JP2000500741A - Microporous pathogen killing composition - Google Patents

Abstract

  (57) [Summary] A microporous material having pores sized to allow entry of pathogen particles but exclude blood cells, where a singlet oxygen production system binds to the microporous material.

Description

DETAILED DESCRIPTION OF THE INVENTION                         Microporous pathogen killing composition                                Field of the invention   Hepatitis and AIDS threats stem from the presence of viral antigens, antibodies, and surrogate markers Blood and blood product screening, The safety of transfusions and blood products is still an issue. The present invention relates to microporous materials. Viruses and other diseases lethal to the associated singlet oxygen production system Provide microporous material that isolates protozoa.                                Background of the Invention   Aspects of the present invention derive from several heterogeneous technologies discussed below.   Photodynamic oxygenation activity: The bactericidal effect of photodynamic action has been known for a century (R aab, 19900). Recently, photochemical-based methodologies have been introduced in blood (Matthews et al., 1998; Sieber et al.). 1989; Neyndorff et al., 1990; O'Brien et al., 1990; Horowitz et al., 1991) and hematopoiesis. (Lin et al., 1989; Prodouz et al., 1991; Dodd et al., 1991; Margolis-Nunno et al., 1992) Developed to inactivate viruses. Light at the appropriate wavelength (hue) The combination of sensitizing molecules and light (hν) is a powerful acid that can kill pathogenic microorganisms. Oxygen molecules (O_Two(For a general review, see Mohr et al. , 1995) invented a practical application for this killing method. Has been almost unsuccessful. In another example, the light is activated by ultraviolet A rays. It has been reported that sensitized psoralen inactivates vesicular stomatitis virus (M argolis-Nunno et al., 1992) and hypericis activated by fluorescent light. It has been reported that hypericin inactivates HIV-1 (Lavie et al., 1995). ).   Photosensitizers may use either type I or type II mechanisms (Schenck and K och, 1960). The following equations explain two types of mechanisms, and use the following terms: use. The "spin multiplicity" of an atom or molecule is defined by the expression | 2S | +1. It is a spectroscopic representation of the total spin quantum number (S) of the molecule as defined. Reactant I.e., the photosensitizer and O_TwoThe spin multiplicity of is described by the superscript number at the front And an asterisk is used to indicate an electronically excited molecule You.   A photosensitizer in the S = 0 state has singlet multiplicity; | 2 (0) | + 1 = 1 (singlet,¹ Photosensitizer molecule). The excited product is in an excited state of electrons but still If they have the same spin number, they maintain the singlet. If a photosensitizer is If they have the same structure and symmetry, intersystem crossing can occur; Changing the spin quantum number (S) of the excited electrons by a singlet photosensitizer molecule Can relax to an electronically lower energy excited state. So relaxed Where S changes from 0 to 1 and the multiplicity, | 2 (1) | + 1 = 3, is the electronically excited triplet (^ThreePhotosensitizer^*Changes to:               ¹Photosensitizer^*  ―Intersystem crossing →^ThreePhotosensitizer^*                (1)   This first event is the same for either type I or type II mechanisms. This The first event of is the ground state singlet multiplicity dye (¹Photosensitizer) absorbs light photons And electronically excited singlet state (¹Photosensitizer^*Is converted to:               ¹Photosensitizer + ray (hν) →¹Photosensitizer^*                  (2)   In a type I reaction, the electron is divided into two radicals (the reduced doublet photosensitizer Excited triple from reduced substrate, yielding nonions and oxidized doublet substrate cations) Transition to the term photosensitizer:               ^ThreePhotosensitizer^*+¹Substrate →^TwoPhotosensitizer^-+^TwoSubstrate⁺             (3) Alternatively, electrons transfer from the excited triplet photosensitizer molecule to the substrate.               ^ThreePhotosensitizer^*+¹Substrate →^TwoPhotosensitizer⁺+^TwoSubstrate^-             (4)   The produced radicals can react with other radicals in the annihilation reaction, or It can react with higher multiplicity molecules in radical multiplication reactions.   For example, photosensitizer radicals produce oxygen radicals that can kill pathogens Can be utilized via a type II reaction. Reaction of oxygen with biomolecules is very ergogenic However, such reactions do not occur spontaneously. Biomolecules are typical In general, singlet multiplicity, but the oxygen we breathe is paramagnetic with triplet multiplicity It is a body molecule. According to Wigner's spin conservation law, this form of biomolecules and oxygen Reactions are less likely to occur (Allen, 1994). However, ground state triplet acid Element (^ThreeO_Two) Is a singlet acid in a type II reaction using an electronically excited triplet photosensitizer. Can be converted to a prime:               ^ThreePhotosensitizer^*+^ThreeO_Two→¹Photosensitizer +¹O_Two ^*                  (5)   This triplet-triplet annihilation proceeds via the singlet reaction interface and is Bottom state singlet photosensitizer molecule and singlet oxygen molecule (¹O_Two ^*) Occurs. O_TwoElectronic Excited form¹Δ_gIs the ground state O_Two ^ThreeΣ_g22.5kcal / mol energy It is. In the singlet state, O_TwoIs the allowed spin with singlet multiplicity biomolecules Can be directly involved in the reaction. in this way,¹O_Two ^*Electrophilic oxygenation activity of bacteria and Can be directed to killing other pathogens or to inactivating the virus.               ¹O_Two ^*+ Microorganism → killing or inactivating microorganisms. (6)   Photosensitizer-mediated inactivation of blood virus is essentially due to such a type II singlet Both the type I and type II mechanisms occur through the oxygen mechanism, whereas the red blood cell Contributes to bleb damage. (Rywkin et al., 1992).   