties following post-treatment storage for 5 days in a standard platelet incubator at 22 ± 2°C; and

4. 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. The results show that under the same conditions: 1) the coumarin-based photosensitizers A, C and D are superior to the psoralen- based photosensitizer B; 2) the non-halogenated coumarin-based

photosensitizers are not suitable for photoactivated inactivation of virus; and 3) the methylated coumarins, photosensitizers D and E, appear to be the most efficient photosensitizers for viral inactivation.

Example 14 describes the synthesis of photosensitizer D. The procedure follows the synthetic scheme depicted in Figure 13. Following this general procedure, believed to be novel, one skilled in the art may also synthesize photosensitizer E and other photosensitizers of the present invention. (See, e.g., Sethna (1945) Chem. Rev. 36:10 ; Sethna et al. (1953) Organic Reactions 7:1 ).

Example 15 describes the results of a series of experiments showing the effectiveness of the present invention in inactivating the non-enveloped

Porcine Parvovirus in plasma. Manipulation of the operating conditions -- particularly ionic strength, pH and freeze/thaw cycles -- make it possible to significantly inactivate Porcine Parvovirus with photosensitizer A and irradiation.

The 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.

PHASE I

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

1. Platelet + Saline + Orbital Shaking + 30 min ambient

2. Platelet + Saline + Orbital Shaking + 30 min UVA

3. Platelet + 100 μg/mL Photosensitizer B + Orbital shaking + 30 min ambient

4. Platelet + 100 μg/mL Photosensitizer B + Orbital Shaking + 30 min UVA

5. Platelet + 300 μg/mL Photosensitizer B + Orbital Shaking + 30 min ambient

6. Platelet + 300 μg/mL Photosensitizer B + Orbital Shaking + 30 min UVA

Sample Set 2

7. Platelet + Saline + Orbital Shaking + 60 min ambient

8. Platelet + Saline + Orbital Shaking + 60 min UVA

9. Platelet + 100 μg/mL Photosensitizer B + Orbital Shaking + 60 min ambient

10. Platelet + 100 μg/mL Photosensitizer B + Orbital Shaking + 60 min UVA

11. Platelet + 300 μg/mL Photosensitizer B + Orbital Shaking + 60 min ambient

12. Platelet + 300 μg/mL Photosensitizer B + Orbital Shaking + 60 min UVA

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.

Variables Under Investigation

1. Dose of Photosensitizer (Dose Response)

2. UVA Exposure Time (Kinetics of Inactivation)

Fixed Parameters

1. Light Source: UVA

2. Photosensitizer: B

3. Virus: HIV-1

4. Suspending medium: Plasma

5. Light intensity (including distance of sample from the light source)

6. Rotational speed for sample platform

7. Viral Titer: 2 X 10⁷

8. Post-photosensitizer incubation time: 10 minutes

9. UVA Reactor: Orbital shaker

PHASE III

Elimination of HIV in Platelet Concentrate - Experimental Conditions

• Dose Response

• Kinetics of Inactivation Sample Set 1

13. Platelet + Virus + Saline + Orbital Shaking + 5 min ambient

14. Platelet + Virus + Saline + Orbital Shaking + 5 min UVA

15. Platelet + Virus + 50 μg/mL Photosensitizer B + Orbital Shaking + 5 min UVA

16. Platelet + Virus + 100 μg/mL Photosensitizer B + Orbital Shaking + 5 min UVA

17. Platelet + Virus + 200 μg/mL Photosensitizer B + Orbital Shaking + 5 min UVA

18. Platelet + Virus + 300 μg/mL Photosensitizer B + Orbital Shaking + 5 min UVA

18A. Platelet + Virus + 400 μg/mL Photosensitizer B + Orbital Shaking + 5 min UVA Sample Set 2

19. Platelet + Virus + Saline + Orbital Shaking + 15 min ambient

20. Platelet + Virus + Saline + Orbital Shaking + 15 min UVA

21. Platelet + Virus + 50 μg/mL Photosensitizer B + Orbital Shaking + 15 min UVA

22. Platelet + Virus + 100 μg/mL Photosensitizer B + Orbital Shaking +

15 min UVA

23. Platelet + Virus + 200 μg/mL Photosensitizer B + Orbital Shaking + 15 min UVA

24. Platelet + Virus + 300 μg/mL Photosensitizer B + Orbital Shaking + 15 min UVA

24A. Platelet + Virus + 400 μg/mL Photosensitizer B + Orbital Shaking + 15 min UVA

Sample Set 3

25. Platelet + Virus + Saline + Orbital Shaking + 30 min ambient 26. Platelet + Virus + Saline + Orbital Shaking + 30 min UVA

27. Platelet + Virus + 50 μg/mL Photosensitizer B + Orbital Shaking + 30 min ambient

28. Platelet + Virus + 50 μg/mL Photosensitizer B + Orbital Shaking + 30 min UVA

29. Platelet + Virus + 100 μg/mL Photosensitizer B + Orbital Shaking + 30 min ambient

30. Platelet + Virus + 100 μg/mL Photosensitizer B + Orbital Shaking + 30 min UVA

31. Platelet + Virus + 200 μg/mL Photosensitizer B + Orbital Shaking + 30 min ambient

32. Platelet + Virus + 200 μg/mL Photosensitizer B + Orbital Shaking +

30 min UVA

33. Platelet + Virus + 300 μg/mL Photosensitizer B + Orbital Shaking + 30 min ambient

34. Platelet + Virus + 300 μg/mL Photosensitizer B + Orbital Shaking + 30 min UVA

34A. Platelet + Virus + 300 μg/mL Photosensitizer B + Orbital Shaking + 30 min UVA

Sample Set 4

35. Platelet + Virus + Saline + Orbital Shaking + 60 min ambient 36. Platelet + Virus + Saline + Orbital Shaking + 60 min UVA

