WO2001049328A1

WO2001049328A1 - Photodynamic inactivation of pathogens in blood by phenothiazines and oxygen

Info

Publication number: WO2001049328A1
Application number: PCT/US2000/035435
Authority: WO
Inventors: Stephen J. Wagner; Andrey Skripchenko; Leonard I. Friedman
Original assignee: The American National Red Cross
Priority date: 2000-01-05
Filing date: 2000-12-28
Publication date: 2001-07-12
Also published as: AU2461001A

Abstract

The invention provides a method for reducing the level of active pathogenic contaminants, such as viruses, bacteria and parasites, found in whole blood and blood components. The method comprising adding to whole blood or blood components at least one phenothiazin-5-ium dye in an amount effective to reduce the level of active pathogenic contaminants and irradiating the whole blood or blood components with light of an appropriate intensity and wavelength for a time sufficient to reduce the level of the active pathogenic contaminants in the whole blood or blood components, wherein the partial pressure of oxygen in the whole blood or blood components is maintained at a level during the irradiation such that the amount of active pathogenic contaminants in the whole blood or blood components is reduced.

Description

PHOTODYNAMIC INACTIVATION OF PATHOGENS IN BLOOD BY PHENOTHIAZINES AND OXYGEN

Background of the Invention

Field of th e In vention

This invention is directed to methods for reducing the level of active pathogenic contaminants, such as viruses, bacteria and parasites, frequently found in whole blood and blood components.

Related Art Among the risks inherent in handling or being transfused with blood, blood proteins or other blood components is the risk of infection from pathogenic contaminants, including, e.g., human immunodeficiency virus (HIV), serum hepatitis, cytomegalovirus, Epstein-Barr virus, herpes simplex, infectious mononucleosis, syphilis and malaria. Virucidal methods, including heat, solvent- detergent and gamma irradiation, have been used to produce non-infectious plasma derivatives, but such methods are generally ineffective or too harsh to be routinely used for effective decontamination of whole blood, red cells and/or platelets. Indeed, any virucidal treatment that damages or introduces harmful or undesirable contaminants into the product is unsuitable for decontamination of a product intended for transfusion into an animal, particularly a human. Due to the critical need for transfusable red blood cells, it is of great importance to develop efficient methods that can be readily used to decontaminate cellular blood components and whole blood without substantially or irreversibly altering or harming them. Efficient decontamination treatments that inactivate contaminating pathogens but do not harm the cellular fractions of blood are not readily available. Common decontamination treatments include the use of photo sensitizers, which in the presence of dissolved oxygen and upon exposure to light that includes wavelengths absorbed by the photosensitizer, inactivate viruses (EP 0 196 515). Typically such photochemicals are dyes or other compounds that readily absorb UV or visible light in the presence of dissolved oxygen. These compounds include merocyanine 540 ("MC 540") (U.S. Patent No. 4,775,625), porphyrin derivatives (U.S. Patent Nos. 5,536,238 and 4,878,891) as well as other photosensitizers. Red blood cell decontamination methods using such photochemicals have previously encountered a problem due to the absorbency, by hemoglobin, of light at wavelengths necessary to activate such compounds. As such, increased virucidal activity of these compounds is realized when the absorption spectrum of the photosensitizer does not significantly overlap the absorption spectra of pigments present in the blood, such as hemoglobin.

It is well known that viruses, such as hepatitis or HIV virus, may be resident within human blood. The viruses residing in blood may be "intracellular," i.e., contained within one of the cellular components of blood, such as white blood cells, or they may be "extracellular," i.e., freely existing in the plasma. Regardless of where the virus resides, the presence of the virus in the bloodstream poses the risk of infection and disease not only to the host, but also, if the blood or a blood component is collected and transfused, to a recipient. It is therefore preferable if the photodynamic treatment inactivates both extracellular and intracellular viruses as well as proviruses. In order to minimize cellular damage, it is preferable that the photosensitizer be non-toxic to cellular blood components and selectively bind to a component of the virus either that is not present in red cells or platelets or, if present therein, that is not essential to red cells' or platelets' function. For example, because blood platelets, erythrocytes and plasma proteins do not contain genomic nucleic acids, compounds that specifically target nucleic acids would desirably result in the targeting of viruses. White blood cells do contain nucleic acids and, therefore, will also be targeted by these compounds. However, since many of these white blood cells may be infected with unwanted viruses and white blood cells are typically filtered from blood products before being administered to patients, the targeting of white blood cells by such compounds is not undesirable. It is further preferable that the virucidal activity of the photosensitizer be uninhibited by the presence of plasma proteins, such as coagulation proteins, albumin and the like.

Treatment with known photochemicals, however, frequently does damage cellular blood components. For example, photochemicals such as the porphyrins

(U.S. Patent No. 4,878,891) and MC 540 (U.S. Patent No. 4,775,625) cause membrane damage in the presence of light and oxygen, which significantly reduces the survivability of the phototreated red cells during storage. Similarly, treatment of red blood cells using phthalocyanine 4 with type 1/type 2 quenchers caused red cell damage even under optimized conditions — about 2% of the cells hemolyze after 21 days of storage (the current FDA guideline for hemolysis is < 1% after 6 weeks of storage at 1-6°C) (Transfusion 35:361-10 (1995)).