Most singlet electronically excited molecules have relatively short lifetimes (eg, 10^-8Seconds) You. Their short-lived nature limits their involvement in chemical reactions. Those luck Life is to return to the ground state by photon emission. Fluorescence is their singlet basis It is the energy product of a singlet excited molecule that relaxes to a state. Triplet electronically excited molecule Has a relatively long lifetime (eg, 10^-2~Ten^TwoSeconds). Phosphorescence is a singlet basis It is the energy product of a triplet excited molecule that relaxes to a state. Excitation by intersystem crossing The transition of Equation 2 from the singlet state to the excited triplet state is a low likelihood event. So Thus, the excited triplet is in a metastable state and can participate in chemical reactions. Singlet^*→ One Unlike triplet transition fluorescence, triplet^*→ Singlet transition phosphorescence is chemically quenched And especially O_TwoVery sensitive to quenching. Relatively long triplet excited state Long lifetimes are the result of low likelihood of spin transitions.   Oxygen is unusual in that its ground state is a triplet. Singlet excited state Relaxation from the state to the triplet ground state also requires intersystem crossing, in the opposite direction You. The low probability of this transition is¹O_Two ^*Responsible for the relative metastability of In aqueous solution¹O_Two ^* Has been reported to be in the microsecond range (Merkel and Kearns, 197 2). in this way,¹O_Two ^*Can act as a chemical reactant, but due to its short lifespan, The response radius is limited to within 0.1-0.2 micrometers of the point of formation (Lindig and And Rodgers, 1981).   Haloperoxidase monooxygenation activity: Haloperoxidase (eg, myelo Peroxidase (MPO) and eosinophil peroxidase (EPO) Or bromide can catalyze the oxidation of hydrogen peroxide to hypochlorous or hypobromite (Al len, 1975a, b):             Cl^-+ H_TwoO_Two―MPO → HOCl + H_TwoO. (7)   Reaction of singlet multiplicity of hypohalous acid with additional singlet multiplicity of hydrogen peroxide. Proceeds through the singlet reaction interface:             HOCl + H_TwoO_Two→ Cl^-+ H_TwoO_Two+¹O_Two ^*, (8) And, thus, all of the oxygen-containing reaction products are singlet multiplicity . Thus, haloperoxidase is the same counterpart that can be generated by photosensitizers. A reaction product,¹O_Two ^*To provide an enzymatic system for generating   Carbohydrate-lectin mechanism in microbial infection and defense  Virus feels cells The mechanism of staining is lectin (which binds to specific sugars on glycoproteins or glycolipids). (A family of proteins that bind tightly). Influue Example of a virus, how viral lectins are involved in the infection process I will explain. Influenza virus is required to enter the cytoplasmic fraction of infected cells. The hemagglutinin protein on the surface of the virus particles binds to sialic acid on the cell membrane. (Weis et al., 1988). Thus, influenza hemagglutination Nitrogen is a lectin that specifically binds sialic acid.   Similarly, HIV-1 and HIV-2 may also be required for cell binding and infection Lectin-like activity. Two Enzymes of Human Immunodeficiency Virus (HIV-1) It appears that the envelope glycoprotein has lectin-like activity. HIV-1 Both the outer membrane protein, gp120, and the transmembrane protein, gp41, , Derived from cleavage of the gp160 precursor, and both proteins Act as lectin and N-acetylglucosaminyl (GlcNAC) binding lectins (Ha ihar et al., 1992a and b). Α-D-Mannosyl residue of glycoprotein on host cell membrane You And α-D-glucosyl residues serve as anchoring sites for these HIV-1 proteins. Can act, thus facilitating virus entry into cells. This mechanism is non-CD-4 cells And / or may assist in CD4-dependent infection mechanisms.   Other lectins can bind to bacteria and viruses. Mannosyl-specific serum Kuching, a mannan binding protein (MBP), has a broad spectrum of bacterial recognition and And acute phase proteins involved in complement activation (Holmskov et al., 1994). Manno Salmonella montevideo containing high-enriched lipopolysaccharide shows high MBP binding levels (Van Emmerick et al., 1994). MBP also binds to the HIV-1 envelope protein. Inhibit HIV-1 infection of cells in vitro (Lifson et al., 1986; Robins on et al., 1987; Ezekowitz et al., 1989; Hansen et al., 1989). α-D-mannosyl residue and Concanava, a plant lectin with affinity for α-D-glucosyl residues Phosphor A (ConA) is also a viral gp120 enzyme that attaches to HIV-1 but binds CD-4. Does not interfere with the envelope glycoprotein (Gattegno et al., 1992). But lymphoid cells Of Con-A-conjugated HIV-1 on HIV decreases in a carbohydrate-specific, dose-dependent manner (Gattegno et al., 1992).   Many bacteria are known to bind to and aggregate with red blood cells, and this The collection is inhibited in some cases by specific monosaccharides. This is It has been shown that bacterial lectins mediate this binding. Mannose specific surface Lectins include Escherichia coli, Klebsiella pneumonia, Salmonella species, Serrat Present on ia marcescens, Shiglla, Enterobacter, and Erwinia species (Du guid and Old, 1980; Speert et al., 1984). Binding to N-acetylglucosamine E. coli lectins with specificity have also been reported (Vaisanen-Rhen et al., 1983). ). With various carbohydrate specificities, these and other lectins are Provides an immobilization mechanism that mediates bacterial binding to and is required during early infection Found in the appendages of filamentous bacteria called fimbriae or pili . E. coli type I ciliary binding to various mannose derivatives and complex type mannose The relative specificity of cutin binding has been reported (Firon et al., 1983). Lectin knot The case was directed to the short oligomannose chain of the N-linked glycoprotein. Like that Unique structures are common to mucosal cell surfaces (Sharon and Lis, 1982). Same lek A pattern of tin specificity was observed from several bacteria of the same bacterial genus. this child That mannose can play a major role in infection by a wide variety of bacteria Suggests. (Firon et al., 1984).   Binding and fungicide properties of haloperoxidase.： Haloperoxidase in white blood cells Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) A cationic glycoprotein that selectively binds and kills bacteria (Allen, 199 2, US Patent Application No. 07 / 660,994; Allen, 1995, US Patent No. 5.389,369). MPO The binding and killing specificity of EPO and EPO to certain Gram-negative microorganisms Includes a chin-carbohydrate binding mechanism. MPO and EPO are both mannose and And N-acetylglucosamine (Yamada et al., 1981; Miyasaki et al., 1986; Olsen et al. 1985; Allen, unpublished finding of lectin binding specificity). Thus, MPO and E PO is Escherichia coli, Klebsiella pneumonia, Salmonella species, Serratia mar Many gram shades including cescens, Shigella species, Enterobacter species, and Erwinia species (Duguid and Old, 1980; Speert et al., 1984). E.co with N-acetylglucosamine binding specificity The li lectin has also been reported (Vaisenen-Rhen et al., 1983). These recti The binding of MPO and EPO to bacteria that carry Yes, and is essentially independent of ionic strength.   