37. Platelet + Virus + 50 μg/mL Photosensitizer B + Orbital Shaking + 60 min ambient

38. Platelet + Virus + 50 μg/mL Photosensitizer B + Orbital Shaking + 60 min UVA

39. Platelet + Virus + 100 μg/mL Photosensitizer B + Orbital Shaking +

60 min ambient

40. Platelet + Virus + 100 μg/mL Photosensitizer B + Orbital Shaking + 60 min UVA

41. Platelet + Virus + 200 μg/mL Photosensitizer B + Orbital Shaking + 60 min ambient

42. Platelet + Virus + 200 μg/mL Photosensitizer B + Orbital Shaking + 60 min UVA

43. Platelet + Virus + 300 μg/mL Photosensitizer B + Orbital Shaking + 60 min ambient

44. Platelet + Virus + 300 μg/mL Photosensitizer B + Orbital Shaking + 60 min UVA

Note: Ambient means ambient laboratory light (Non-UVA light Source).

METHODS - PHASE I

Selection of uniform UVA exposure area:

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

10 mM phosphate buffered saline (PBS) such that the final concentration is 40 mg/mL. Next, prepare 4 working solutions, having final concentrations as indicated, from solution A:

Solution B: 5 mg/mL

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

G into 15 mL centrifuge tubes labeled for both control and test samples (100 and 300 μg/mL). Step 10: Pipette 71 μl 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 μl of phosphate buffered saline (PBS) to the control samples and incubate as described above.

Step 11 : Place 3.0 mL aliquots of treated and untreated samples from Step 10 in covered 35 mm petri dishes and irradiate samples according to the experimental conditions as outlined in Phase IA.

Step 12: After irradiation, pour platelet samples into 5 mL test tubes and test control and treated samples for (1) cellular toxicity for viral assay system; and (2) viral interference for assay system. METHODS-PHASE II:

Selection of uniform UVA exposure area: Using the same area of uniform radiation distribution as in Phase I (i.e., Steps 1-7), prepare stock solution B.

Step 8: Preparation of Platelet Concentrates with HIV-1 for

UVA Irradiation

Add 8 mL of HIV- 1, at 2 x 10⁷ PFU of HIV-l/mL, to the remaining 132 mL of platelet concentrate from step 8 of Phase I such that the final HIV-1 titer is about 1.1 x 10⁶ (platelet-HIV suspension H). Divide platelet suspension H into the following aliquots for use in the different sample sets of the Phase II viral elimination studies:

27 mL of Suspension H for Sample Set 1

27 mL of Suspension H for Sample Set 2

39 mL of Suspension H for Sample Set 3

39 mL of Suspension H for Sample Set 4

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 Infectivity Assay:

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.

Buffer Toxicity /V iral Interference

Twenty-four well cluster plates are seeded with MT-4 cells in a total immunoassay for the detection of p24 antigen of HIV in plasma, serum or tissue culture media. This assay uses a murine monoclonal antibody (anti- HIV core antigen) coated onto microwell strips and binds the present antibody to the antibody-coated microwells. The bound antigen is recognized by biotinylated antibodies to HIV which react with conjugated streptavidin horseradish peroxidase, and develop color from the reaction of the peroxidase with hydrogen peroxide in the presence of

tetramethylbenzidine substrate. The intensity of the color developed is directly proportional to the amount of HIV antigen present in the sample. The p24 assay negative control is RPMI 1640 and the positive control is antigen reagent.

Culture fluid from each well is analyzed by the HIV p24 assay and the absorbance value is compared to the cut off value for a positive result. The cut off value for a positive result is determined by adding the mean absorbance value of the ELISA negative control to a predetermined factor of 0.055. The expected range of the cut off value is 0.055 to 0.155. If the absorbance value for the well exceeds the cut off value, then the well is considered positive for HIV p24 antigen. The level of HIV p24 in each well is not quantitated. The TCID_S0 of the sample is determined from the sum of the percentage of wells positive for HIV p24 antigen at each dilution using the standard formula, as stated above.

Materials

Positive Control and Spiking Virus: Human immunodeficiency virus type l

Strain: IIIB

Lot No.: VP012 H.1/8/93

Titer: 10^7.5 TCID₅₀/ml

Source: Advanced Biotechnologies, Inc.,

Columbia. Maryland volume of 1.0 ml/well. Each test dilution is inoculated into 3 wells at 0.1 ml/well and the cultures are incubated at 36°C ± 1 °C. Observations for cytotoxicity and, if necessary, an estimation of the percentage of cells affected in each culture are performed on day 5 and day 7 post-inoculation. Viral Inactivation Assay

The samples are spiked with Human Immunodeficiency Virus- 1. The spiked samples are then carried through the inactivation process. All samples are tested undiluted or diluted in RPMI medium (negative control) at various dilutions. Retained samples are stored frozen at -60°C or below. Titration of Samples for the Presence of HIV- 1

Twenty-four well cluster plates are seeded with MT-4 cells in a total volume of 1.0 ml/well. Ten fold serial dilutions are made in culture medium from the spiked sample or positive control,. At each dilution step, in quadruplicate, 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 TCID₅₀ On days 7, 14 and 28, 1.0 ml of each culture is removed for analysis by HIV-1 p24 antigen capture ELISA.

The formula for the final titer calculation of TCID₅₀ is based on the Karber method:

negative logarithm of the endpoint titer = A - (S₁/100-0.5) x B wherein, A = negative logarithm of the highest concentration inoculated, S, = sum of the percentage positive at each dilution, and B = log₁₀ (of the dilution factor). The values are then converted to TCID₅₀/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

Source: Microbiological Associates, Inc.,

Rockville, Maryland

Test System: MT-4 cells (L013-T)

Source: National Institute of Health,

Bethesda, Maryland (Human T cells isolated from a patient with adult T cell leukemia; HTLV-I transformed)

Results

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 log₁₀ plaque forming units (PFU)/mL. Photosensitizer is then added to the virus spiked plasma at either 100 or 300 μg/mL final concentration.