Additionally, both MC 540 and porphyrin derivatives apparently bind to blood components, such as albumin (Transfusion 29A2S (1989); Biochim. Biophys. Ada 7255:428-436 (1995)). For example, the effect of MC 540 on platelets and the influence of albumin on MC 540's virucidal activity has been studied. In the presence of light and MC 540, the platelets aggregated. Albumin, however, prevented aggregation and inhibited the inactivation of viral contaminants by MC 540 plus light. Similarly, because of such competitive inhibition reactions with blood and/or plasma components, some dyes are not suitable for decontaminating blood, cellular blood components, or any blood- derived products containing high plasma concentrations (as plasma concentration increases, the percentage of viral inactivation substantially decreases).

The phenothiazin-5-ium dyes such as methylene blue, toluidine blue O, thionine, azure A, azure B, and azure C have been shown to inactivate animal viruses (U.S. Patents Nos. 4,407,282, 4,402,318, 4,305,390 and 4,181,128). Light-induced activation of photochemical agents such as methylene blue is believed to result in the production of singlet oxygen which enhances the reactions that inactivate virus. One target for virus inactivation is viral nucleic acids (Abe et al, Photochem. Photobiol. 67:402-409 (1995)). Methylene blue and visible light damage guanine residues of nucleic acids (Simon et al, J. Mol. Biol 4:488-499 (1962)), and methylene blue and white light produce 8-hydroxy- guanine in DNA (Floyd et al, Arch. Biochim. Biophys. 275:106-1 11 (1989)). Based on this activity, phenothiazin-5-ium dyes have been employed for inactivation of extracellular enveloped viruses in blood and blood components

(U.S. Patent No. 5,545,516) because the dyes absorb light at wavelengths that are not substantially absorbed by hemoglobin.

These particular phenothiazin-5-ium dyes, however, have certain drawbacks that limit their usefulness for inactivating pathogens in whole blood or blood components. For example, red cells readily take up or bind such dyes

(Sass et al, J. Lab. Clin. Med. 75:744-752 (1969)). In addition, photosensitized oxidation of biological membranes is deleterious to membrane structure and function (methylene blue cross-links the membrane protein, spectrin, in red cells exposed to visible light and oxygen) (Girotti, Biochim. Biophys. Ada. (502:45-56 (1980)). Also, methylene blue treated red blood cells have been shown to bind to plasma proteins, such as IgG and albumin (Wagner etal. , Transfusion 55:30-36 (1992)).

As described in U.S. Serial No. 009,892, issued as U.S. Patent No. 6,030,767 and assigned to the assignee of the present application, other specific phenothiazin-5-ium dyes such as dimethylmethylene blue have been shown to possess highly effective pathogen inactivation activity in red blood cells. For a given fixed set of experimental conditions, however, the extent of virus inactivation has been found to be variable between different blood units, even though for a given blood unit, the extent of inactivation is highly reproducible. While the prior art methods represent progress in the inactivation of contaminants, such as viruses, in biological fluids, such as blood or blood components, there is still room for improvements. For example, one of the prime areas of concern is to better assure reproducible and efficient inactivation of a substantial portion of the pathogenic contaminant with the goal of 100% inactivation. In addition, it is desirable that exposure of the phenothiazin-5-ium dye-containing whole blood or blood components to a light source provide maximum viral inactivation with minimal damage to the blood components. Also, it is desirable that exposure of the whole blood or blood components to a light source be of short duration to increase efficiency of treatment and reduce the cost. Consequently, there remains an acute need for an efficient, safe, effective, reproducible and rapid method for reducing the level of active pathogenic contaminants, particularly HIV and hepatitis, in whole blood or blood components without rendering the blood or blood components unsuitable for transfusion.

Summary of the Invention

Accordingly, the present invention provides a reproducible and rapid method of reducing the level of both intracellular and extracellular active pathogenic contaminants in whole blood and blood components, including cellular blood components and liquid blood components, with increased efficiency and minimal or no damage to the blood components. Other features and advantages of the present invention will be set forth in the detailed description of preferred embodiments that follows, and in part will be apparent from the description or may be learned by practice of the invention. These advantages of the invention will be realized and attained by the methods particularly pointed out in the written description and claims hereof.

It has been found that the rate of virus photoinactivation in red cell suspensions is directly related to the partial pressure of oxygen. Although it is generally known in the art that the photodynamic action of phenothiazine dyes such as methylene blue or dimethylmethylene blue requires the presence of dissolved oxygen for biological activity via singlet oxygen production, it has been assumed that red blood cell suspensions possess sufficient oxygen for maximal generation of singlet oxygen. However, it has been unexpectedly discovered that there is a direct correlation between the kinetics of virus inactivation and the partial pressure of oxygen.

