JP3373201B2 - Inactivation method of blood contaminants of viruses and bacteria - Google Patents

Description

FIELD OF THE INVENTION The present invention relates to the general field of biochemistry and medical science, and in particular to lyophilized or reconstituted blood cell compositions containing red blood cells, platelets, etc. or protein fractions. Inactivation of viral / bacterial contaminants (coutaminant).

Background of the Invention A significant concern regarding the use of stored or donated allogeneic blood or plasma protein preparations derived from human blood is the potential for viral and bacterial contamination.

Virus inactivation due to severe sterilization is not acceptable.
This is because it can also break down functional components of blood, especially red blood cells and labile plasma proteins. Viable red blood cells (RBCs) can be characterized by one or more of the following. ATP synthesizing ability; cell morphology; P ₅₀ value; oxyhemoglobin value, methemoglobin value and hemichome value; MCV value, MCH value, and MCHC value; cell enzyme activity; and survival in vivo. Thus lyophilized,
The reconstituted, virus-inactivated cells are then damaged to the extent that these cells are unable to metabolize or synthesize ATP, or the cell circulation is impaired, which in turn impairs their utility in transfusion medicine. .

There is an immediate need to develop protocols for the inactivation of viruses that may be present in human red blood cell supplies.
For example, most recently, non-hepatitis A, non-hepatitis B tests have been developed, but such screening methods reduce the incidence of viral transmission but do not completely secure the blood supply, and Don't be virus free. Current statistics show that transfusion risk per unit of transfusion is as high as 1: 100 for non-hepatitis A and non-B hepatitis, depending on geographical location, and for HIV ranges from 1: 40,000 to 1: 1,000,000.
Clearly, it would be desirable to develop methods for inactivating or removing viruses from blood indiscriminately.

Contamination problems are also seen with blood plasma protein fractions such as plasma fractions containing immunoglobulins and coagulation factors. For example, new cases of non-A hepatitis, non-B hepatitis occurred in hemophilia patients who received a protein fraction containing Factor VIII treated for viral inactivation according to an approved method. Therefore, there is a need for improved viral inactivation treatment of blood protein fractions.

Thus, the present invention provides a method of inactivating viral and bacterial contaminants present in blood and blood protein fractions.

SUMMARY OF THE INVENTION The present invention allows cells or protein fractions to be effective in transfusion conditions while maintaining relatively high cell viability, ATP synthesis and oxygen transport in the case of cellular components, and protein fractions. A method for viral / bacterial inactivation of dried or reconstituted cells (red blood cells, platelets, hemosomes and other cellular or cellular components) that maintains therapeutic efficacy in the case of fractions is provided.

The lyophilization medium and reconstitution medium of the present invention can be used to lyophilize and reconstitute proteins, especially blood plasma protein fractions. The protein fraction can be virus / bacteria inactivated by mixing with a chemosensitizer, lyophilized and then irradiated. When the lyophilized medium of the present invention is used, it is envisioned that the components of the medium can also be utilized to provide some degree of protection of dried proteins during irradiation.

A preferred embodiment reduces viral and bacterial contamination of dry or reconstituted cells with a wash solution containing a polymer or polymer mixture having a molecular weight in the range of about 1K to 360K, followed by a pH in the range of about 7.0-7.4. Dextrose-
Including one or more additional wash cycles using a saline solution wash. The dextrose-saline solution also contains a polymer having a molecular weight in the range of about 1K-40K, preferably about 2.5K. Further, the composition of reconstituted cells preferably contains a monosaccharide.

The cells are preferably previously lyophilized using a lyophilization solution buffered in the pH range 7.0-7.4 (preferably with a phosphate buffer solution). A typical phosphate buffered lyophilized solution contains monobasic and dibasic potassium and sodium phosphates (usually in the range of 1-10 mmol respectively) and 5-10 mmol of adenine. This solution keeps the pH around 7.2.