After a 15 minute room temperature incubation, samples of photosensitizer treated virus spiked plasma is placed in a ultraviolet (UV) irradiator and exposed to 24 J/cm² of UVA energy. Treated samples are then assayed for residual infectious virus by plaque assay. Virus reduction (V_R) is calculated by the equation

v_s-v_f=v_R

wherein, V_s is the starting virus titer, and V_f is the virus titer after treatment.

Example 3: Inactivation of Cytomegalovirus in Human Platelet

Concentrate

Inactivation of Cytomegalovirus (CMV) in human platelet

concentrate is conducted under ambient oxygen tension using a

photosensitizer and long wavelength ultraviolet radiation (UVA) at 22 ±

2°C. Dose response and kinetics studies are conducted in order to determine the optimal conditions for inactivation of CMV in human platelet concentration. Four to six logs of CMV virus are added to standard units of human platelet concentrate. The contaminated platelet concentrate is incubated at ambient non-UVA laboratory light for 60 ± 5 minutes with different concentrations of suspension F (100 - 300 μg/mL). Following incubation, the platelet concentrates are exposed to UVA at various fluences

(14 - 43 J/cm²). Inactivation of CMV virus is then evaluated by an infectivity assay using MRC-5 cells. Complete inactivation of CMV is obtained at 100 μg/mL using Photosensitizer B and a UVA fluence of 21.6 J/cm². Example 4: Inactivation of Vesicular Stomatitis Virus in Platelet

Concentrates

Inactivation of Vesicular Stomatitis (VSV) in human platelet concentrate is conducted under ambient oxygen tension using a

photosensitizer and long wavelength ultraviolet light (UVA) at 22 ± 2°C. Dose response and kinetics studies are conducted in order to determine the optimal conditions for inactivation of VSV in human platelet concentrate. Six logs of VSV are added to standard units of human platelet concentrate. The contaminated platelet concentrate is incubated at ambient non-UVA laboratory light for 10 ± 5 minutes with different concentrations of photosensitizer B (30 and 150 μg/mL). Following incubation, the platelet concentrate is exposed to UVA at various fluences (4.20-8.40 J/cm²).

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/cm².

Example 5: Herpes Simplex Virus Type 1 Inactivation in Calf Serum

Inactivation of Herpes Simplex Virus type 1 (HSV-1) in calf serum is conducted under ambient oxygen tension using a photosensitizer and long wavelength ultraviolet light (UVA) at 22 ± 2°C. Using a fixed

concentration of photosensitizer B (30 μg/mL) 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/cm²). 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/cm². Example 6: Measurement of Photosensitizer Mutagenicity by Ames

Mutagenicity Test

The Ames Mutagenicity test is based upon the use of five specially constructed strains of Salmonella typhimurium containing a specific mutation in the histidine operon. These genetically altered strains, TA98, TA100, TA1535, TA1537 and TA1538, cannot grow in the absence of histidine. When they are placed in a histidine-free medium, only those cells which mutate spontaneously back to their wild type state -- non-histidine- dependent by manufacturing their own histidine - are able to form colonies. The spontaneous mutation rate, or reversion rate, for any one strain is relatively constant, but if a mutagen is added to the test system, the mutation rate is significantly increased. Each test strain contains, in addition to a mutation in the histidine operon, two additional mutations that enhance sensitivity to some mutagens. The rfa mutation results in a cell wall deficiency that increases the permeability of the cell to certain classes of chemicals, for example, those chemicals containing large ring systems that are otherwise excluded. The second mutation is a deletion in the uvrB gene resulting in a deficient DNA excision-repair system. Test strains TA98 and TA100 also contain the pKM101 plasmid that carries the R-factor. It has been suggested that the plasmid increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process. TA98, TA1537 and TA1538 revert from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutations. TA100 reverts by both frameshift and base substitution mutations and TA1535 reverts only by substitution mutations.

EXPERIMENTAL DESIGN FOR AMES MUTAGENICITY TEST

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:

Based on the above assumptions, if a patient receives 5 Units of platelet concentrates the final concentration of photosensitizer B in the body is derived as follows:

A Salmonella/mammalian microsome mutagenicity test is conducted to determine whether a plasma test solution of photosensitizer B in platelet concentrate causes mutagenic changes in histidine-dependent mutant strains of Salmonella typhimurium. The Ames mutagenicity test system has been widely used as a rapid screening procedure for the determination of mutagenic and potential carcinogenic hazards of pure compounds, complex compounds and commercial products.

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 cm² 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. After exposure to photosensitizer B, the cells are microscopically examined at approximately 100 x, and scored for cytotoxic effects (CTE) at the end of the 24, 28 and 72 hours of incubation. Presence (+) or absence (-) of a confluent monolayer, vacuolization, cellular swelling and the percentage of cellular lysis are also recorded. CTE is scored as either Nontoxic (N), Intermediate (I) or Toxic (T). These data are shown in Table 2 and the evaluation criteria are shown below:

CTE SCORE MICROSCOPIC APPEARANCE OF CELLS

Nontoxic (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.

Intermediate (I) Cells may show marked vacuolization, crenation or swelling. Cytolysis (0-50%) of cells that results in "floating" cells and debris in the medium may be present. The remaining cells are still attached to the flask.

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.