Therefore, one embodiment of the present invention is directed to a method for decontaminating whole blood or blood components which comprises adding to whole blood or blood components at least one phenothiazin-5-ium dye in an amount effective to reduce the level of active pathogenic contaminants and irradiating the phenothiazin-5-ium dye-containing whole blood or blood components with light of an appropriate intensity and wavelength for a time sufficient to reduce the level of active pathogenic contaminants therein, wherein the partial pressure of oxygen in the whole blood or blood components is maintained at a level during the irradiating of the whole blood or blood components such that the amount of active pathogenic contaminants is reduced.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the invention as claimed.

Brief Description of the Figures

Figure 1 displays the oxygen dependence of Vesicular Stomatitis Virus (VSV) inactivation. The mean log₁₀ inactivation of VSV is plotted as a function of mean oxygen partial pressure.

Detailed Description of th e Preferred Embodiments

Definitions

Unless defined otherwise, all technical and scientific terms used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the relevant art. All patents and publications mentioned herein are expressly incorporated by reference. As used herein, the term "pathogenic contaminant of whole blood or blood components" is intended to mean a contaminant that, upon handling or transfusion into a recipient may cause disease in the handler or recipient, respectively. Examples of such pathogens include, but are not limited to, retroviruses, such as HIV, hepatitis viruses and other pathogenic viruses not yet known.

As used herein, the term "blood components" is intended to mean one or more of the components that may be separated from whole blood and include, but are not limited to: cellular blood components, such as red blood cells and platelets; blood proteins, such as blood clotting factors, enzymes, albumin, plasminogen, and immunoglobulins; and liquid blood components, such as plasma and plasma-containing compositions.

As used herein, the term "cellular blood component" is intended to mean one or more of the components of whole blood that comprises cells, such as red blood cells or platelets.

As used herein, the term "blood protein" is intended to mean one or more of the proteins that are normally found in whole blood. Illustrative examples of blood proteins found in mammals (including humans) include, but are not limited to, coagulation proteins (both vitamin K-dependent, such as Factor VII or Factor IX, and non-vitamin K-dependent, such as Factor VIII and von Willebrands factor), albumin, lipoproteins (high density lipoproteins and/or low density lipoproteins), complement proteins, globulins (such as immunoglobulins IgA, IgM, IgG and IgE), and the like.

As used herein, the term "liquid blood component" is intended to mean one or more of the fluid, non-cellular components of whole blood, such as plasma

(the fluid, non-cellular portion of the blood of humans or animals as found prior to coagulation) or serum (the fluid, non-cellular portion of the blood of humans or animals after coagulation).

As used herein, the term "composition containing a cellular blood component and/or a blood protein" is intended to mean a composition that contains a biologically compatible solution, such as ARC-8, Nutricell (AS-3), ADSOL (AS-1) or Optisol (AS-5), and one or more cellular blood components, one or more blood proteins, or a mixture of one or more cellular blood components and/or one or more blood proteins. Such compositions may also contain a liquid blood component, such as plasma.

Compositions containing a cellular blood component and/or a blood protein may optionally be leukodepleted. As used herein, the term "leukodepleted" is intended to mean that the concentration of leukocytes in the composition has been reduced by a specified amount, such as a factor of 10⁵. It is not necessary that compositions be leukodepleted before application of the methods of the present invention.

As used herein, a "transfusable composition" is intended to mean a composition that can be transfused into the blood stream of a mammal. Transfusable compositions may contain whole blood, one or more blood components, such as one or more cellular blood components, one or more blood proteins, and one or more liquid blood components, or mixtures of whole blood and one or more blood components, such as red blood cells, clotting factors or plasma.

As used herein, "decontamination" is intended to mean a process whereby the level of active pathogens, such as viral or bacterial contaminants, in a given composition is reduced. Such reduction may occur by rendering the pathogens inactive and/or noninfectious or by reducing the number of pathogens in the composition. A composition containing whole blood or a blood component that has been "decontaminated" can be transfused or manipulated without harming or infecting anyone exposed thereto.

As used herein, the term "pathogen" is intended to mean any replicable agent that may be found in or infect whole blood or blood components. Such pathogens include the various viruses, bacteria, and parasites known to those of skill in the art to generally be found in or infect whole blood or blood components. Illustrative examples of such pathogens include, but are not limited to: bacteria, such as Streptococcus species, Escherichia species and Bacillus species; viruses, such as human immunodeficiency viruses and other retro viruses, heφes viruses, paramyxoviruses, cytomegaloviruses, hepatitis viruses (including hepatitis A, hepatitis B, and hepatitis C), pox viruses and toga viruses; and parasites, such as malarial parasites, including Plasmodium species, and trypanosomal parasites.

The ratio of the titer of the control sample to the titer of virus in each of the treated samples is a measure of viral inactivation. As used herein, the term "log₁₀ inactivation" is intended to mean the log₁₀ of this ratio. Typically, a log₁₀ inactivation of at least about 4 indicates that the treated sample has been decontaminated.

As used herein, the term "fluence" is intended to mean a measure of the energy per unit area of sample and is typically measured in joules/cm² (J/cm²). As used herein, the term "fluence rate" is intended to mean a measure of the amount of energy that strikes a given area of a sample in a given period of time and is typically measured as milliwatts (mW)/cm² or as joules/cm² per unit time of exposure.