As discussed above, HIV-1 and HIV-2 produce α-D-mannosyl and N-acetate. Binding via an envelope protein with tylglucosaminyl carbohydrate structure I do. Thus, both viruses can selectively bind to MPO and EPO. MPO and E PO is known to inactivate viruses, including HIV-1 (Klebanoff and And Belding, 1974; Klebanoff and Coombs, 1991). In addition, prepare with ExOxEmis NIH-sponsored contract studies using MPOs and EPOs have shown high HIV-1 resistance to AZT Level inactivation was demonstrated.   Molecular and particle sieving (gel filtration): Regularly arranged at regular intervals There are microporous materials that interweave with subcellular channels. Groove network May comprise a substantial proportion of the total amount of microporous material. Porous substances have a crystalline structure From aluminum silicate or aluminum phosphate, and cross-linked dextran Includes a composed gel.   Porath and Flodin (1959) provide a tool for rapid separation of macromolecules based on size. Cross-linked dextran beads (ie Sephadex (Pha rmaca)) was introduced. Generally referred to as gel filtration The procedure does not require the use of a gel and is not a true filtration. Three of beads A dimensional molecular network is composed of many open pores of a certain size range. If the pore size is too small to allow a large molecule to enter, the molecule is excluded. On the other hand, molecules smaller than the pore size can enter the pore matrix. Follow Thus, molecules that cannot enter the pore matrix are excluded from this section of the gel, and Runs off the gel at the same rate as the aqueous solution. A smaller part of the maze in the hole Very large volumes of solution are required to elute the pups. Smaller to penetrate the hole Molecules can be retained by the equilibrium of molecules entering and leaving the pore. Small molecule size and The shape and the size and shape of the pores determine the equilibrium conditions. In this way physical The degree of specific binding determines the delay in elution time.   Gel filtration techniques have been used to isolate and purify viruses. example Crude bovine warts by gel filtration using Fine Chemicals, Uppsala, Sweden). It has been purified from the extract. Hjorth and Moreno-Lopez, J.A. Virol. Methods, Barley yellow dwarf virus can be used to It is refined from wheat. Hewish and Shukula, J.M. Virol. Methods, 7: 223-228 (1983). In both cases, purity (as determined by SDS-PAGE) and isolated The yield of the virus obtained was high. Therefore, this reduces virus particles in solution. 9 illustrates the selectivity and ability of a gel filtration solid support to sequester. these The result is that virus particles are easily separated from larger particles by gel filtration technology Demonstrate what can be done.   Various molecules can be covalently linked to the chemically activated sieve. Many laps Known techniques are available for these purposes (e.g. Affinity Chromatography (Pharmacia Fine Chemicals) Provided).                                Summary of the Invention   The present invention provides pores sized to allow entry of pathogen particles but exclude blood cells. And a microporous material having the same. Here, the singlet oxygen production system has its microporosity Binds to substance. A typical microporous material for this purpose is a controlled pore glass And carbohydrate polymers. In an exemplary embodiment, the pore is a virus particle. Size in the range of about 0.1 μm to about 1.0 μm to allow penetration of Or The pores can be up to about 3 μm in size to allow bacterial entry. Singlet oxygen The production system can be a photoactivated photosensitizer or an enzymatic system. Appropriate Photosensitizers include phthalocyanine, porphyrin, methylene bull , Hypericin, fluorescein derivatives, and psoralen. Singlet Preferred enzyme systems for the system for producing oxygen include oxidase and Contains haloperoxidase. The oxidase is preferably present in the blood The substrate can be oxidized. A suitable oxidase for this purpose is lactate oxidase , Oxalate oxidase, glucose oxidase and cholesterol oxy And Dase. Haloperoxidase, myeloperoxidase, eosinophils Selected from the group consisting of peroxidase and fungal chloroperoxidase Can be Singlet oxygen production systems can be microporous, either covalently or non-covalently. May bind to sexual substances. Microporous materials include beads, wafers, gel filtration matrices Configure or form devices such as filters, filters, bags, or tubes I can do it.   In a particularly preferred embodiment, the ligand that binds to the pathogen particle is a microporous It also binds to substances. Ligands include high mannose glycans, N-glucosamine, N-acetylglucosaminyl nucleus of glycans, mannosyl nucleus of complex N-linked glycans, Mannan, α-methyl mannoside, haloperoxidase, sulfated polysaccharide, low content Dextran sulfate, and lectins. Good for this purpose Preferred ligands are the haloperoxidase myeloperoxidase and lekoperoxidase. Includes chin concanavalin A. In an exemplary embodiment, the microporous material is Concanana to which lactate oxidase and myeloperoxidase are bound respectively Takes the form of a carbohydrate polymer carrying valine A.                             BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows microporous material in contact with blood, and a pore diameter of 1 μm To eliminate red blood cells (RBC) as efficiently as possible Is shown in the figure.   Figure 2 shows that myeloperoxidase (MPO) and oxidase are covalently linked. The pores (lumens) of the microporous material (sieving material) are illustrated. Regan on MPO Close to the virus particles that have been captured by the Schematic illustration of oxidase and MPO-catalyzed reactions producing singlet oxygen in close proximity I do.   FIG. 3 shows that the virus killing activity of singlet oxygen is limited by its short lifespan Illustrate valid radii (overlapping red circles) within a range. According to reports, The lifetime of singlet oxygen is in the microsecond range, so its radius of reactivity is Its production is limited to 0.1 μm to 0.2 μm.   