Example 8: Measurement of Photosensitizer Cytotoxicity Using

Chinese Hamster Ovary (CHO) Hybridoma Cells and AE- L Cells

Chinese hamster ovary and AE-L cells are grown to confluency in 25 cm² culture flasks using sterile Eagles Minimum Essential Medium

(EMEM) supplemented with 2 mM L-glutamine, 1% proline and 5% calf serum treated with various concentrations of photosensitizer B (30-150 μg/mL) in the presence of UVA. 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 viability of CHO cells as well as the expression of rhCg proteins on recombinant CHO cell lines, is not affected. Note that upon UVA exposure of 30 minutes in the presence of 30 μg/mL of photosensitizer B, there are no adverse effects to the growth supporting functions of the treated sera or the expression of rhCg antigens.

Example 9: Synthesis of 3-Bromo-7-(r-Triethylammonium Propyloxy)

Coumarin Bromide (Photosensitizer A)

Place 15 g of 7-hydroxycoumarin, 15 g of potassium carbonate, 500 mL of tetrahydrofuran (THF) and 70 mL of 1,3-dibromopropane into a 1000 mL round bottom flask containing a 2.5 cm stirring bar. After stirring at reflux for 72-96 hours, the solution is filtered, the solids washed 4 times with 50 mL of dichloromethane and the combined filtrate concentrated by rotary evaporator. 50 mL of ethyl acetate is added to the concentrate followed by concentration under reduced pressure (25 in Hg.). Another 50 mL of ethyl acetate is added to the concentrate followed by filtration. The solids are washed 3 times with 10 mL of a 1:1 mixture of ethyl acetate and hexane. After drying, 15-20 g of the crude product is dissolved in 150-200 mL of dichloromethane and purified by flash chromatography (130-150 g SiO₂ (70 - 230 mesh), 35 mm O.D. column, approximately 60 cm in length), using dichloromethane as the eluting solvent. The fractions are collected in

50-250 mL beakers and monitored by TLC (developing solvent: 4:6 mixture of ethyl acetate.hexane). The fractions containing product are combined, concentrated by rotary evaporator and dried.

Synthesis of 3-bromo-7-(γ-bromopropyloxy)coumarin.

Place 13 g of 7-(γ-bromopropyloxy)coumarin and 120-150 mL of THF into a 500 mL round bottom flask containing a 2.5 cm stirring bar . When the 7-(γ-bromopropyloxy)coumarin is completely dissolved, 3 mL of bromine is added by syringe. After stirring for 2-5 hours at room

temperature, the solution is concentrated by rotary evaporator. 50 mL of a 1 : 1 mixture of ethyl acetate and hexane is added to the concentrate and the mixture is stirred for 30 minutes at room temperature. The solution is then filtered and the solids washed 3 times with 1 : 1 ethyl acetate and hexane and then dried for 2 hours. To obtain additional product, the filtrate is concentrated by rotary evaporator and 30 mL of a 1 :1 mixture of ethyl acetate and hexane is added to the concentrate. 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 SiO₂ (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.

Synthesis of 3-hromo-7-( γ-triethylammoniumpropyloxy) coumarin bromide.

Place 11 g of 3-bromo-7-(γ-bromopropyloxy)coumarin, 150-200 mL of THF and 60-70 mL of triethylamine into a 500 mL round bottom flask containing a 2.5 cm stirring bar . After stirring at reflux for 72-96 hours, the solution is filtered and the solids washed 3 times with 10 mL of acetone, 3 times with 10 mL of hexane and then dried for 1 hour. The product is transferred to a 600 mL beaker and 80 mL of acetone is added. The mixture is stirred for 30 minutes, filtered, washed 3 times with 10 mL of acetone and dried for 3-5 hours. The product is checked by TLC (ethyl acetate :hexane (4:6)).

It has been found that photosensitizer A fluoresces when treated with UV radiation. The absorption spectrum of photosensitizer A in water is shown in Figure 10. The fluorescence spectrum of photosensitizer A is shown in Figure 11.

Example 10: Measurement of Photosensitizer Migration in

Solution

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 μM photosensitizer solution either in phosphate buffered saline (PBS) or plasma. 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. Next, the solutions are removed from individual chambers and absorbance is determined at 350 nm using a spectrophotometer. For experiments involving liposomes, 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. Example 11: Irradiation of Platelet Concentrates and

Photosensitizer

Twenty-four hour old random donor platelet concentrates are obtained from American Associate of Blood Banks accredited blood banks.

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/cm² fluence. During the irradiation, samples are placed on a linear shaker. After UVA exposure, the samples are stored in a platelet incubator with shaking for an additional 4 days. During storage 3 mL aliquots from each unit are collected and subsequently analyzed for platelet in vitro properties.

Example 12: Comparison Study of Photosensitizer B, 8-Mop, and

Amt

Full units of one day old human platelet concentrate are collected in Cyrocyte bags (PL 269, Fenwal, Deerfield, IL) according to standard blood banking procedures (AABB Technical Manual (1989) 13th Ed., p. 136).

Platelet units are spiked with 6 logs of bacteriophage Φ6. Equimolar concentrations (60 μM) 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/cm². 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. In vitro platelet properties are evaluated using (1) aggregation response to collagen; (2) hypotonic shock response; and (3) morphology according to method described by Kunicki et al. ((1975) Transfusion 15:414). Data represent the mean ± standard deviation of n=3.

Example 13: Comparison Study of Photoinactivation of Sindbis

Virus in Human Plasma

A comparison study is performed to evaluate the viral inactivation properties of photosensitizers A, B, D, and E of the present invention. Also examined are non-halogenated forms of photosensitizer A (hereinafter referred to as photosensitizer AX), photosensitizer C (hereinafter referred to as photosensitizer CX), and photosensitizer D and E (hereinafter referred to as photosensitizer DX). The TC1D₅₀ 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 10⁷. 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/cm². 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.