As used herein, the term "appropriate wavelength and intensity" is intended to mean light of a wavelength and intensity that can be absorbed by the dye, but does not damage the blood or blood components present. It is well within the level of skill in the art to select such wavelength and intensity empirically based on certain relevant parameters, such as the particular dye employed and its concentration in the composition.

As used herein, the term "phenofhiazin-5-ium dye" is intended to mean a compound having the general structure

which is soluble in aqueous solutions and also capable in sufficient amounts to reduce the level of active pathogenic contaminants in whole blood or blood components upon irradiation with light of a suitable intensity and wavelength. This class of compounds includes, but is not limited to, 1,9- dimethylmethylene blue, methylene blue, toluidine blue O, thionine, azure A, azure B and azure C. One skilled in the art may determine the suitability of a particular substituent group or groups empirically using any of the standard assays for determining the level of active intracellular pathogenic contaminants.

Illustrative examples of organic moieties include, but are not limited to, alkyl groups, alkenyl groups, alkynyl groups, hydroxy groups, methoxy groups, alkoxy groups, aryl groups, heteroaryl groups, aryloxy groups, heteroaryloxy groups, nitro groups, amine groups, amide groups, alkoxycarbonyl groups, alkylcarboxyl groups, arylcarboxyl groups, aralkyl groups, cyano groups, azide groups, haloalkyl groups, haloaryl groups. Preferable organic moieties include alkyl groups, such as methyl, ethyl, and propyl, alkenyl groups, such as ethenyl, alkynyl groups, such as acetenyl, and amine groups, such as monomethylamine and dimethylamine.

Illustrative examples of suitable inorganic moieties include, but are not limited to, sulfur, selenium and tellurium. Preferred inorganic moieties include sulfur and selenium.

As used herein, the term "alkyl group" is intended to mean a straight or branched chain hydrocarbon radical having from 1 to 10 carbon atoms, preferably 1 to 5 carbon atoms, and more preferably 1 or 2 carbon atoms, such as methyl, ethyl and the like. As used herein, the term "alkenyl group" is intended to mean a straight or branched chain hydrocarbon radical having from 2-10 carbon atoms and at least one double bond, such as ethenyl or propenyl and the like.

As used herein, the term "alkynyl group" is intended to mean a straight or branched chain hydrocarbon radical having from 2-10 carbon atoms and at least one triple bond, such as ethynyl or propynyl and the like. As used herein, the term "aryl group" is intended to mean a monocyclic or bicyclic aromatic hydrocarbon radical having from 6-12 carbon atoms in the ring(s), such as phenyl or naphthyl and the like.

As used herein, the term "aralkyl group" is intended to mean a straight or branched chain hydrocarbon radical having from 1 to 6 carbon atoms bound to a monocyclic or bicyclic aromatic hydrocarbon radical having from 6-12 carbon atoms in the ring(s), such as benzyl or 2-phenylethyl and the like.

As used herein, the term "heteroaryl group" is intended to mean a monocyclic or bicyclic aromatic radical having from 4-1 1 carbon atoms and at least one heteroatom (i. e. an oxygen atom, a nitrogen atom and/or a sulfur atom) in the ring(s), such as thienyl, furyl, pyranyl, pyridyl, quinolyl and the like.

As used herein, the term "effective amount" is intended to mean an amount sufficient to provide a concentration of dye in the composition that is acceptable for transfusion and which is effective to reduce the level of active pathogens in the composition when irradiated with light of an appropriate intensity and wavelength.

As used herein, the term "a biologically compatible solution" is intended to mean an aqueous solution in which cellular blood components may be exposed or stored, such as by being suspended therein, and remain viable, i. e. , retain their essential biological and physiological characteristics. Such biologically compatible solutions preferably contain an effective amount of at least one anticoagulant.

As used herein, the term "a biologically compatible buffered solution" is intended to mean a biologically compatible solution having a pH and osmotic properties (e.g., tonicity, osmolality and/or oncotic pressure) suitable for maintaining the integrity of the cell membrane of cellular blood components. Suitable biologically compatible buffered solutions typically have a pH between 5.0 and 8.5 and are isotonic or only moderately hypotonic or hypertonic. Biologically compatible buffered solutions are known and readily available to those of skill in the art. Illustrative examples of suitable solutions include, but are not limited to, those listed in Table I below.

Table I Common Biologically Compatible Buffered Solutions

Preferred Embodiments

In one aspect of the invention there is provided a highly efficient method for reducing the level of active pathogenic contaminants in whole blood or blood components which comprises adding to the whole blood or blood components at least one phenothiazin-5-ium dye in an amount effective to reduce the level of active pathogenic contaminants and irradiating the whole blood or blood components with light of an appropriate intensity and wavelength for a time sufficient to reduce the level of said active pathogenic contaminants in the whole blood or blood components, wherein the partial pressure of oxygen in the whole blood or blood components is maintained at a level during the irradiating of the whole blood or blood components such that the amount of active pathogenic contaminants is reduced.