A preferred phosphate buffer solution for use as a lyophilization buffer comprises nicotinic acid, reduced glutathione, glutamine, inosine, adenine, monopotassium phosphate, magnesium chloride, disodium phosphate, all of which are about
Available as salt buffer at pH 7.2. In addition, the lyophilization buffer contains about 26% (w / v) final concentration of monosaccharides, preferably 1.7 molar glucose, and about 3.0% (w / v) final concentration of polyvinylpyrrolidone (average of 360K). Molecular weight), as well as hydroxyethyl starch (average molecular weight of 500K) at a final concentration of about 15% (w / v).

The term freeze-drying freezes the substance, then the solvent,
That is, it is broadly defined as that the concentration of water is lowered to a level that sustains a biological reaction or a chemical reaction by sublimation or desorption. Usually, the drying process is performed under high vacuum. However, regarding the storage of cells, especially red blood cells,
The degree of dryness (amount of residual water) is important to the ability of cells to withstand long-term storage at room temperature. Using the procedures described herein, cells can be lyophilized to a residual water content of less than 10% by weight, preferably less than 3% by weight and reconstituted into transfused therapeutically effective cells. Cells with a water content of about 3% by weight using this procedure do not decompose and
It can be stored at room temperature for up to 2 weeks and at 4 ° C for more than 8 months. This is the current AABB for frozen storage of erythrocytes for 6 weeks at 4 ° C without degradation or less than 1 day at room temperature.
Far beyond the standard. These dried cells can be inactivated using the chemosensitizers described herein.

According to a preferred embodiment of the present invention, the washed and concentrated red blood cells are mixed with a chemosensitizer and then washed to remove excess sensitizer that is not bound to viral or bacterial nucleic acids and then treated cells are Lyophilized. The mixture of dried cells and sensitizers is then sufficient to destroy the virus (especially single-stranded or double-stranded RNA / DNA viruses) without appreciably adversely affecting the recovery and efficacy of the cells. Over a period of time, typically about 3K gamma
Irradiated with an intensity of 50K rad. Other wavelengths of electromagnetic radiation such as X-rays may be used.

In another preferred embodiment, the chemosensitizer may be added to the liquid protein formulation and then lyophilized and irradiated. Particularly preferred, but not limited to, blood protein formulations including plasma proteins, blood protein extracts, coagulation factors (eg Factor VIII and Factor IX), immunoglobulins and serum albumin.

The dried (lyophilized) cell or protein fraction can be mixed directly with the chemosensitizer and then irradiated.

From the above description, it is appreciated that the present invention may be used to selectively bind metal atoms or chemosensitizers containing metal atoms to blood-borne viruses, bacteria, or parasites. Also, a monoclonal or polyclonal antibody directed against a particular viral antigen (coat protein or envelope protein) may be covalently coupled to a metal atom or a chemosensitizer compound containing a metal atom, whereby It may increase the effective cross-sectional area of contaminants for transmitted or other forms of radiation energy.

Since cell compositions also include various proteins, the method of decontaminating cells described herein also includes protein fractionation, particularly blood plasma protein fractionation (coagulation factors (eg,
Factors VIII and IX), including but not limited to fractions containing serum albumin and / or immunoglobulins). Viral and bacterial inactivation can be achieved by treating the protein fraction with the sensitizers described herein. The lyophilized and reconstituted protein fraction can be sensitized and irradiated to inactivate potential contaminants. It is envisioned that liquid protein fractions and frozen protein fractions may also be decontaminated by the present invention.

Depending on the nature of the expected radiolytic mechanism of reaction of the virus with the sensitizer, other types of radiation, such as X-rays, may be used, but the strength and power used adversely affect cells. Provided that it is sufficient to inactivate the viral contaminants. Mature human red blood cells and platelets are deficient in nucleic acids, so sensitizers that bind nucleic acids selectively target contaminating viruses and bacteria. Although described with respect to viruses, it is understood that the methods of the invention are also generally beneficial to any contaminants found in stored blood or blood products, including bacteria and blood-borne parasites.