Example 14: Synthesis of 3-Bromo-7-(T-Triethylamino 8-Methyl)

Coumarin (Photosensitizer D)

Preparation of 7-hydroxy-8-methyl coumarin

2-methyl resorcinol (.161 mmol, 20.024 g) and malic acid (.165 mmol, 22.129 g) are dissolved in concentrated sulfuric acid. The reaction mixture is stirred at 80°C for 24 h. The resulting solution is then poured over crushed ice, and the precipitate is collected by vacuum filtration. The precipitate is then washed with 5% NaHCO₃ and again collected by vacuum filtration yielding an orange-yellow solid in a 20.64% yield.

Preparation of 7-3'-bromopropyIoxy-8-methyl coumarin

7-hydroxy-8-methyl coumarin (0.286 mol, 5.045 g) and K₂CO₃ (.0317 mol, 4.379 g) are added to 50 ml of dibromopropane. The reaction is stirred at reflux for 24 h. The excess dibromopropane is removed by distillation. The remaining slurry is taken up in CHCl₃ and gravity filtered to remove the K₂CO₃. The CHCl₃ is dried and removed in vacuo. The final solid is washed with hexanes giving a pale-yellow product in a 76.0% yield. Preparation of 3 bromo-7-3'-bromopropyloxy-8-methyl coumarin

7-3'-bromopropyloxy-8-methyl coumarin (6.75 mmol, 2.01 g) is dissolved in THF and cooled to -76°C. Approximately 3 ml of Br₂ is slowly added to the solution. The mixture is stirred for 3h and then allowed to warm to room temperature. The resulting solution is dissolved in CHCl₃ and washed with a 10% Na₂S₂O₄ solution, a 10% NaHCO₃ solution and finally water.

The CHCl₃ is dried and removed in vacuo. The resulting pale-yellow solid is washed with hexanes and collected by vacuum filtration to give a quantitative yield. Preparation of 3-bromo-7-(γ-triethylamino-propyloxy)-8-methyl coumarin

7-3'-bromopropyloxy-8-methyl coumarin (0.17 mmol, 0.064 g) is dissolved in 30 ml of CHCl₃. Approximately 10 ml of triethylamine is added to the solution. By following TLC, the reaction requires refluxing for

48 h to ensure completion. The CHCl₃ and Et₃N are removed in vacuo. The precipitate is washed with hexane, ethyl acetate and acetone several times. The resulting white solid is dried under high vacuum and obtained in a 65.4% yield. Example 15: Inactivation of Parvovirus in Plasma

This set of experiments identifies the precise combination of factors and levels that optimize the conditions for the inactivation of Porcine Parvovirus (PPV, a small non-lipid enveloped single stranded DNA virus) in human plasma using different photosensitizers (photosensitizers B, A, D and E) in the presence of long wavelength ultraviolet radiation (UVA).

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

Family Parvoviridae

Genome ss DNA

Lipid Envelope None

Size (nm) 18-2 6 Shape Icosahedral

Resistance to Physioco-Chemical Reagents High

This study involves the increase of PPV susceptibility to inactivation using various photosensitizer and UVA combinations. Three different factors are manipulated to achieve such increased susceptibility , including pH (5.5), freeze-thaw cycle (-30°C), freeze-thaw duration (2 to 8°C) and low and high ionic strength. In order to obtain low and high ionic strength plasma, fresh frozen plasma is thawed at 4°C and then centrifuged to remove any cryo-precipitate. Following centrifugation, the resulting 40 mL of supernatant plasma is diluted with either distilled water or 2 M sodium chloride solution to obtain a low- and high ionic strength plasma

respectively. The following experimental conditions are employed: Sample Set 1: Normal Plasma Samples at pH 7.0 - 7.5

1. Plasma + PPV + pH 7 + Saline + ambient (50-60 minutes)

2. Plasma + PPV + pH 7 + 300 μg/mL + 30 J/cm²

Sample Set 2: Normal Plasma: Freeze - at -30°C and Thaw at

+37°c

3. Plasma + PPV + pH 5.5 + Saline + ambient (50-60 minutes)

4. Plasma + PPV + pH 5.5 + 300 μg/mL + FT + 30 J/cm²

Sample Set 3: Normal Plasma Diluted 1:1 with Saline at pH 5.5-

6.0

5. Plasma + PPV + pH 5.5 + Saline + ambient (50-60 minutes)

6. Plasma + PPV + pH 5.5 + 600 μg/mL Photosensitizer B + FT 4°C at

30 J/cm²

7. Plasma + PPV + pH 5.5 + 600 μg/mL Photosensitizer A + FT 4°C at 30 J/cm²

8. Plasma + PPV + pH 5.5 + 600 μg/mL Photosensitizer D + FT 4°C at 30 J/cm² 9. Plasma + PPV + pH 5.5 + 600 μg/mL Photosensitizer E + FT 4°C at 30 J/cm²

Sample Set 4: Normal Plasma Diluted 1:1 with Distilled Water

10. Plasma + PPV + pH 5.5 + 600 μg/mL Photosensitizer B + FT 4°C at 30 J/cm²

11. Plasma + PPV + pH 5.5 + 600 μg/mL Photosensitizer A + FT 4°C at 30 J/cm²

Sample Set 5: Normal Plasma Diluted 1:1 with 2 M Sodium

Chloride

12. Plasma + PPV + PH 5.5 + 600 μg/mL Photosensitizer B + FT 4°C at

30 J/cm²

13. Plasma + PPV + pH 5.5 + 600 μg/mL Photosensitizer A + FT 4°C at 30 J/cm²

Variables under Investigation

1. Concentration of Sensitizer (0, 300 and 600 μg/mL)

2. Temperature of processing (freeze-thaw: -30°C + 37°C vs. -30°C to + 4°C)

3. Ionic strength (Normal vs. Low vs. High ionic strength)

4. Types of Sensitizers (Photosensitizers B, A, D and E) Fixed Parameters

1. Volume of plasma for irradiation (5 mL)

2. Light source (UVA, 320-400 nm with peak intensity at 365 nm)

3. Post-Sensitizer incubation time (60 minutes)

a. Shaker speed: 70 ± 5 rpm b. Sample location: six samples

c. Temperature during irradiation (22°C-29°C) 4. UVA Fluence (30 J/cm²)

The selections of the variables under investigation are based on a preliminary review of the manufacturing processes for the various plasma fractions as well as current literature on the inactivation of non-enveloped viruses.