As a first step when practicing any of the embodiments of the invention disclosed herein, whole blood is preferably drawn from a donor into a suitable biologically compatible buffered solution containing an effective amount of at least one anticoagulant. Suitable anticoagulants are known to those skilled in the art, and include, but are not limited to, lithium, potassium or sodium oxalate (15 to 25 mg/10 mL blood), sodium citrate (40 to 60 mg/10 mL blood), heparin sodium (2 mg/ 10 mL blood), disodium EDTA ( 10 to 30 mg/ 10 mL whole blood),

ACD-Formula A solution (1.0 mL/10 mL blood) or citrate-phosphate-dextrose solution (CPD) (1.0 mL/10 mL blood).

The whole blood so collected may then be decontaminated according to the methods of the present invention. Alternatively, the whole blood may first be separated into blood components, including, but not limited to, plasma, platelets and red blood cells, by any method known to those of skill in the art.

For example, the blood can be centrifuged for a sufficient time and at a sufficient centrifugal force to sediment or pack the red blood cells. Leukocytes collect primarily at the interface of the red cells and the plasma-containing supernatant in the buffy coat region. The supernatant, which contains plasma, platelets, and other blood components, may then be removed and centrifuged at a higher centrifugal force, whereby the platelets sediment.

Human blood normally contains about 7 x 10⁹ leukocytes per liter. The concentration of leukocytes, which form a layer on top of the sedimented red cells, can be decreased by filtering whole blood prior to centrifugation through a filter that decreases their concentration by selected orders of magnitude. Leukocytes can also be removed from each of the components by filtration through an appropriate filter that removes them from the solution.

In a preferred embodiment of this invention, the whole blood or blood component to be decontaminated is obtained in, prepared in or introduced into gas permeable blood preservation bags which are sealed and flattened to a width sufficiently narrow to permit light to irradiate the contents, such that any pathogenic contaminant present in the blood or blood component in the bag will be irradiated. Preferably, the whole blood or blood component to be decontaminated is obtained in, prepared in or introduced into gas permeable blood preservation bags which are flattened to a width of about 4 mm or less thick, more preferably about 2 mm or less. Any such blood bag known to those of skill in the art may be used provided that the bag is transparent to the selected wavelength of light. The method of the present invention may be practiced in a static system or in a flow system. Examples of systems which may be used in the present invention are disclosed, for example, in U.S. Patent Nos. 5,304,113 and 5,030,200.

The whole blood or blood component composition that is to be decontaminated may also include any suitable biologically compatible buffered solution known to those of skill in the art. Examples of such buffers include, but are not limited to, ADSOL, ARC-8 and Nutricell.

The phenothiazin-5-ium dyes used in the methods of the present invention may be prepared according to the methods and techniques known to those skilled in the art. Suitable synthetic methods are described, for example, in U.S. Patent

No. 4,962,197.

An effective amount of at least one selected dye is introduced into the composition. Any amount sufficient to provide a concentration of dye in the composition that is acceptable for transfusion and which is effective to reduce the level of active pathogens in the composition when irradiated with light of an appropriate intensity and wavelength may be used. Preferably, the selected dye is non-toxic and the effective concentration is acceptable for transfusion so that the treated blood or blood component does not require additional manipulation to remove the dye. The effective concentration of dye to be used can be determined empirically by one of skill in the art. Preferably, the effective concentration of the dye is from about 0.2 μM to about 50 μM, more preferably from about 1 μM to about 25 μM.

In a preferred embodiment, the phenothiazin-5-ium dye has the formula

wherein each of R„ R,', R₂, and R₂' is independently selected from the group consisting of a methyl group, an ethyl group, a propyl group, an alkenyl group, an alkynyl group, a nitrile, an aralkyl group, a hydroxy group, an alkoxy group, an amine group, and a hydrogen atom, each of R₃, R₃', R₄, R₄', R₅ and R₅' is independently selected from the group consisting of an alkyl group, an alkenyl group, an alkynyl group, a nitrile, an azide, an aryl group, an aralkyl group, a heteroaryl group, an alkoxy group, an aryloxy group, an amine group, and a hydrogen atom, or any two of R„ R,', R₂, R₂', R₃, R₃', R₄, R₄', R₅ and R₅' together form an aryl or heteroaryl ring;

Y is selected from the group consisting of S, Se and Te; and

X is a counter-ion; with the provisos:

(i) that if each of R,, R,', R₂ and R₂' is independently a hydrogen atom or an alkyl group, then at least one of R₃, R₃', R₄, R₄', R₅ and R₅' is other than a hydrogen atom,

(ii) that if one of R₄ and R₄' is an alkyl group, then at least one other of R₃, R₃', R₄, R₄', R₅ and R₅' is other than a hydrogen atom, and

(iii) that if any two of R₃, R₃', R₄, R₄', R₅ and R₅' together form an aryl or heteroaryl ring, each of R„ R,', R₂, R,' is other than a hydrogen atom.