Detailed Description of the Invention Cells are immersed in a large number of red blood cells, platelets and / or hemosomes, etc. in a physiological buffered aqueous solution containing a carbohydrate and one or more biocompatible polymers, preferably biocompatible polymers having amphipathic properties. It is preferably prepared by The term amphipathic means that there are hydrophobic and hydrophilic moieties in a single molecule. Following this immersion, freeze the solution and dry the frozen solution.
New lyophilized red blood cells containing less than 10% by weight of water, preferably less than about 3% by weight, are reconstituted to yield a significant proportion of viable transfusionally beneficial red blood cells, platelets or hemosomes. A preferred method for reconstituting a lyophilized composition is described below. Although described with respect to red blood cells, it is understood that these methods are also generally useful for lyophilizing platelets, hemosomes, and blood protein fractions.

The carbohydrates used to prepare the red blood cell, platelet and / or hemosome compositions of the present invention are biocompatible with red blood cells, platelets or hemosomes, i.e., non-dissociative to cells or hemosome membranes, and It is or can penetrate the membrane of red blood cells, platelets or hemosomes. It is also advantageous to stabilize proteins, especially labile blood proteins, with carbohydrates during freeze-drying and irradiation according to the invention. The carbohydrate may be selected from the group consisting of monosaccharides. This is because it is clear that the disaccharide does not permeate the membrane to a large extent. The monosaccharides pentose and hexose have a final concentration of about 7.0 to 37.5% by weight, preferably about 26% by weight, in phosphate buffered saline (PBS) or phosphate buffer.
Is preferred. Xylose, glucose, ribose, mannose and fructose are used with particular advantage.

It is understood that cells can be lyophilized and irradiated using other protocols, as described below. Failure to follow the described freeze-drying procedure results in virus inactivation, but at the expense of retaining a significant proportion of viable and valid red blood cells.

In the following, the present invention will be described with reference to red blood cells (referred to as RBCs), but the present invention is not only applicable to platelets, hemosomes or other blood cell types or biological cells, but also protein fractions, especially plasma protein fractions. It is understood that it is also applicable to.

Erythrocytes are preferably prepared by centrifugation of whole blood, removal of plasma supernatant and resuspension of cells in PBS or phosphate buffer or commercially available dextrose-saline solution. This wash cycle can be repeated 2-3 times, preferably using a dextrose-saline solution, and then the concentrated cells are diluted with the lyophilization buffer described above, so that the final diluted concentrations of carbohydrate and polymer are required. Be kept within a range.

Commercially available concentrated blood cells may also be used, which are typically prepared in CPDE (commercial solution containing citrate, phosphate, dextrose and adenine).

After lyophilization to a water content of less than 10%, preferably less than 3%, the lyophilized cells are such that when reconstituted for use as transfused cells, there is no significant degradation of their desirable properties. , Or in the absence of its decomposition,
It may be maintained at room temperature under vacuum in an airtight container or under a nitrogen or other inert gas atmosphere for extended periods of time.
In using the preferred freeze-drying method disclosed herein, a particular advantage of the present invention is that the freeze-dried cells can be stored at room temperature for extended periods of time, thus preserving liquid CPDE-preserved red blood cells prepared by prior art methods. Eliminating the need for cryogenic refrigeration required for storage. The present invention is also directed to the very low temperature (-80%) of red blood cells in glycerol.
Eliminates the need for frozen storage (° C).