Fig. 4 shows a sieve substance containing Concanavalin A to which MPO is non-covalently bound. Is exemplified. Oxidase is bound to the sieve material. The virus is conca In the pore by ligand-receptor interaction with navalin A, MPO or both Selectively retained. Singlet oxygen produced by the immobilized enzyme kills the virus Wound or inactivate.                       Detailed Description of the Preferred Embodiment   The invention relates to biological fluids of pathogen particles such as viruses and bacteria (eg, Blood) is provided. Microporous substances are pathogen particles To allow entry of desired cells (eg, cells of blood) in a biological fluid. Having pores that are sized to exclude the sexual element). According to the present invention, singlet oxygen production The system is coupled to the microporous material. Microporous substances are found in blood and other raw materials. Pathogens (eg, viruses, bacteria, prions, or other similar Form a sieving matrix where the size of the pathogen particles) is removed and inactivated. To achieve. In an exemplary embodiment, the enzyme component of the singlet oxygen production system comprises It is connected to the groove of the gel. Pathogen binding ligand molecules also Can be combined.   The nature of the sieving gel, and especially its pore size, depends on the molecules held by the substance. Alternatively, the size of the particles is specified. Generally, gel particles are made of natural polymers and synthetic It is a polymerizable substance including a polymer. Polymers are linear or branched long Are often covalently linked to each other to form High molecular weight compounds consisting of smaller subunits that also have interchain bonds between the units It is. The polymerizable material used in gel filtration is a natural polysaccharide (eg, , Agarose, dextran, and cellulose) and synthetic polymers (eg, Polyacrylamide, polytrisacrylamide, and hydroxylated vinyl Polymer). These polymers include particles having various pore sizes (and Can be formed into beads). The size of the pores in the matrix is It can be controlled by the degree of crosslinking. Generally, highly crosslinked polymers are: It has a smaller pore size than the lightly crosslinked polymer.   For the present invention, the pore sieving matrix is a crosslinked carbohydrate (eg, poly Dextran, controlled pore glass, or other suitable materials). Frame Allyldextran crosslinked with bridged agarose and bisacrylamide Many of these materials are commercially available, including psala, Sweden). these In gels, the polymeric material is crosslinked to various degrees using bisacrylamide. Polysaccharide.   A variety of devices are available for sieving materials (eg, beads, tubes, flat porous surfaces, backing Plastic blood bag, filter, hollow cellulose tube filter, And gel filtration matrices). Sieve substance is a groove Communicates with pores or external liquids and allows the passage of liquid or gaseous substances Void region or void region within the solid material. The diameter of the groove is the pathogenic virus Large enough to allow the entry of bacteria, bacteria, or other similarly sized pathogens. Can get. As shown in FIG. 1, the pore diameter is sufficient for the desired cells, especially for the cellular components of blood. Small enough to eliminate in minutes.   As shown in Table 1, the virus particles contaminating the blood had a diameter of 20 nm (Parbow Ls) to 150 nm (0.02 μm to 0.15 μm). With the least cellular elements of the blood Some platelets have a free diameter of about 3000 nm (3 μm). Red blood cells, 7.2-7. The next smallest molecule in size with a diameter of 9 μm. Thus, about 155n with pore sizes ranging from about m (about 0.15 μm) to about 2,500 nm (or less than 3 μm) Any substance that removes virus particles while eliminating blood platelets and other cellular components Physically house the offspring.   The pore size of about 2.5 μm is for non-viral with a diameter smaller than the platelet diameter. It is particularly useful for isolating sexual pathogens. Bacteria are typically 2 μm in diameter And most or all bacteria have pores with a diameter of about 2-3 μm. Can get into it. Therefore, the size of the pores just slightly smaller than the platelet diameter of 3 μm Kasa contains most pathogens that are expected to contaminate the blood supply.   In a first embodiment of the present invention, a photosensitizer (eg, a fluorescein derivative) Or a phthalocyanine derivative or other suitable molecule) can be The covalent bond binds to the surface of the groove present in the pores of the sieve material. Sometimes, "photodynamic A "photosensitizer", also known as a "dye", transfers the energy of the excited state to another compound. Or a compound or molecule that can be transferred to a molecule. Photosensitizers absorb photons of light rays After that, the state becomes electronically excited, and then the energy of the excited state is transferred to another compound. Or transfer to molecules. The excited energy is converted to oxygen molecules (ie, Oxygen at the bottom,^ThreeO_Two), Thereby causing the excited oxygen molecules (ie, excited Oxygen in the state,¹O_Two ^*The photosensitizer that produces) is a singlet oxygen photosensitizer.   The photosensitizer is converted to a triplet excited state when illuminated with light of the appropriate wavelength. Characteristics. The excited triplet photosensitizer molecule is then Involved in a type II reaction with triplet oxygen in the ground state of Reactive singlet oxygen,¹O_Two ^*To produce   The photosensitizers contemplated by the present invention include visible light (about 350 nm to about 700 nm) and near- It is a photosensitizer that absorbs infrared rays (about 700 nm to about 1000 nm). Activate photosensitizer Purify the blood because the light needed to be absorbed is not absorbed by red blood cells Preferred photosensitizers for the red region (about 600 nm to about 700 nm) and the near infrared region Includes compounds that absorb visible light. Exemplary families of photosensitizers suitable for the present invention Lee is acridine, fluorescein, xanthine, rhodamine, porphyrin , Cyanines, and phthalocyanines. Specific useful in compositions of the invention Photosensitizers include methylene blue, hypericin, hematoporphine, and fluorescein. And derivatives of fluorescein (eg, rose bengal) , Eosin, and erythrosine).   