In a preferred embodiment of the present invention, each of R,, R,', R₂ and R₂' is independently a methyl group, an ethyl group or a hydrogen atom. In a more preferred embodiment, each of R,, R,', R₂, R₂', R₃ and R₃' is a methyl group. In another preferred embodiment of the present invention, each of R₃ and R₃' is independently an alkyl group.

In yet another preferred embodiment of the present invention, each of R„ R,', R₂, and R₂', is independently a lower alkyl group (i.e. an alkyl group having about 1 -6 carbon atoms), a lower alkynyl group, or a hydrogen atom, and each of R₃, R₃', R₄, R₄', R₅ and R₅' is independently a lower alkyl group, a lower alkynyl group, or a hydrogen atom. More preferably, the lower alkyl group is a methyl group or an ethyl group.

Preferably, Y is a sulfur atom or a selenium atom. More preferably, Y is a sulfur atom.

The counter ion X may be any anion, monovalent or polyvalent, sufficient to balance the charge on the phenothiazin-5-ium dye. Illustrative examples of suitable counter ions include inorganic moieties, such as halides (preferably chloride or bromide), sulfates and phosphates, and organic moieties, such as acetate, citrate and tartrate.

In a particularly preferred embodiment of the present invention, 1 ,9-dimethylmethylene blue

is employed as the phenothiazin-5-ium dye. Preferably, the 1 ,9-dimethylmethylene blue is introduced into the whole blood or blood component to be decontaminated at a concentration of about lμM to about 25 μM.

In the inventive method, the partial pressure of oxygen in the phenothiazin-5-ium dye-containing whole blood or blood components is maintained at a level during the irradiating step of the inventive method such that the amount of active pathogenic contaminants contained in the whole blood or blood components is reduced. Oxygen has been identified as playing an important role in the reproducible and rapid inactivation of pathogenic contaminants in red cell suspensions. More specifically, it has been found that the rate of virus photoinactivation in red cell suspensions is directly related to the partial pressure of oxygen.

Although it is generally known by those of skill in the art that the photodynamic action of phenothiazine dyes such as methylene blue or dimethylmethylene blue requires the presence of oxygen for biological activity via the production of singlet oxygen, it has previously been assumed that red blood cell suspensions possessed ample oxygen for maximal generation of singlet oxygen. Through extensive experimentation and analysis of a multitude of hematology measurements, it was unexpectedly discovered that there is a direct correlation between the kinetics of virus inactivation and the partial pressure of oxygen. By maintaining the partial pressure of oxygen at a sufficient level, reproducible pathogen inactivation is thereby achieved.

The partial pressure of oxygen in whole blood or blood components can be maintained at a sufficient level by any method or instrument which is capable of exposing air or oxygen gas mixtures to thin films of blood to achieve a reproducible partial pressure of oxygen. For example, the whole blood or blood components can be agitated, air or oxygen can be injected into a blood bag containing the whole blood or blood components or a blood oxygenator instrument can be used in the inventive method. Pure oxygen or any oxygen gas mixture, e.g., ambient air, may be used in the inventive method provided that the partial pressure of oxygen in the whole blood or blood components is maintained at a level during the irradiating procedure such that the amount of active pathogenic contaminants contained in the whole blood or blood components is reduced.

The normal partial pressure of oxygen in whole blood or blood components is typically from about 20 mm Hg to about 40 mm Hg at the time of blood collection. In a preferred embodiment, the partial pressure of oxygen in the whole blood or blood components is maintained at a level of at least about 40 mm Hg during the irradiating of the phenothiazin-5-ium-containing whole blood/blood components, more preferably between about 40 mm Hg and about 400 mm Hg. In a particularly preferred embodiment, the oxygen partial pressure is maintained at a level between about 100 mm Hg and about 350 mm Hg.

High levels of extracellular oxygen obtained by maintaining the partial pressure of oxygen above normal levels during the irradiating of the whole blood or blood components to promote pathogen inactivation may also result in even higher levels of oxygen in red blood cells. Such elevated levels of oxygen may promote phenofhiazin-5-ium and light induced, oxi dative red cell damage.

In another embodiment of the invention, the red blood cells are equilibrated with a hemoglobin blocking agent that blocks oxygen uptake. If such a hemoglobin blocking agent were added, then mixture with a highly oxygenated additive solution would result in similar or greater levels of oxygen extracellularly than present in the red blood cells, thereby promoting pathogen inactivation while protecting red blood cells from photodynamic oxidative damage. Any agent that binds hemoglobin and blocks oxygen uptake can be used in the invention. In a preferred embodiment, the hemoglobin blocking agent is carbon monoxide. In yet another embodiment of the invention, the hemoglobin blocking agent is removed from the whole blood or blood components after irradiation. For example, following the irradiation step, the red blood cell suspension could be exposed to oxygen and/or carbon dioxide to remove the blocking agent from hemoglobin. The mixture of the whole blood and/or blood component and phenothiazin-5-ium dye is then irradiated with light of an appropriate wavelength (or mixture of wavelengths) and intensity. The term "appropriate wavelength and intensity" is intended to mean light of a wavelength and intensity that can be absorbed by the dye, but does not damage the blood or blood components present. It is well within the level of skill in the art to select such wavelength and intensity empirically based on certain relevant parameters, such as the particular dye employed and its concentration in the composition. For example, one having skill in the art would know that if the intensity of the light source is decreased, a greater concentration of dye and/or longer exposure time should probably be used. An appropriate wavelength is preferably selected based on the absoφtion profile of the dye (or dyes) employed and is most preferably one that does not result in substantial damage to one or more of the cellular blood components in the composition being decontaminated.