By using the preferred reconstitution method disclosed herein, a further advantage is that freeze-dried red blood cells are at room temperature, ie temperatures above about 4 ° C. and up to about 37 ° C., which are normal human body temperatures. Corresponding), preferably at room temperature (about 22 ° C.). Reconstitution medium is approximately 12-30% (w /
v) present at a concentration in the range of about 2.5K to 360K, preferably 5
A solution containing a polymer or polymer mixture having a molecular weight of K to about 360 K is preferred. This polymer may be the same polymer used to freeze-dry the red blood cells above. Therefore, a polymer selected from polyvinylpyrrolidone, hydroxyethyl starch, and dextran is particularly preferred, with about 19% in the reconstitution solution.
Most preferred is polyvinylpyrrolidone present at a concentration of (w / v), preferably with a molecular weight of about 10K. The reconstituted solution is typically re-buffered with a phosphate buffer solution containing the above monopotassium phosphate and disodium phosphate.
Keep the pH within the range of about 7.0-7.4. An especially most preferred polymer is polyvinylpyrrolidone with an average molecular weight of about 10K. The most preferred reconstitution buffer also contains adenosine triphosphate (ATP) at a final concentration of about 5 millimolar.

The polymer may be present in various solutions at final concentrations of from about 3.6 Kwt% to saturation and has a molecular weight ranging from about 2.5K to about 360K. The polymer preferably has a molecular weight in the range of about 2.5K to about 500K, most preferably about 2.5K to about 50K, and a concentration of from about 3.6% by weight to the limit of solubility of the polymer in solution. Exists in.
Polymers selected from the group consisting of polyvinylpyrrolidone (PVP) and polyvinylpyrrolidone derivatives, as well as dextran and dextran derivatives offer important advantages. In the range of 3-20% (w / v) in solution before lyophilization
Most preferably, polyvinylpyrrolidone (an amphiphilic polymer) with an average molecular weight in the range of 2.5 to 360K is used.
Amino acid-based polymers (ie proteins), dextran or hydroxyethyl starch may also be used. Hydroxyethyl starch (M-HES) with an average molecular weight of about 500 in lyophilization buffer is used at a final concentration of 15% (w / v). Other amphipathic polymers such as poloxamers can be used in any of their various forms. The use of a carbohydrate-polymer solution during lyophilization of red blood cells
It allows the recovery of intact cells, with a significant proportion of the cells containing bioactive hemoglobin.

The most preferred reconstitution buffer is a solution containing monopotassium phosphate, disodium phosphate and ATP, all of which form a basic salt buffer at a pH of about 7.2, which also contains about 19%. It contains (w / v) polyvinylpyrrolidone (average molecular weight of about 10K).

In addition, the reconstituted solution may optionally contain monosaccharides, which are preferably present in a concentration range of about 7.0-37.5% (w / v). Preferred monosaccharides are xylose, glucose, ribose, mannose and fructose.

In the most preferred embodiment, the freeze-dried red blood cells are
It can be reconstituted and mixed by mixing with an equal volume of reconstitution buffer at a temperature of about 37 ° C. The term "equal" means that its volume is the same as the starting volume before lyophilization. After the initial reconstitution, the solution is preferably diluted 1: 1 with an additional 1 to 4 volumes of reconstitution buffer at a temperature of about 37 ° C and mixed until fully hydrated.

Then, the rehydrated cells are preferably washed by the following procedure. However, once the cells have been reconstituted with reconstitution buffer, they are found to be in the hydrated effective form, but the following wash combinations are particularly preferred for clinical purposes.

After separating the cells from the reconstitution buffer by centrifugation,
The resulting enriched cells contain nicotinic acid, inosine, adenine, glutamine, and magnesium chloride, all of which are present at about 0.4-10 millimolar, and are each about 30
A wash buffer containing 10 mM sodium chloride and potassium chloride (pH 7.2 with 10 mM disodium phosphate).
Resuspended at room temperature (in approximately the same volume used for the initial reconstitution). This wash buffer contains monosaccharides at a concentration of about 20 millimolar,
Preferably it further comprises glucose and a polymer of molecular weight 40K, preferably polyvinylpyrrolidone, which is present at a concentration of about 16% (w / v). Separation by centrifugation completes the first post-rehydration step, washing step.