In a related embodiment, to assist in capturing or retaining the pathogen particles, On the surface of the pore, the virus or bacterial binding composition may also be covalent or non-covalent It is connected associatively. The enhanced capture of the virus results in ligands ( (E.g. concanavalin A or other lectins). Can be achieved by Further capture of pathogens can be Luic acid, mannose, N-acetylglucosamine, haloperoxidase (eg, Myeloperoxidase or eosinophil peroxidase), or And other known to have the ability to bind to molecules present on the surface of bacteria (Any substance). This embodiment An important advantage is that ligand-mediated binding of the pathogen is itself and its own. It serves only to partially remove the pathogen particles from the blood.   The main advantage of using a photosensitizer attached to a microporous sieving material is The approach prevents the problem of photosensitizers binding to important blood components, the circulatory system Eliminating the toxicity associated with introducing photosensitizers into cells, and also cellular components To minimize damage to the The relative disadvantage of this approach is that That is, a light beam is needed to drive the objective action. But O_TwoElectronic excitation of^Three Σ_g→¹Δ_gIs a relatively low energy transition requiring about 23 kcal / mol. blood For the purification of liquids, in the red and near infrared which are not absorbed by hemoglobin Even enough conversion molecules can be used to drive this process.   In a second preferred embodiment of the present invention, the sieve material is chemically HOCl or And¹O_TwoIs covalently or non-covalently linked to an enzyme system that can produce One This Systems such as halides and H2 as substrates to produce singlet oxygen_TwoO_Two Consisting of the aforementioned haloperoxidase which requires Haloperoxidase is salt Hydrogen peroxidase oxidation of bromine or bromine to hypochlorous or hypobromite. Mediate. The amount of chlorine normally present in the blood is determined by myeloperoxidase. Provide sufficient halide for the catalyzed reaction (Allen, US Pat. No. 5,389,369) issue). Therefore, hypohalous acid is an additional component of hydrogen peroxide. A reaction with offspring produces singlet oxygen. Directly around the reactive singlet oxygen molecule The virus or bacteria present on the side is inactivated or killed. Hole too large Blood components that cannot enter the_TwoO_TwoOr significant singlet oxygen I can't. Haloperoxidases suitable for the present invention include myeloperoxidase, Includes eosinophil peroxidase, and fungal chloroperoxidase.   Oxidases that can act on the substrates normally present in the blood are readily available To provide the hydrogen peroxide substrate required by haloperoxidase. Such oxidases include lactate oxidase, oxalate oxidase, choles Use terol oxidase, glucose oxidase, or substrates in the blood Other oxidases that have the ability to For example, insufficient amounts of lactic acid or If glucose is present, these substrates may not be sufficient for the selected oxidase. Can be added to blood or blood collection bags to ensure proper hydrogen peroxide production . Lactate oxidase reacts with H_TwoO_TwoProduces:             Lactic acid + O_Two→ Pyruvate + H_TwoO_Two                          (9)   In equation (9), the peroxide is converted from lactic acid to L-lactic acid: O_TwoOxidoreductase E Produced by C 1.1.3.x (Naka, 1993). Lactic acid is typically relatively present in the blood It is an excellent substrate present at high concentrations. The venous blood of a normal and healthy adult is 1 mm (9 mg). / dL). Lactic acid is the end product of glycolytic metabolism, It is a major product of Lucose metabolism. Each molecule of metabolized glucose is 2 molecules To produce lactic acid. Lactic acid continues to be produced by erythrocytes during blood bank storage You. Thus, the peroxide produced by lactate oxidase is a sieve-bound halo. Provides a substrate for peroxidase.   Other oxidases (eg, oxalate oxidase (oxalate: O_Two  Oxide Reductase, EC1.2.3.4), cholesterol oxidase (cholesterol: O_Two Oxidoreductase, EC1.1.3.6.), Glucose oxidase, etc. Substrates are also present in blood and can be used in a similar manner. These, and other Oxidases are well known, and some of these are disclosed in U.S. Patent No. 5,389,369. It is described in some detail in the issue.   Figure 2 shows a surface with lactate oxidase and myeloperoxidase bound. 1 schematically shows a cross-sectional view of a virus-containing hole. This hole has a diameter of about 0.2μm ~ 0.3μm Yes, including the size of two viral particles of HIV (ie, about 0.1 μm in diameter). Larger pores can be used to cover bacterial and even cellular pathogens. Apart from the advantages of capturing pathogen particles, the isolated environment of the pores also Acts to concentrate and ligate substrates and products required for interaction . FIG. 2 further shows that the oxidation of lactate to pyruvate is close to the captured virus particles. Touched¹O_Two ^*How the production of is driven. FIG.¹O_Two ^*Activity Shows the extent to which gender is limited due to its short lifetime (overlapping circles).   For the enzyme system of the present invention, concanavalin A has a haloper Oxidases can be used for non-covalent attachment. Concanavalin A molecule It is a protein lectin derived from beans. Concanavalin A molecule is carbohydrate It has four sites that can be non-covalently attached to an object and thus has the ability to aggregate various animal cells Having. As illustrated in FIG. 4, the tetravalent molecule concanavalin A is a microporous matrix. "Platform" to guide some components of the components in close proximity within the pores of Trix Can act as " To achieve this multi-component proximity, concanavalin A One of the reactive sites is first covalently bound to the microporous substrate itself, and then its lectin Reactive site binds haloperoxidase and mannosylated oxidase Is allowed to do so. Under empirically controlled stoichiometric conditions, lectin molecules Defines a reactive site available for binding to a receptor present on a virus or bacterium. Still have. This strategy is illustrated in FIG. This is because MPOs Shows covalently attached concanavalin A that is covalently bound. In this example, The oxidase is directly linked to the sieving molecule, whereas in other applications the oxidase is It can be nonsylated and non-covalently linked to Concanavalin A.   