Model viral systems are known to those of skill in the art which may be used to test the selected dye and light source for their efficacy. Such model viral systems include, but are not limited to, the enveloped bacteriophage Φ6, vesicular stomatitis virus (an animal virus that contains its genome encoded in RNA), and Pseudorabies virus (an animal virus that contains its genome encoded as DNA). Based on the effective values of parameters such as wavelength and light intensity measured for such model systems, one of skill in the art can routinely select suitable values for these parameters for use in practice of the present invention.

In another embodiment, the partial pressure of oxygen in the whole blood or blood components is reduced after the irradiating step. If high oxygen levels cause red cell damage during storage, such a deoxygenating step can be performed subsequent to irradiation to reduce oxygen levels. An instrument similar to an oxygenator can be utilized to reduce the partial pressure of oxygen to non-damaging levels, except that the instrument would contain an inert gas, e.g., nitrogen, rather than air or oxygen.

In a preferred embodiment of this invention, red blood cells, which have optionally been leukodepleted with a five log filter, are first suspended in

Nutricell at a hematocrit of about 15% to 65%, then introduced into gas permeable blood preservation bags in an amount such that the filled bag has a thickness of about 1 mm to about 4 mm, and finally treated with 1,9- dimethylmethylene blue at a concentration of about 1 μM up to about 25 μM. The partial pressure of oxygen in this mixture is then increased to a pO₂ between about 100 mm Hg and about 350 mm Hg and irradiated with red light of wavelength of about 560 to 800 nm at a sufficient intensity for a sufficient time, such as 5.0-6.0 mW/cm² for about 5-10 seconds, to reduce the level of active pathogenic contaminants in the red blood cells and Nutricell solution. Preferably, the active pathogenic contaminants in the treated sample are inactivated to a log₁₀ inactivation of at least about 4, more preferably at least about 6.

Following treatment in accordance with the method of this invention, the whole blood or composition containing a cellular blood component and/or blood protein may be stored or transfused. Alternatively, after treatment of compositions such as red cell preparations or platelet-rich plasma, the composition can be centrifuged at a force sufficient to pellet the cellular components. The supernatant can be removed following centrifugation and the cells resuspended to reduce the concentration of residual photosensitizer and any reaction products. The following examples are illustrative only and are not intended to limit the scope of the invention as defined by the appended claims. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods of the present invention without departing from the spirit and scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. All patents and publications referred to herein are expressly incoφorated by reference.

Example 1

Citrate-phosphate-dextrose anticoagulated whole blood was centrifuged at room temperature, and platelet rich plasma and buffy coat (50mL) were successively expressed from the packed red cells. The additive solution, AS-3

(Nutricell) was added to packed red cells to yield a hematocrit of 60-65%, and resulting suspension was leukoreduced using a Pall BPF-4 filter at room temperature. Vesicular stomatitis virus (VSV) was added to 60-65% hematocrit red cells in such a way that the volume of the virus spike was £10% of the final sample volume, and AS-3 containing dimethylmethylene blue (DMMB) was added to the resulting solution to yield a final hematocrit of 45% and a final DMMB concentration of 25mM. 2-mL aliquots of the VS V/red cells were placed in 60 mm diameter petri dishes and samples were either agitated for 15 minutes at 100 φm or kept stationary prior to measurement of the partial pressure of oxygen using a blood gas analyzer (Corning model 238). Samples were then exposed to 8 seconds of red light (Q-Beam LED, 670 nm, Quantum Devices) at a fluence rate of 5.3-5.6mW/cπr and subsequently titered for virus. Results are given in Table 2.

Table 2

* Stored at 1-6°C in a gas permeable container for 2 weeks prior to inactivation.

+ Stored at 1-6°C in a gas permeable container for 5 days prior to inactivation.

Example 2

Citrate-phosphate-dextrose anticoagulated whole blood was centrifuged, and platelet rich plasma and buffy coat (50mL) were successively expressed from the packed red cells. The cold additive solution, AS-3 (Nutricell) was added to packed red cells to yield a hematocrit of 60-65%, and resulting suspension was leukoreduced using a Pall BPF-4 filter. Leukoreduced red cells were cooled to 1-8°C. All subsequent steps were performed at 1-8°C except for sample illumination. Cold VSV was added to 60-65% hematocrit red cells in such a way that the volume of the virus spike was £ 10% of the final sample volume, and cold

AS-3 containing dimethylmethylene blue (DMMB) was added to the resulting solution to yield a final hematocrit of 45% and a final DMMB concentration of 25mM. 2-mL aliquots of the VSV/red cells were placed in 60 mm diameter petri dishes and agitated an orbital shaker at 200 or 250 φm for various times (2.5, 5, 7.5, 10, 12.5, or 15 minutes). Agitated samples were either measured for the partial pressure of oxygen using a Rapidlab 860 (ChironDiagnostics) instrument or illuminated for 5 seconds with a Q-Beam LED red light (670 nm, Quantum Devices) at a fluence rate of 5.3-5.6mW/cπr and titered for virus. Five experiments were conducted on different units of blood. The mean log,₀ inactivation of VSV is plotted in Figure 1 as a function of mean oxygen partial pressure.