After the washing step, the rehydrated cells may be suspended in dextrose-saline transfusion buffer at room temperature, which buffer
It is preferred to include polyvinylpyrrolidone with an average molecular weight of 2.5K at a final concentration of 10% (w / v). The cells can be used as is or returned to autologous plasma. An additional wash step in phosphate buffered dilution buffer may further remove virus, but this step is optional for the preparation of rehydrated transfused cells.

The above reconstitution and washing almost always gives a log reduction of about 4 of viral and cellular contamination, with a log reduction of 1 being obtained by drying and a log reduction of 3 being obtained by washing. Of course, heterologous viruses may respond differently, potentially resulting in a log reduction of greater than 4 of contamination.

The reconstituted cells have properties that make them transfusionable and effective for therapeutic purposes in that their properties are similar to those of new (ie, not previously lyophilized) red blood cells. Erythrocytes typically reconstituted according to the invention have an oxyhemoglobin content greater than about 90% of the oxyhemoglobin content in normal erythrocytes. Hemoglobin recovery before the wash step is typically 30-
It is in the range of 85%. The total cell hemoglobin recovery including the post-hydration wash step is about 20-30%. The morphology of cells reconstituted according to the present invention (by scanning electron microscopy) typically shows no discrepancies or voids and shows predominantly discocytic morphology, including some lipoid morphology. Show. Measure the oxygen holding capacity of new red blood cells (50 oxygen molecules
% (Measured by P _50, which is the partial pressure of oxygen bound), it is about 26 to 28 (average 26.7), and the average Hill coefficient (a measure of cooperative binding of oxygen molecules to natural hemoglobin) is 1.95.
Met. Typical P ₅₀ of the lyophilized reconstituted red blood cells according to the present invention is about 27.5 (average), the average Hill coefficient is 2.08. The assay for ATP in reconstituted cells is
ATP levels suggesting normal ATP to ADP metabolism are shown. Also, normal hemagglutination with readily available blood typing antisera of red blood cells made according to the invention is typically seen.

This lyophilization and reconstitution procedure is performed for cell-like substances (eg hemosomes), and virus / protein in protein fractions.
Bacterial contamination is advantageously and significantly reduced. The contamination may be due to radiation sensitization (radiation sensitizinization, especially while the cell or protein fractions are dry).
g) and can be further reduced by treatment.

The starting packed red blood cells or proteins, which may initially be in liquid or lyophilized form, are in sufficient quantity (based on the total wet weight of the cells) of a chemosensitizer. Mixed with. About 0.1-1 mg in a composition of concentrated red blood cells (about 10% hematocrit)
It is preferred that the chemosensitizer of is used per ml of concentrated cells. The mixture is preferably irradiated with gamma radiation in the range of 3K to 50K rads, typically about 3K rads.
When using gamma radiation, the preferred exposure is 1 to 10 minutes. In addition, ultraviolet rays (320n
m) can be used. When using UV light, the preferred exposure is 1 to 10 minutes, preferably 3 minutes. This irradiation in the presence of sensitizers results in about a 6 log reduction in viral and bacterial contamination, based on the impurities present prior to washing and irradiation.

The present invention provides a selective method of generating free radicals derived from chemosensitizers only in the vicinity of viral RNA or DNA. Indiscriminate radiolysis of blood containing hydrated virus produces hydroxyl radicals. However, hydroxyl radicals not only damage both erythrocytes and contaminating proteins, but also viral targets. Thus, viral inactivation is achieved at the expense of red blood cell viability. Therefore, there is a need for sensitizers that can be selected to bind DNA and / or RNA and generate radicals after irradiation. Since the radiolysis is carried out in the dry state (preferably less than 10% residual water content), the generation of hydroxyl radicals from water is greatly reduced.
In this way, indiscriminate radical damage is further prevented.
Examples of these compounds include the following compounds:

The preparation of these compounds is known. Martin, RF
And Kelly, DP, Aust, JC-hem., 32, 2637-47 (1979);
Firth, W. And Yielding, LW, J.Org.Chem., 47 , 3002 (198
See 2). Other radical generating reagents that generate radicals after irradiation are described in Rlatz et al., Proc.SPIE-Int.Soc.Op.
t. Eng. 847 , 57-60 (1988) and Kanakarajan et al., JACS 11
0 6536-41 (1988).