H in the pore environment_TwoO_TwoLimiting the production of has several advantages. This is the pathogen Maximize contact between body particles and pore-bound myeloperoxidase,_TwoO_TwoDilution , And H_TwoO_TwoMake sure that they do not come into contact with red blood cells H by catalase_TwoO_TwoTo avoid the conversion). The blood cellular component is also H_TwoO_TwoBreaking Protected by limiting catastrophic effects to the pore environment. Most importantly, this This limits peroxide production by oxidase at the site of MPO. Restrictions on pore space Is also HOCl (H_TwoO_TwoDependent MPO-catalyzed product of chloride oxidation) but additional H_TwoO_TwoAnd anti In response¹O_Two ^*Is produced. In the pore space¹O_Two ^*Enzymatic production of High level of focus without the need for exogenous energy sources Ensure anti-pathogen activity. Unlike systems based on photosensitizers,¹O_Two ^*The enzyme Production has the advantage that no external light source is required. Spatial restrictions are Reaction radius less than 0.2 μm required for maximum virus lethality or bactericidal action Limited lifespan by localizing pathogens within¹O_Two ^*The reaction potential of You.   The present invention provides for the use of pathogen particles, particularly viruses, from blood or other liquids. Compositions useful for removing bacteria, bacteria, and prions. For example, The microporous composition of the present invention is a drug produced by cultured eukaryotic cells or prokaryotic cells. Can be used to remove viruses from biological preparations. In addition, the composition is pure It can be applied to remove pathogens from virtually any fluid that requires a degree.   The composition of the present invention is such that pores are opened on surfaces that interact with the liquid to be purified. In all or a part of the microporous material. Typical minute Porous materials include controlled pore glass and polymers (eg, crosslinked carbohydrates) I do. These holes allow entry of pathogen particles (eg, viruses and bacteria). It is the appropriate size to work. The method for producing holes of the desired diameter is It is known in the field. The size of the pores in the crosslinked polymer may be, for example, The pores in the glass beads can be controlled, but can be controlled by manipulating the amount of the agent. Can be established by etching. About 0. Pores ranging in size from 1 μm to about 1.0 μm, and about Pores up to 2.5 μm in size are particularly useful. Holes less than about 2.5 μm in diameter It offers the advantage of excluding cellular components of, for example, blood) from the interstices of the porous material.   The compositions of the present invention also employ one or more ligand-receptor pairs. obtain. Generally, a ligand is a receptor molecule or a receptor molecule on the surface of a cell or virus. A molecule or group of molecules that binds to a site. One such pair is one of the pair One member binds to the microporous material, and the other member is a microorganism (eg, Microorganisms, if present on the surface of viruses or bacteria) Adhere to the microporous material by the force of the non-covalent bond between the two members of the Consists of two molecules with sufficient affinity for each other. Composition of the present invention For substances, members of the ligand-receptor pair that bind to the microporous surface are: Ligands referred to as "ligands" and on the surface of a virus or bacterium A molecule is a “receptor”. Concanavalin A is a virus (eg, HIV- 1) Ligand capable of binding to the above mannan oligosaccharide receptor (s) This is an example.   Various substances, including carbohydrate moieties, glycoproteins, lectins, etc. Can form a composition of a do-receptor pair. Suitable ligands include high mannose Can, mannosylated surface of complex N-linked glycans, mannosyl Nucleus, N-acetylglucosamine and its derivatives, mannan, sulfated polysaccharides (eg, For example, heparin or low molecular weight (MW) dextran sulfate (DS), lectin (eg, , Concanavalin A and α-D-mannosyl, α-D-glycosyl residues, and N- Other lectins sharing concanavalin A affinity for cetyl glucosamine ). Concanavalin A adheres to HIV-1 (Gattegno et al., 1992) and And also binds to EPO, MPO, and mannosylated oxidase.   The compositions of the present invention provide that the ligand is spread over the microporous material, and particularly over the material. Need to be bonded to the hole. These types of molecules are covalently bonded to the polymer surface. Methods are widely available for interlocking.   The microporous material of the present invention can be configured into various devices. Such devices are , Beads, gel filtration matrices, filters, bags, tubes, and hollow cellulose Includes the filters that make up the iris tubes. For example, to remove microorganisms from blood For example, blood may be filtered ex vivo through a filter or column containing microporous material. Let through. The blood can be filtered before being placed in the storage bag, or Is Can be filtered when removed from the bag, or both . Inactivates particulate virus and / or bacteria while blood is stored in the bag In order for the blood to be stored, the inner surface of the bag where the blood is stored is a thin layer of microporous material. Substances or beads that can be coated or consist of microporous substances A tube lined with wafers or wafers can simply be placed inside the bag. These And other devices so that equilibrium can be established between pathogens within and outside the pores. Meet the criteria that the microporous material comes into contact with the liquid to be purified.                                  Example1. A microporous material with controlled pores.   Any composition having pores of the required size is a microporous material (ie, Sieve material). Several different types of sieve materials are commercially available Have been. Sephacryl S-1000 (Pharmacia, Sigma S-1000) is a 40-105 μm Diameter and 1x10⁹Allyl dextran and dalton, which have a Dalton pore size exclusion limit And a cross-linked copolymer of N, N'-methylenebisacrylamide. Sepharose  CL2B (Pharmacia, Sigma CL-2B-300) also has a bead diameter of 60-200 μm. And 0.2x10⁹Cross-linked agarose that excludes Dalton pores and is used against small viruses. Can be used. Controlled pore glass heats the borosilicate glass being processed And rubbing out boric acid with an acid. With controlled holes Glyceryl Glass (Sigma GG3000-200) has a pore size of 0.3 μm (300 nm). Available in 20-200 mesh (75-125 μm diameter).   