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

7, Tube 2 - pH 5.5). 1 unit of fresh frozen plasma is obtained and prepared according to the recommendation of the American Association of Blood Banks (AABB). This unit of fresh frozen plasma is placed in a 37°C waterbath and allowed to thaw.

The thawed plasma is centrifuged at 3000 g for 20 minutes. Using a

50 mL pipette, all the supernatant is transferred into a sterile 500 mL plastic bottle. With a 50 mL syringe, 40 mL aliquots of plasma are transferred into tubes 1 and 2. The pH of the plasma in Tube 1 is adjusted to a value between 7 and 7.5 by dropwise addition of 2 M hydrochloric acid.

Similarly, the pH of plasma in Tube 2 was adjusted to a value between 5.5 and 6.

Sample Set 1 9 mL of pH 7 plasma into Tube 1

Sample Set 2 9 mL of pH 5.5 plasma into Tube 2

Sample Set 3 12 mL of pH 5.5 plasma into Tube 3

Sample Set 4 4.5 mL of pH 5.5 plasma into Tube 4

Sample Set 5 4.5 mL of pH 5.5 plasma into Tube 5

12 mL of 0.9% sodium chloride solution is added to tube 3 sample set 3; 4.5 mL of sterile distilled water to tube 4 sample set 4; and 4.5 mL of 2 M sodium chloride solution to tube 5 sample set 5. Each tube is agitated to ensure adequate mixing of the contents of the tubes. 1 mL of parvovirus (PPV) is added to tubes 1, 2, 4 and 5 for sample sets 1, 2, 4 and 5, respectively; 2.67 mL of PPV to tube 3, sample set 3. The contents of each tube are agitated to ensure homogenous dispersion of the virus. Thirteen 50 mL sterile plastic tubes are labeled 1-12 corresponding to the samples outlined above. 5 mL of PPV-contaminated plasma is transferred into the labeled tubes accordingly:

-5 mL of PPV-Plasma is transferred from sample set 1 into tubes 1 and 2

-5 mL of PPV-Plasma is transferred from sample set 2 into tubes 3 and 4

-5 mL of PPV-Plasma is transferred from sample set 3 into tubes 5 and 9

-5 mL of PPV-Plasma is transferred from sample set 4 into tubes 10 and 11

-5 mL of PPV-Plasma is transferred from sample set 5 into tubes 12 and 13

Saline or photosensitizer solution is added accordingly:

-1 mL of 0.9% sodium chloride solution to sample tube 1

-1 mL of 1.8 mg/mL of Photosensitizer B solution to sample tube 2 -1 mL of 0.9% sodium chloride solution to sample tube 3

-1 mL of 1.8/mg/mL of Photosensitizer B solution to sample tube 4 -1 mL of 0.9% sodium chloride solution to sample tube 5

-2.5 mL of 1.8 mg/mL of Photosensitizer B solution to sample tube 6

-2.5 mL of 1.8 mg/mL of Photosensitizer A solution to sample tube 7 -2.5 mL of 1.8 mg/mL of Photosensitizer D solution to sample tube 8 -2.5 mL of 1.8 mg/mL of Photosensitizer E solution to sample tube 9 -2.5 mL of 1.8 mg/mL of Photosensitizer B solution to sample tube

10

-2.5 mL of 1.8 mg/mL of Photosensitizer A solution to sample tube

11

-2.5 mL of 1.8 mg/mL of Photosensitizer B solution to sample tube

12

-2.5 mL of 1.8 mg/mL of Photosensitizer A solution to sample tube

13

All samples are placed on a shaker and incubated at room

temperature (22-24 °C) for 60 minutes. At the end of the incubation period, samples 4, 6-13 are frozen at temperatures between -20°C and -40°C.

Sample 4 is thawed at 37°C (about 10-15 minutes at 37°C) and samples 6- 13 at temperatures between 2 and 8°C. Freeze-thaw cycles are repeated 10 times over 24 hour period. At completion of the freeze-thaw cycles, 6 mL of each sample were exposed to UVA at 30 J/cm². At the end of the 60 minute incubation, 6 mL of the contents from tubes 1, 2, 3 and 5 are transferred to appropriately labeled petri dishes for photoinactivation treatment. The results of this experiment are shown in Table 17.

While the above description contains many specificities, these specificities should not be construed as limitations on the scope of the invention, but rather an exemplification of the preferred embodiment thereof. That is to say, the foregoing description of the invention is exemplary for purposes of illustration and explanation. Without departing from the spirit and scope of this invention, one skilled in the art can make various changes and modifications to the invention to adapt it to various usages and conditions. As such, these changes and modifications are properly, equitably, and intended to be within the full range of equivalence of the following claims. Thus, the scope of the invention should be determined by the appended claims and their legal equivalents, rather than by the examples given.

l

Claims

WE CLAIM:

1. A method of inactivating viral, bacterial and other parasitic contamination from a biological solution, comprising the steps of:

a. mixing said biological solution with a photosensitizer capable of binding to said viral, bacterial, or parasitic contaminant, wherein said photosensitizer is capable of inactivating said viral, bacterial or parasitic contaminant upon absorption of radiation; and

b. irradiating said mixture of said biological solution and said photosensitizer under conditions whereby substantially all of said viral, bacterial and parasitic contamination are inactivated and the physiological activity of said composition is substantially unimpaired.