Radiation sensitizing compounds, which can also be modified to have metal atom substituents, can also be selected from the class of DNA-binding agents, these are netropsin, BD peptide (a common peptide from HMG-1). , S2 peptide, and the like, but are not limited thereto. These DNA-binding agents and other DNA-binding agents are described in Pjura, PE, Grzeskowiak, K. and
Dickerson, RE (1987), J. Mol. Biol. 197 , 267-271, and Tengi, M. Usman, N., Frederick, CA and Wang. AHJ.
(1988), Nucleic Acids Res. 16 , 2671-2690.

Also, the radiation sensitizing compound, which may also have metal atom substituents, may include proteins and / or polypeptides and / or peptides of the type which bind DNA. Examples of this type of protein and / or polypeptide and / or peptide that bind DNA are Churchill,
MEA and Travers, AA (1991) Trends in Biochemica
I Sciences 16 , 92-97. Specific examples of peptides that bind DNA include the SE and BD peptides disclosed in this document.

DNA binding specificity can be obtained by covalently coupling a radiosensitizing compound and / or a metal atom to a DNA binding agent or a protein or polypeptide or peptide that binds DNA.

Subsequent sensitizers were specially designed molecules that formed triplet DNA, such as Youngquist and Dervan PNA.
S 82 2565 (1985); Van Dyke and Dervan, Science 225 1
122 (1984), Van Dyke and Dervan, Nuc.Acids Res. 11 5
555 (1983); Barton and Raphael, PNAS 82 6460 (198
5); Barton et al., JACS 106 2172 (1984); and Barton PN.
Includes molecules disclosed in AS 81 1961 (1984). These molecules have reactive moieties that bind site-specifically to DNA and RNA and, optionally, generate free radicals in the vicinity of DNA or RNA.

R−I + e ⁻ → R · I ⁻ R−I ^+. + Guanine → R−I + guanine ^+. Without wishing to be bound by theory, it is believed that the emitted electrons are trapped by that site with the most favorable electron affinity, which site is probably the second molecule of sensitizer in the sample. Electron trapping by RI (or R-Br) results in RX dissociation with formation of radicals. The radicals thus generated are C-from the sugar moiety of the nearby nucleic acid.
The H hydrogen atom is abstracted, which in turn causes DNA or RNA cleavage and virus inactivation.

The radical cation (R−X ^+. ) Of the sensitizer will eventually withdraw an electron from that component of the sample that has the most favorable oxidation potential. This is probably guanine. The electron transfer reaction produces a guanine radical cation. This material reacts with oxygen after reconstitution with aerated water. This process also results in DNA cleavage and virus inactivation. Unreacted material and reaction byproducts are removed during the washing step involved during reconstitution of lyophilized cells (Table 2).
checking). This process also further removes the virus that has not been inactivated by the above treatment.

Compounds (1) and (2) are firmly attached to DNA and RNA by intercalation and / or by electrostatic interaction between the positively charged ammonium ion group and the negatively charged phosphate group of the nucleic acid target. Join. Red blood cells do not contain nucleic acids and therefore do not bind such compounds by intercalation.

The best way to use the invention is to add a sensitizer to a potentially contaminated blood solution and expose it to gamma or X-rays. The liquid solution of blood is preferably exposed to 3000 rads and the dried lyophilized solid formulation contains 10,000
It is preferably exposed to the radiation of Rad. Red blood cells are known to survive these doses of radiation in the absence of sensitizers. Lyophilized blood can withstand higher dose levels of radiation than hydrated blood.