Many technologies use chemically activated sieve materials and activated materials. Available for covalently bound proteins (Jakoby and Wilchek, 1 974). Below is CNBr activation of Sephacryl, Sepharose, and glyceryl glass And MPO, EPO, lactate oxidase, to activated sieve substances And examples of concanavalin A binding.2. Preparation of sieve material   The following preparation steps were applied separately: 20 ml stock suspension in 20% ethanol Sepharose 2B (Sigma CL-2B-300); 20 ml stock suspension in 20% ethanol Sephacryl S-1000 (Sigma S-1000); Solid 10 ml (10 cc) Glyceryl-Glass Cotrolled Pore (Sigma GG3000-200).   In the first step, the sieve material is leached to 50 ml with distilled water and er) on top for 30-60 minutes. Next, the substance is allowed to settle, and the aqueous supernatant is decanted. I did it. The material was washed with a second 50 ml of distilled water as before. second time After decanting the wash water, 2M phosphate buffer (PB), pH 11.4, (536 g Na_TwoHP O_Four-7H_TwoO / L H_TwoAdjusted to pH 11.4 with O and NaOH) until a final volume of 50 ml Was added. Place under vacuum for 1 hour to degas, then place in ice bath to cool placed.3. The sieve material can be activated using bromocyan (CNBr) (Sigma C-6388). When.   Highly buffered alkaline bromo cyanide (CNBr) method (Porath, 1974; Par. ikh et al., 1974) for bead activation and protein coupling. Was. Add 2 g of CNBr crystals to 20 ml of cold distilled water and mix to dissolve. Was. (Only 50% of the crystals were dissolved after 30 minutes.) A total of 4 ml of CNBr (200 mg total at approximately 50 mg / ml) was added over 0.5 ml over the interval. . 4 volumes of H at 4 ° C_TwoWash the beads with a 0.2 μm filter using O. Was.4. Covalent linkage of protein to cyclic imino carbonate on beads.   MPO-lactate oxidase (MPO-LOx), EPO-LOx, and concanavalin A Prepared for the coupling as follows:   (A.)MPO-LOx (And lactate oxidase): 5ml myeloperoxidase ( 10 mg, porcine, ExOxEmis, Lot 1899201, total 70 nmol), 3.2 mg And Pediococcus sp. (3.2 mg, 108 units, Sigma L-06380).   (B.)EPO-LOx (And lactate oxidase): 5 ml of eosinophil peroxidase ( 10 mg, porcine, ExOxEmis, Lot 1929201, 150 nmol), 3.2 mg (Pediococcus sp., 3.2 mg, 108 units, Sigma L-06380).   (C.)Con-A: 10 mg of concanavalin A, type IV, Canavalin ensiformis (Sigma  C-2010) was dissolved in 5 ml of distilled water.   Similarly, oxidases that can use substrates present in the blood were prepared concomitantly. Obtained and allowed to bind to the activated beads.   The beads activated above were treated with 0.25 M bicarbonate buffer (BB), pH 9.0 (pH was adjusted with NaOH). 21g NaHCO_Three/ L H_TwoResuspended in O) to a final volume of 30 ml. Se above Contains each of pharose 2B, Sephacryl S-1000, and Glyceryl Glass Divide each 30 ml bead suspension into equal volumes into three tubes, for a total of nine tubes did. For each bead preparation tube: 1.75 ml MPO-LOx, 1.75 ml EPO-LOx, and And 1.75 ml of ConA were added. Then place the tube on a rocker to mix gently. -Place on a rocker table and react at 22 ° C overnight (12 hours) .   Upon completion of the coupling, a 1 M glycine solution (75 g glycine / l) was prepared. And added to each bead suspension to a final volume of 50 ml. Glycine, Provided to react with any unreacted imino carbonate remaining on the column.   The successful binding of haloperoxidase to the beads indicates that the derivatized This was evident from the coloration of the dose. Once settled, the bead preparation is MPO specific A pale green color and an EPO orange-brown color. After the beads have settled UV-visible spectroscopy was performed to quantify the protein remaining in the supernatant. The amount of MPO bound to each type of beads was 78 mM^-1cm^-1Use the millimolar extinction coefficient of 4 By measuring the reduced (dithionite) -oxidized difference spectrum at 75 nm And quantified. The results showed that 23.3 nmol of MPO bound to each type of bead.   The amount of EPO bound to the beads was 125 mM^-1cm^-1At 449 nm using extinction coefficient Original (dithionite)-quantified as a difference spectrum to be oxidized. The results indicated that 50 nmol of EPO bound to each type of bead.   Vibration to which either MPO or EPO is bound with Dulbecco's Phosphate Buffer pH 7.4 Washes once more and a final volume of 10 in Dulbecco's Phosphate Buffer was used. It was adjusted to ml.   To determine the maximum amount of MPO and EPO that can bind to each type of activated bead Was not performed. However, the results not shown in this example indicate that the binding capacity of the beads The force suggests that it is much greater than the amount bound in this experiment. Join The amount is such that further increases in concentration do not result in an increase in the amount of protein binding to the beads. Higher concentrations of MPO and EPO during the binding step until the It can be maximized by incremental additions.5. Bacteriophage model of pores: lactate-oxidase-haloperoxidase Virus inactivation.   Bacteria, like eukaryotic cells, are susceptible to viral infection and lysis. This kind of The virus is called bacteriophage or phage. Bacteriophage Are comparable in size to animal viruses, and their presence is Plaques that form on bacterial "lawn" (ie, lyse or clarify (cl earing) can be measured by their ability to form an area. Bacteria off The number of pages can be measured as the number of plaque forming units per sample volume .   Bacteriophages are not covered by the outer membrane and are the target HI for blood infectivity. It shows a different surface than V and hepatitis virus. But bacteriophage Due to the size and ease of detection, the pores of the present invention: oxidase-haloperoxy It is possible to test the size-specific inactivation quality of the sidase preparation.   The bacteriophage suspension is added to the prepared MPO and EPO conjugated beads described above. Make contact. Bacteriophages enter the pores of the beads and are Interacts non-covalently with concanavalin A and / or EPO or MPO You. After the beads have settled, the supernatant is collected and the bacteriophage in the supernatant is collected. Is determined using standard bacteriological techniques. As a control, Make sure that the bacteriophage suspension was not added with MPO and EPO during the binding step. Except for (1) and (2), the beads are brought into contact with the beads that have been subjected to all the preparation steps described above. Bind to beads The efficiency of phage killing by the MPO and EPO enzymatic systems It is determined by comparing the trawl titer with the test titer.6. Pore bacterial model: lactate-oxidase-haloperoxidase inactivation.   