2. The method of claim 1 wherein said biological solution is comprised of a component selected from the group consisting of blood, a blood component, a cell culture and a component of a cell culture.

3. The method of claim 1 wherein said biological solution is comprised of whole plasma.

4. The method of claim 1 wherein said biological solution is comprised of a cellular fraction prepared from whole blood.

5. The method of claim 4 wherein said cellular fraction prepared from whole blood is selected from the group consisting of red blood cells, platelets, white blood cells and stem cells.

6. The method of claim 1 wherein said biological solution is comprised of plasma protein fractions.

7. The method of claim 6 wherein said plasma protein fraction is selected from the group consisting of serum albumin, immune globulins and clotting factor.

8. The method of claim 1 wherein said photosensitizer is comprised of a lipophilic moiety, a hydrophilic moiety and a photoreactive moiety.

9. The method of claim 8 wherein said lipophilic moiety comprises an intercalator, said hydrophilic moiety comprises an ammonium or

phosphonium group and said photoreactive moiety comprises a halogen.

10. The method of claim 1 wherein said photosensitizer is an intercalator comprising at least one halogen atom.

11. The method of claim 1 wherein said photosensitizer is an intercalator comprising at least one non-hydrogen bonding ionic moiety.

12. The method of claim 1 wherein said photosensitizer is an intercalator comprising at least one halogen atom and at least one non-hydrogen bonding ionic moiety.

13. The method of claim 12 wherein said photosensitizer is an

intercalator comprising one halogen atom selected from the group consisting of F, Cl, Br and I.

14. The method of claim 12 wherein said photosensitizer is an

intercalator comprising one non-hydrogen bonding ionic moiety selected from the group consisting of ammonium and phosphonium.

15. The method of claim 12 wherein said photosensitizer has a psoralen chemical backbone structure.

16. The method of claim 12 wherein said photosensitizer has a coumarin chemical backbone structure.

17. The method of claim 1 wherein said photosensitizer is selected from the group of compounds of the formula:

wherein μ is an integer from 1 to 6; X is an anionic counterion; Z is N or P; R₁, R₂, R₃, R₄, R₅ and R₆ are independently halo; H; linear or branched alkyl of 1-10 carbon atoms; linear or branched alkoxy of 1-10 carbon atoms;

(CH₂)_pZ•R^,,R%R'" or -O(CH₂)_nZ•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 by H; and at least on one of R₁, R₂, R₃, R₄, R₅ or R₆ is (CH₂)_m O(CH₂)_pZ•R',R",R'" or -

O(CH₂)_nZ•R',R",R"'.

18. The method of claim 17 wherein R₄

is -O(CH₂)_nN•R',R",R"'.

19. The method of claim 17 wherein R₆, R₅, R₂ and R₁ are H and R₃ is a halogen atom.

20. The method of claim 17 wherein said photosensitizer is comprised of the formula:

wherein X is selected from the group consisting of F, Cl, Br and I.

21. The method of claim 1 wherein said photosensitizer is selected from the group of compounds of the formula:

wherein μ is an integer from 1 to 6; X is an anionic counterion; Z is N or P; R₁, R₂, R₃, R₄, R₅ and R₆ are independently halo; H; linear or branched alkyl of 1-10 carbon atoms; linear or branched alkoxy of 1-10 carbon atoms;

(CH₂)_PZ•R',R",R'" or -O(CH₂)_nZ•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 by H; and at least on one of R₁, R₂, R₃, R₄, R₅ or R₆ is (CH₂)_m O(CH₂)_pZ•R',R",R"' or -

O(CH₂)_nZ•R',R",R'".

22. The method of claim 21 wherein R₂ is

OCH₂CH₂CH₂N'(CH₂CH₃)₃.

23. The method of claim 21 wherein R₆ is selected from the group consisting of F, Cl, Br and I.

24. The method of claim 21 wherein said photosensitizer is comprised of the formula:

wherein X is selected from the group consisting of F, Cl, Br and I; and Y is H or CH₃.

25. The method of claim 1 wherein said photosensitizer is a fluorescent intercalator comprising at least one halogen atom.

26. The method of claim 25 wherein said photosensitizer has a psoralen chemical backbone structure.

27. The method of claim 25 wherein said photosensitizer has a coumarin chemical backbone structure.

28. The method of claim 25 wherein said photosensitizer is further comprised of at least one non-hydrogen bonding ionic moiety.

29. The method of claim 25 wherein said photosensitizer is selected from the group of compounds of the formula:

wherein μ is an integer from 1 to 6; X is an anionic counterion; Z is N or P; R₁, R₂, R₃, R₄, R₅ and R₆ are independently halo; H; linear or branched alkyl of 1-10 carbon atoms; linear or branched alkoxy of 1-10 carbon atoms; (CH₂)_PZ•R',R",R'" or -O(CH₂)_nZ•R',R",R'" wherein n, m and p are independently integers from 1 to 10 and R',R", and R'" arc independently II 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 by H; and at least on one of R₁, R₂, R₃, R,, R₅ or R₆ is (CH₂)_m O(CH₂)_pZ•R',R",R"' or - O(CH₂)_nZ•R',R",R"'.

30. The method of claim 25 wherein said photosensitizer is comprised of the formula:

wherein X is selected from the group consisting of F, Cl, Br and I; and Y is

H or CH₃.

31. The method of claim 1 wherein said contaminant is now an enveloped virus and further comprising the step of:

adjusting the operating conditions of said mixture in order to increase the permeability of the capsid of said viral contaminants prior to irradiation of said mixture.

32. The method of claim 31 wherein said operating condition adjusted is the ionic strength of said mixture.

33. The method of claim 31 wherein said operating condition adjusted is the pH of said mixture.

34. The method of claim 31 wherein said operating condition adjusted is the addition of solvent detergent.

35. The method claim 31 wherein said operating condition adjusted is the addition of a chaotrophic agent.

36. The method of claim 31 wherein said operating condition adjusted is the addition of a reducing agent.

37. The method of claim 31 wherein said operating condition adjusted is the performance of freeze-thaw cycles of said biological solution prior to or subsequent to mixing with said photosensitizer.

38. The method of claim 31 wherein said operating condition adjusted is the temperature of said mixture.

39. The method of claim 31 wherein said operating condition adjusted is the osmolality of said mixture.

40. The method of claim 31 wherein said operating condition adjusted is the addition of one or more organic solvents.

41. The method of claim 31 wherein said operating condition adjusted is the addition of one or more polyols.

42. A method of inactivating non-enveloped viral contamination from a biological solution, comprising the steps of:

a. mixing said biological solution with a photosensitizer capable of binding to said non-enveloped viral contaminant, wherein said

photosensitizer is capable of inactivating said non-enveloped viral contaminant upon absorption of radiation;

b. adjusting the operating conditions of said mixture of said biological solution and said photosensitizer in order to increase the permeability of the capsid of said viral contaminant; wherein said operating conditions adjusted are selected from the group consisting of ionic strength of said mixture, the pH of said mixture, the addition of solvent detergent, the addition of a chaotrophic agent, the performance of freeze-thaw cycles, the temperature of said mixture and the osmolality of said mixture; and c. irradiating said mixture of said biological solution and said photosensitizer under conditions whereby substantially all of said non- enveloped viral contaminants are inactivated and the physiological activity of said composition is substantially unimpaired.

43. The method of claim 42 wherein said biological solution is comprised of a component selected from the group consisting of blood, a blood component, a cell culture and a component of a cell culture.

44. The method of claim 42 wherein said biological solution is comprised of a plasma protein fraction.

45. The method of claim 44 wherein said plasma protein fraction is selected from the group consisting of serum albumin, immune globulin and clotting factor.

46. The method of claim 42 wherein said photosensitizer has a chemical backbone less hydrophilic than psoralen.

47. The method of claim 46 wherein said photosensitizer contains a halogen atom.

48. The method of claim 46 wherein said photosensitizer has a coumarin chemical backbone.

49. The method of claim 1 wherein said viral contaminant is HIV- 1.

50. The method of claim 49 wherein said biological solution is human platelet concentrate.

51. The method of claim 1 wherein said viral contaminant is Sindbis Virus.

52. The method of claim 51 wherein said biological solution is human plasma.

53. The method of claim 1 wherein said viral contaminant is

Cytomegalovirus.

54. The method of claim 53 wherein said biological solution is human platelet concentrate.

55. The method of claim 1 wherein said viral contaminant is Vesicular Stomatitis.

56. The method of claim 55 wherein said biological solution is human platelet concentrate.

57. The method of claim 1 wherein said viral contaminant is Herpes

Simplex Virus.

58. The method of claim 1 wherein said viral contaminant is Parvovirus.

59. A photosensitizer capable of binding viral, bacterial, or parasitic contaminants in a biological solution, and further capable of inactivating said viral, bacterial or parasitic contaminant upon irradiation without substantially impairing said biological solution, comprised of the formula:

/

wherein μ is an integer from 1 to 6; X is an anionic counterion; Z is N or P; R₁, R₂, R₃, R₄, R₅ and R₆ are independently halo; H; linear or branched alkyl of 1-10 carbon atoms; linear or branched alkoxy of 1-10 carbon atoms; (CH₂)_PZ•R',R",R'" or -O(CH₂)_nZ•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 by H; and at least on one of R₁, R₂, R₃, R₄, R₅ or R₆ is (CH₂)_m O(CH₂)_pZ•R',R",R"' or - O(CH₂)_nZ•R',R",R"'.

60. The photosensitizer of claim 59 wherein R₆ is selected from the group consisting of F, Cl, Br and I.

61. The photosensitizer of claim 60 wherein R₆ is Br.

62. The photosensitizer of claim 60 wherein R₆ is Cl.

63. The photosensitizer of claim 59 wherein R₂ is

OCH₂CH₂CH₂N•(CH₂CH₃)₃ Br.

64. The photosensitizer of claim 59 wherein R₁ is CH₃ or H.

65. The photosensitizer of claim 59 having the formula:

66. The photosensitizer of claim 59 having the formula:

67. The photosensitizer of claim 59 having the formula:

68. The photosensitizer of claim 59 having the formula:

69. A photosensitizer capable of binding viral, bacterial, or parasitic contaminants in a biological solution, and further capable of inactivating said viral, bacterial or parasitic contaminant upon irradiation without substantially impairing said biological solution, comprised of:

a. an intercalating chemical backbone structure;

b. at least one halogen atom; and

c. at least one non-hydrogen bonding ionic moiety.

70. The photosensitizer of claim 69 wherein said chemical backbone structure is psoralen.

71. The photosensitizer of claim 69 wherein said chemical backbone structure is coumarin.

72. The photosensitizer of claim 69 wherein said non-hydrogen bonding ionic moiety is ammonium or phosphonium.

73. A photosensitizer capable of binding viral, bacterial, or parasitic contaminants in a biological solution, and further capable of inactivating said viral, bacterial or parasitic contaminants upon irradiation without substantially impairing said biological solution, comprised of:

a. an intercalating chemical backbone structure; and

b. at least one halogen atom;

wherein said photosensitizer is fluorescent.

74. The photosensitizer of claim 73 further comprising at least one non- hydrogen bonding ionic moiety.

75. The photosensitizer of claim 74 wherein said non-hydrogen bonding ionic moiety is ammonium or phosphonium.

76. The photosensitizer of claim 73 wherein said chemical backbone structure is psoralen.

77. The photosensitizer of claim 73 wherein said chemical backbone structure is coumarin.