Gamma or X-rays are mainly absorbed by the heavy atoms of the sensitizer, which is bound to the viral DNA or RNA. Radiation ionizes the sensitizer as described below.

R-I + γ-ray (X-ray) → R-I ^+. + E ^-In some cases, especially if the sensitizer and red blood cells are left together for more than a few minutes, the sensitizer may diffuse into the red blood cells prior to lyophilization.

Antioxidants such as glutathione (an excellent hydrogen atom donor) can be added to the formulation to enhance red blood cell protection against free radical initiated damage. Contamination of cells with sensitizers may also include intracellular viruses, particularly those thought to be inside white blood cells (most concentrated red blood cell units contain residual white blood cells), or intracellular blood parasites (eg, Malaria parasite that infects red blood cells)
It is understood that it enables the deactivation of.

Sensitizers are removed from reconstituted blood serum or protein fractions by the wash protocol described above for lyophilized cells.

Gamma or X-ray radiolysis preferably occurs in lyophilized blood (or protein), virus, and sensitizer formulations that have been dried rather than in a wet liquid substance for several reasons. First, dry matter is not sensitive to radiation, and even larger doses of γ can be obtained without damaging red blood cells.
It may be exposed to rays or other transmitted radiation (see Table 1). This increases the degree of radiolysis of the sensitizer. Second, the sensitizer radicals, which are bound to DNA or RNA in the dry state, cannot dissociate from the virus due to the lack of diffusion in the solid material. It reacts sensitizer radicals with viral RNA or DNA. Third, the solid state condition is probably the quantum mechanics tunnel effect.
g) promotes the hydrogen atom transfer reaction between the sensitizer radical and the viral nucleic acid. Fourth, the reconstitution and wash protocols used for lyophilized blood or protein fractions can be used to remove unreacted material or reaction by-products and further remove virus not affected by treatment (Table 2). checking).

Other types of radiation, which generally include ionizing radiation such as X-rays, can be used. In one embodiment, the metal atom can be a substituent of a chemoradiosensitizer that binds to nucleic acids, thereby targeting embodiments such as bacteria, parasites and viruses. The metal atom substituents of the chemosensitizer for this purpose include Br, I, Zn, Cl, Ca and F. The x-ray source is preferably a tunable source so that radiation can be limited to narrow wavelengths and energy bands if desired. The tunable feature allows optimization of energy absorption by metal atoms, thereby guiding the absorbed penetrating radiation energy to the generation of radicals by the chemosensitizer bound to the nucleic acid.

The present invention can be applied to contaminants containing single-stranded or double-stranded nucleic acid chains containing RNA and DNA, and viruses, bacteria or other parasites containing RNA and / or DNA.

To illustrate the present invention, red blood cells were lyophilized, irradiated and tested for measured red blood cell properties as described above. The results are shown in Table 1. The same procedure was used except that bacteriophage T4 (in dextrose saline) was mixed with the cells and then washed successively with 4 wash buffers. The results are shown in Table 2.

Example 1 Human concentrated red blood cells purified from donated whole blood were washed to remove the anticoagulant stock solution (commercially available CPDA with citrate / phosphate / dextrose / adenine), 10
% Hematocrit suspended in dextrose-saline. Approximately 10 ml of washed concentrated red blood cells are placed in a quartz chamber and exposed to UV light at 2 minute intervals, preferably at 320 nm for a total exposure of 10 minutes. Mix the suspensions every 2 minutes,
A small (10 μl) sample of red blood cells is removed and diluted in 2 ml of water for spectrophotometric analysis of hemoglobin. The temperature of the red blood cell suspension irradiated in each step is measured to ensure that the suspension was not overheated. The suspension did not exceed 26 ° C at any time point (normal body temperature is 37 ° C). Untreated red blood cells usually contain a high proportion of functional oxyhemoglobin (oxyHb), in the range of 96% and above.
including. Oxidative damage is a metastable methemoglobin species (meth
Hb), which is normally reduced back to oxyhemoglobin by cytoreactive enzymes. Hemichrome represents a more severely damaged form, which may be reversible. Normal red blood cells can tolerate modest levels of methemoglobin. Hemichromolysis can produce free heme, the iron-porphyrin component of native hemoglobin, which damages cell membranes. Thus, it is desirable to minimize hemichrome levels. Each hemoglobin species can be detected at a particular wavelength using a conventional spectrophotometer.