The functional capacity of untreated and MPO-LOx binding sieving material (porous beads) is approximately 0.5μ Uses Escherichia coli, a short gram-negative bacillus with a cell width of m And tested. E. coli in trypticase soy broth medium for about 16 hours at 37 ° C And cultured. Bacteria are concentrated by centrifugation and the final concentration is measured turbidimetrically 10 per milliliter by (titration)⁶~Ten⁷Bacteria.   The binding-killing ability of the different sieve preparations described above was tested as follows. Less than Was placed in each test tube:                 (1) 100 µl (microliter) sieve material (beads) suspension                 Suspension;                 (2) 100 μl of E. coli suspension (about 10⁶Bacteria);                 (3) 100 μl of 0.1 M lactic acid (finally 10 mm lactic acid); and                 (4) 700 μl of normal saline (0.85% NaCl)   The materials were mixed gently and allowed to incubate at 24 ° C. for 1 hour. Mix the ingredients To obtain a homogeneous suspension, diluted and pre-plated on trypticase soy broth. I did it. The number of E. coli colony forming units (CFUs) was It was measured after the incubation.   The results were as follows:         Sieve substance E.coli (final CFU / μl)         None 4,520,000         Sepharose CL 2B                 -3,000,000 unprocessed                 -MPO-LOx combination 830,000         Sephacryl S1000                 -Untreated 2,920,000                 -MPO-LOx combination 200,000         Glyceryl-Glass                 -Untreated 3,160,000                 -MPO-LOx combination 91,000   The slight decrease in E. coli CFU observed with untreated sieving material was It is thought to reflect the binding and capture of bacteria without. In particular, different MP E. coli removal and / or killing by O-LOx binding sieve Example. Three different MPO-LOx binding sieves were tested. Among these, gly The pore size of ceryl-glass is larger than the pore size of Sephacryl S1000, while The smallest pore in this group was that of Sepharose CL-2B. Therefore, MPO-LOx combined Se pharose CL-2B was not the most effective of the three sieves tested . This latter material also bound the least amount of MPO-LOx.   Having illustrated and described preferred embodiments of the invention, the spirit and scope of the invention have been described. It can be appreciated that various modifications can be made without departing from the scope. In particular, The compositions and methods described herein can be used to transform pathogenic microbes from any form of liquid. It can be applied to remove objects.   The aforementioned publications are incorporated herein by reference.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 31/5415 A61K 31/54 602 31/70 31/70 38/00 47/06 Z 38/44 C12N 7 / 02 47/06 7/04 C12N 7/02 11/10 7/04 A61K 37/50 11/10 37/02 (81) Designated country EP (AT, BE, CH, DE, DK, ES, FI, FR , GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG) ), AP (KE, LS, MW, SD, SZ, UG), UA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA , BB, BG, BR, BY, CA, C , CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG , US, UZ, VN

Claims (1)

[Claims] The embodiments of the invention for which exclusive ownership or privilege is claimed are defined below: 1. Micropores with pores large enough to allow entry of pathogen particles but exclude blood cells A composition comprising a microporous material, wherein the singlet oxygen production system comprises the microporous material. A composition that binds to a substance. 2. From the group consisting of controlled pore glass and carbohydrate polymers wherein the microporous material is controlled The composition according to claim 1, which is selected. 3. The pores allow for entry of virus particles in the range of about 0.1 μm to about 1.0 μm. The composition of claim 1, wherein the composition is large. 4. The pores are up to about 3 μm in size to allow entry of bacteria. A composition according to claim 1. 5. 2. The composition of claim 1, wherein said singlet oxygen production system is a photosensitizer. 6. The photosensitizer is phthalocyanine, porphyrin, methylene blue, hypericin Fluorescein derivatives, and psoralens. Item 6. The composition according to Item 5. 7. 2. The method of claim 1, wherein the singlet oxygen production system is an enzymatic system. Composition. 8. The enzymatic system comprises oxidase and haloperoxidase; A composition according to claim 7. 9. 9. The composition of claim 8, wherein said oxidase is capable of oxidizing a substrate in blood. 10. The oxidase is lactate oxidase, oxalate oxidase, Selected from the group consisting of glucose oxidase and cholesterol oxidase The composition of claim 9. 11. The haloperoxidase is myeloperoxidase, eosinophil peroxidase; And a fungal chloroperoxidase. Item 10. The composition according to Item 8. 12. 9. The haloperoxidase retains a ligand of a pathogen particle. A composition according to claim 1. 13. The singlet oxygen production system is covalently linked to the microporous material The composition of claim 1, wherein 14． The singlet oxygen production system is non-covalently linked to the microporous material. The composition of claim 1, which is compatible. 15. 2. The composition of claim 1, wherein the ligand of the pathogen particle binds to a microporous surface. object. 16. The ligand is a high mannose glycan, N-glucosamine, or N-glycol of an oligosaccharide. Cetyl glucosaminyl nucleus, mannosyl nucleus of complex N-linked glycans, mannan, α -Methyl mannoside, haloperoxidase, sulfated polysaccharide, low molecular weight dext 16. The composition of claim 15, wherein the composition is selected from the group consisting of lansulfate, and lectin. object. 17． 17. The haloperoxidase is myeloperoxidase. A composition according to claim 1. 18. 17. The composition of claim 16, wherein said lectin is Concanavalin A. 19. The ligand is concanavalin A, and the singlet oxygen producing cis Wherein the system comprises a myelooxidase non-covalently bound to concanavalin A, A composition according to claim 15. 20. A device comprising the composition of claim 1, wherein the device comprises a bead. Group consisting of beads, wafers, gel filtration matrices, filters, bags, and tubes The device that is more selected.