Claims

What Is Claimed Is:

1. A method of reducing the level of active pathogenic contaminants in whole blood or blood components which comprises:

(a) adding to said whole blood or blood components at least one phenothiazin-5-ium dye in an amount effective to reduce the level of active pathogenic contaminants; and

(b) irradiating said whole blood or blood components with light of an appropriate intensity and wavelength for a time sufficient to reduce the level of said active pathogenic contaminants in said whole blood or blood components, wherein the partial pressure of oxygen in said whole blood or blood components is maintained at a level during the irradiating of said whole blood or blood components such that the amount of active pathogenic contaminants in said whole blood or blood components is reduced.

2. The method of claim 1, wherein the partial pressure of oxygen in said whole blood or blood components is maintained at a level of at least about 40 mm Hg during the irradiating of said whole blood or blood components.

3. The method of claim 2, wherein the partial pressure of oxygen in said whole blood or blood components is maintained at a level between about 40 mm Hg and about 400 mm Hg during the irradiating of said whole blood or blood components.

4. The method of claim 3, wherein the partial pressure of oxygen in said whole blood or blood components is maintained at a level between about 100 mm Hg and about 350 mm Hg during the irradiating of said whole blood or blood components.

5. The method of claim 1, wherein said active pathogenic contaminants in said whole blood or blood components are inactivated to a log,₀ inactivation of at least about 4.

6. The method of claim 5, wherein said active pathogenic contaminants in said whole blood or blood components are inactivated to a log₁₀ inactivation of at least about 6.

7. The method of claim 1, further comprising reducing the partial pressure of oxygen in said whole blood or blood components after the irradiating of (b).

8. The method of claim 1 , further comprising adding a hemoglobin blocking agent prior to the irradiating of (b).

9. The method of claim 8, wherein said hemoglobin blocking agent is carbon monoxide.

10. The method of claim 8, further comprising removing said hemoglobin blocking agent from said blood or blood components after the irradiating of (b).

11. The method of claim 1 , further comprising leukodepleting said whole blood or blood components prior to the addition of (a).

12. The method of claim 1 , wherein at (b) said whole blood or blood components are irradiated in a blood bag about 4.0 mm or less in thickness.

13. The method of claim 12, wherein at (b) said whole blood or blood components are irradiated in a blood bag about 2.0 mm or less in thickness.

14. The method of claim 13 , wherein the time sufficient to reduce the level of said active pathogenic contaminants in said whole blood or blood components at (b) is less than 1 minute.

15. The method of claim 14, wherein said time sufficient to reduce the level of said active pathogenic contaminants in said whole blood or blood components at (b) is less than 30 seconds.

16. The method of claim 15, wherein said time sufficient to reduce the level of said active pathogenic contaminants in said whole blood or blood components at (b) is less than 10 seconds.

17. The method of claim 1 , wherein said appropriate wavelength is between about 500 nm and about 800 n .

18. The method of claim 17. wherein said appropriate intensity is between about 1.0 mW/cm² and about 10.0 mW/cm².

19. The method of claim 18, wherein said appropriate intensity is between about 5.0 mW/cm² and about 6.0 mW/cm².

20. The method of claim 1, wherein said effective amount is an amount sufficient to provide a concentration of said dye in said whole blood or blood components of between about 0.2 μM and about 50 μM.

21. The method of claim 20, wherein said effective amount is an amount sufficient to provide a concentration of said dye in said whole blood or blood components of between about 1 μM and about 25 μM.

22. The method of claim 1, wherein said phenothiazin-5-ium dye is 1 ,9-dimethylmethylene blue.

23. The method of claim 1, wherein said whole blood or blood components are transfusable.

24. The method of claim 1 , wherein said pathogenic contaminants are selected from the group consisting of viruses, bacteria, parasites and leukocytes.

25. The method of claim 24, wherein said viruses are selected from the group consisting of intracellular viruses and extracellular viruses.

26. The method of claim 24, wherein said viruses are enveloped viruses.

27. The method of claim 26, wherein said enveloped viruses include at least one virus selected from the group consisting of RNA viruses and DNA viruses.

28. The method of claim 27, wherein said enveloped viruses include at least one or more viruses selected from the group consisting of human immunodeficiency virus, cytomegalovirus, heφes virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, Vesicular Stomatitis virus, Sindbis virus, and pox virus.

29. The method of claim 24, wherein said bacteria include members of the genus Streptococcus or the genus Escherichia.

30. The method of claim 24, wherein said parasites include members of the genus Trypanosoma.

1. A blood product produced by the method of claim 1.