The data below shows the sensitivity of hemoglobin to damage by increased UV exposure. 3 minutes of exposure
It was determined that it could be used for viral inactivation using radiosensitizers without undue damage to red blood cells.

Example 2 A suspension (0.1 ml) of bacteriophage lambda or bacteriophage phi-X174 at least 10 EV PFU / ml was added,
Separately added to 4 ml of dextrose-saline containing 1 mg / ml of compound I or II or III. Each suspension of radiosensitizing compound and bacteriophage is then exposed to UV light of the preferred wavelength (320 nm) for a preferred time (3 minutes) in a quartz chamber. A control sample of each bacteriophage suspension containing sensitizer is not exposed to UV light. Serial dilutions are performed to quantify the level of infectious titer, then aliquots of various bacteriophage samples are mixed with host bacteria and plated on nutrient agar. Plates are measured for plaques after a normal growth period.
The other bacteriophage suspensions were separately irradiated as described above without the addition of sensitizers to reveal the effect of this dose of UV radiation alone.

These data show that all three compounds tested significantly increase the susceptibility of the double stranded DNA virus (λ) to UV radiation of the preferred exposure. Also,
Compound I is effective against the single-stranded DNA virus phi-X174. Compound I is most preferred and exhibits a high (at least a 6 log reduction) inactivation potency against both single and double stranded DNA viruses.

─────────────────────────────────────────────────── ─── Continuation of the front page (31) Priority claim number 656,254 (32) Priority date February 15, 1991 (February 15, 1991) (33) Country of priority claim United States (US) (72) Inventor Goodrich Raymond Peugenia California, United States 91106 Pasadena 312 South Mento 140 (72) Inventor Van Bolsom Wahlkes Marian The Netherlands Vennendal 3906 Zetker Bafflan 30 (72) Inventor Wong Victoria A United States California 91107 Pasadena 1 South Greenwood 100 (56) References JP-A-60-16930 (JP, A) JP-A-59-155257 (JP, A) JP-A-61-275228 (JP, A) JP-A-4-507403 (JP, A) US Patent 4620908 (US, A) International publication 90/3187 (WO, A1) International publication 88/10087 (WO, A1) (58) Fields investigated (Int.Cl. ⁷ , DB name) A61K 35/14-35/18

Claims (6)

(57) [Claims] 1. A method for reducing viral and / or bacterial contamination in a biological composition, the composition being selected from the group consisting of compounds that bind to DNA and / or RNA. Contacting with at least one chemosensitizer, wherein the chemosensitizer is a compound capable of generating free radicals upon exposure to radiation, lyophilizing the composition, and the sensitizer A method as described above, comprising exposing the composition to radiation of sufficient wavelength and intensity for a period of time sufficient to further reduce viral and bacterial contamination in the composition. 2. The method of claim 1, wherein the radiation comprises gamma radiation. 3. The chemosensitizer comprises a metal atom.
The method described. 4. The method of claim 1 wherein said chemosensitizer is sensitized by penetrating ionizing radiation. 5. The method of claim 1 wherein said chemosensitizer is sensitized by gamma radiation or X-rays. 6. The method of claim 1 wherein one of said chemosensitizers is selected from the group consisting of compounds of the formula: