Methods of and compositions for selectivel;y modifying nucleic acids

The present invention relates to a method for selectively inactivating microorganisms. The invention belongs to the field of bio-organic chemistry, molecular biology, biochemistry, immunology and virology as well as medicine and veterinary medicine. The invention solves the problems associated with the preparation of appropriate methods and also indicates the possibility of creating a composition for the selective chemical modification of nucleic acids contained in centers such as human blood, cellular blood components, blood plasma and plasma biopolymers purified from blood (albumin, clotting factors, gammaglobulin, fibrinogen etc.), in cell culture components such as fetal bovine serum and porcine trypsin, non-blood products derived from normal or cancer cells (e.g., by recombinant DNA technology), each of which is substantially free of infectious viral contaminants and is suitable for therapeutic or diagnostic use.

Transmission of viral diseases (e.g., hepatitis A and B, acquired immune deficiency syndrome (HIV), cytomegalovirus infections) by blood or blood products is a significant problem in medicine. Although the selection of donors and the selection of donor blood for viral markers helps reduce the transmission of viruses to recipients, the selection methods are incomplete or less than 100% sensitive because they are intended for only a few specific viruses, and even if their sensitivity is insufficient. It is desirable to inactivate any virus contained in donor blood or blood products without changing the structure and function of their valuable components, e.g., red cells, platelets, leukocytes and plasma biopolymers, such as proteins, polysaccharides, etc. Similarly, other biological compositions, e.g. mammalian cell lines and hybridomas, cell line products, milk, colostrum and sperm may contain an infectious virus and it will be beneficial to inactivate the viruses while retaining valuable components or products of these compositions. Finally, it is often not known whether blood or blood products or mammalian cell products contain infectious viruses. In this case, it will preferably have compositions and methods for processing such a composition containing cells or biopolymers to inactivate any infectious virus.

The production of fully safe and effective vaccines containing killed microorganisms for use in medicine and veterinary medicine requires methods that fully and reliably cause non-infectiousness of live microorganisms, e.g., viruses and bacteria ("deactivation"), but have minimal impact on their immunogenicity. Ways typically sto4

187 When used to deactivate viruses, such as those useful for making viral vaccines, they generally alter or destroy the function and structure of cells, proteins and other antigens.

Current inactivation methods, including the use of formalin, beta-propiolactone and ultraviolet, have been empirically developed with little reliance on fundamental chemical or structural bases. Ethyleneimine monomers were used to inactivate the muzzle and hoof disease virus (Russian Patent No. SU 1915956). Ethyleneimine monomers have also been used to inactivate Mycoplasma and Acholeplasma (publication WO 92/18161) and avian infections (Romanian Patent No. RO 101400). Double ethyleneimine was used to inactivate the feline intestinal coronavirus, FECV (Patent No. EP 94200383). Polyethyleneimine was used as a plant virus control agent (Japanese Patent No. JP 7882735). The following methods and compounds modify microorganisms, such as viruses and bacteria, nonspecifically, and are difficult to standardize and use in a reproducible manner. In general, many components of the microorganism, including important surface antigenicity factors, e.g. viral capsid proteins are affected by currently used inactivating agents that modify not only nucleic acids, but also other biopolymers such as proteins, carbohydrates and lipids, violating their function. Altered antigens or inactivation of protective epitopes may lead to reduced immunogenicity, i.e. reduced potency (e.g., inactivated polio vaccine) or alteration of antigenicity and strengthening of the disease instead of disease prevention (e.g., respiratory syncytial virus vaccine and inactivated measles vaccine produced by formalin inactivation) . Another example is the preparation of a hepatitis B virus vaccine, where the preparation is usually heated to over 80 ° C and treated with formaldehyde. This treatment not only inactivates infectious viruses, but also damages proteins and other antigens. Carriers added to the vaccine as stabilizers may also inadvertently be modified, resulting in allergic reactions, as in the case of human serum albumin in beta-propiolactone inactivated rabies vaccine. In addition, ignorance of chemical changes giving the microorganism non-infectiousness makes it difficult to use the process in a repetitive manner. The result is periodic attacks of disease resulting from inappropriate inactivation or reversal after inactivation. The main attacks of paralytic poliomyelitis, snout and hoof disease, and Venezuelan equine encephalitis were caused in this way.

Thus, none of the currently available agents used for the production of inactivated viral vaccines is sufficiently selective to completely inactivate infectious viruses while retaining the antigenic properties of the viral particles, at least under the conditions previously used to inactivate the viral genome.

Another problem is that certain blood-contaminating viruses or other biological fluids are contained in cells as complete viruses, viral DNA fragments, or viral nucleic acid integrated into the host genome. For example, the HIV virus is contained in leukocytes. A special concern is the ability to inactivate cell-free and virus-contained forms of cells, while maintaining structural cell integrity.

Problems of inactivation of viruses in biological mixtures differ from problems of inactivation of viruses themselves due to the simultaneous presence of desired biopolymers such as proteins, carbohydrates and glycoproteins in serum. Although it is possible to inactivate the hepatitis B virus using agents such as formaldehyde and oxidizing agents, these methods are not appropriate for inactivating the virus in the blood, since it has been noted that most of these activating agents damage the biological activity of biopolymers in serum or cellular blood components. The use of ultraviolet light has been shown to inactivate viruses in platelet concentrate. However, acute platelet damage occurred at higher intensities. Beta-propiolactone reacts with nucleic acid and protein at a similar rate; thus, although viruses can be inactivated, more than half of factor VIII from serum is lost.

Difficulties in obtaining valuable biopolymers from sources outside the blood are also possible because pathogenic viruses can also contaminate such compositions. These sources include, but are not limited to, colostrum and mammalian milk, ascites fluid, serum, saliva, extracts

187 759 placenta, tissue culture cell lines and their extracts, including, e.g., transformed cells and fermentation products.

The subject of the invention is a method for the selective inactivation of microorganisms in a biological composition containing biopolymers by the selective modification of nucleic acid molecules in this composition containing biopolymers, in which the composition containing the biopolymer is contacted with an ethyleneimine oligomer solution with a suitable pH and ionic strength, biopolymers in this compositions retain all activity at the level of inactivation.

The method preferably uses biopolymers selected from the group consisting of proteins, carbohydrates and lipids.

In a preferred embodiment of the method of the invention, the biological composition comprises one or more proteins selected from the group consisting of fibrinogen, factor VII, factor VIII, factor IX, factor X, immunoglobins, prealbumin, retinol binding protein, albumin, alpha-globulin, gamma-globulin , complementary ingredients, fibronectin, antithrombin III, hemoglobin, interferon, growth factors, plasminogen activator, growth hormone, insulin and erythropoietin.

Also preferably, the biological composition is selected from the group consisting of mammalian whole blood, purified or partially purified blood proteins, blood cell proteins, milk, saliva, blood plasma, platelet rich plasma, plasma concentrate, sediment from any fractionation of such plasma, supernatant from any fractionation plasma, serum, cryoprecipitate, cryosupematant, cell lysate, mammalian cell cultures, placental extracts, fermentation products and proteins induced in blood cells.

Most preferably the method of the invention can be carried out when the biological composition is mammalian and non-mammalian cells.

Also preferably, the biological composition can be selected from the group: leukocyte concentrate, red blood cell concentrate and / or platelet concentrate.

Microorganisms can be selected from the group of cell-free forms of viruses, blood-borne viruses and bacteria and parasites.

Preferably, the viruses are selected from the group of enveloped and uncoated viruses, or the microorganisms are selected from the group of cellular viruses, blood-borne viruses, bacteria and parasites.

Also preferably, the viruses are selected from the group consisting of poxviruses, herpes viruses, adenoviruses, papy viruses, parvoviruses, reoviruses, orbiviruses, rotaviruses, alphaviruses, rubiviruses, flaviviruses, coronaviruses, paramyxoviruses, morbilliviruses, pneumoviruses, livirus bunya, phleboviruses, nairoviruses, hepadnaviruses, arenaviruses, retroviruses, enteroviruses, rhinoviruses and filoviruses.

In the process according to the invention, an ethyleneimine dimer may be preferably used as the ethyleneimine oligomer, optionally also preferably a substituted ethyleneimine oligomer.

The substituted ethyleneimine oligomer has the general formula P-Hal- (CH₂CH₂-Nh) "H where Hal is a halogen atom and n is a number from 2 to i0.

In the method of the invention, the biological composition is incubated with about 0.000i M to about 0.015M ethyleneimine oligomer in the contacting step; at a pH of about 6.5 to about 8.5; in a solution with an ionic strength of from about 0.1 M to about 0.2 M at a temperature of about and 5 ° C to about 30 ° C for about 1 hour to about 500 hours and more preferably the composition can be incubated with about 0.007 M ethyleneimine oligomer; at a pH of about 7.0 to about 8.0; in a solution with an ionic strength of about 0.15 M. Very preferably, ethyleneimine trimer or, optionally, ethyleneimine tetramer may be used as the ethyleneimine oligomer in the process of the invention.

A preferred embodiment of the method of the invention is that the biological composition is suitable for therapeutic use after selective inactivation of microorganisms.

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The method of the invention preferably uses a biological composition selected from the group consisting of mammalian whole blood, leukocyte concentrate, red blood cell concentrate and platelet concentrate.

In the process according to the invention, preferably the ethyleneimine oligomer is selected from the group consisting of ethyleneimine dimer, ethyleneimine trimer and ethyleneimine tetramer, and more preferably said ethyleneimine oligomer is an ethyleneimine dimer. In such a case, most preferably said biological composition is a red blood cell concentrate.

The method of the invention allows the selective modification of nucleic acid in the presence of other valuable biological macromolecules and cells. According to this method, the nucleic acid of viruses, other microorganisms and cells is subjected to selective chemical modification, maintaining the structure and function of non-nucleic acid components.

It has now been discovered that ethyleneimine oligomer inactivating agents can effectively and specifically inactivate infecting viruses in a biological composition, such as a composition comprising cells or a biopolymer. The subject of the invention is therefore a method of selectively modifying the nucleic acid particles of viruses or other microorganisms in a mixed biopolymer composition, comprising contacting the composition with an ethyleneimine inactivating oligomer. It has now been found that although most of the currently available virus inactivating agents alter biopolymers such as blood protein VIII, rendering them biologically inactive, the inactivating ethyleneimine oligomers of the invention used under inactivation conditions do not exert such an effect. It has been discovered that when the biopolymer-containing composition, e.g., blood cell proteins, blood plasma, blood plasma fractionation sediment, blood plasma fractionation supematant, cryo-precipitate, cryosupematant, or part or derivative thereof, or a serum or non-blood product made from normal or transformed cells (e.g. recombinant DNA techniques) are contacted for a sufficient time with the inactivating ethyleneimine oligomer, the viruses present in the composition are inactivated to the desired extent (at least about 6 orders in the measurement of viral inactivation or at least about 20 orders in the calculation) without significant biopolymer damage, such as contained there protein. By contacting a blood protein mixture or concentrate with an inactivating ethyleneimine oligomer, you can inactivate a virus, such as hepatitis A or B or HIV, to the desired degree, e.g., to a measurable degree of inactivation by at least 6 orders or in calculating at least about 20 orders of magnitude . Biopolymers such as proteins, carbohydrates and lipids in the treated composition retain essentially all activity at the pre-inactivation level.

The method of the invention allows the preparation of biopolymer-containing compositions, e.g., blood cell derivatives (e.g., hemoglobin, interferon alpha, human growth hormone, erythropoietin, PDGF, tPA, etc.), blood plasma, blood plasma fraction, blood plasma sediment (e.g. cryoprecipitate, ethanolic supernatant or poly (ethylene glycol) supernatant) that are essentially free of infectious virus while maintaining substantially quantitative activity of the protein present before inactivation. The amount of virus in the composition is referred to as infectivity.

The method of the present invention is described as the processing of plasma, plasma fractions, plasma concentrates or their components. However, the method is also useful for treating lysates or proteins secreted from cells. Treatment of fractions derived from platelets, white cells (leukocytes), red cells, fibroblasts, as well as solutions of interferon, growth hormone, tPA, factor VIII, transmission factor, hemoglobin, growth factors, EPO and DNAse is also contemplated.

The use of inactivating agents and the method of the present invention is also envisaged for the treatment of fresh frozen plasma, thawed frozen plasma, cryoprecipitate, cryosupematants or frozen plasma concentrates as well as their dilution products.

In the same method described herein, the virus present in normal or transformed cell products can be inactivated while maintaining the biopolymeric activity of such products. For example, products made using normal or transformed cells, exudate from normal or transformed cells, hybridomas, and products made by genetic engineering can be inactivated using an ethyleneimine inactivating oligomer. Such processing does not substantially adversely affect the desired biopolymer, such as a specific protein. The cells used to produce the desired protein may, of course, be derived from a mammal as well as not from a mammal.

Compositions that can be prepared based on the method of the invention essentially inactivate all viruses contained in the sample. Methods for determining the level of infectivity are well known to those skilled in the art. See, e.g., Lennette, EH and Schmidt, NJ (ed.) (1985) Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections, ed. 62, American Publisher ^ Assn., Washington. DC According to the invention, the experimentally measured virus inactivation reaches at least about "6-log" (6.0 logio). This means that where the virus is present in the untreated composition at such a concentration that even after dilution 10⁶ times viral infectivity can be detected, in the treated sample, the virus is completely inactivated to the extent determined by infectivity studies, so that after treatment, no virus can be detected in the undiluted sample. More importantly, after obtaining a precise kinetic description of the inactivation process described herein, a calculated reduction in infectivity of the virus-containing compositions of at least about 20 orders of magnitude can be obtained.

In some embodiments, the inactivating ethyleneimine oligomer is a trimer, linear tetramer or branched tetramer. Preferred inactivation conditions include incubating the composition with about 0.0001 M to about 0.010 M inactivating ethyleneimine oligomer; at a pH of about 6.5 to about 8.5; in a solution with an ionic strength of about 0.01 M to about 0.5 M. More preferred reaction conditions are incubating the composition with about 0.001 M to about 0.01 M inactivating ethyleneimine oligomer at a pH of about 6.9 to about 8.5; in a solution with an ionic strength of about 0.1 to less than about 0.5 M. The most preferred inactivation conditions are contact of the cell-containing or biopolymer-containing compositions with an inactivating ethyleneimine oligomer such as a trimer, linear tetramer or branched tetramer at a temperature in the range of about 4 ° C up to 30 ° C.

The inactivating agent for the selective modification of nucleic acids is a biopolymer mixture containing about 0.0001 M to about 0.015 M of ethyleneimine oligomer; with an ionic strength from about 0.1 M to about 0.2 M; at a pH of about 6.5 to about 8.5.

The method of selectively inactivating functional nucleic acids in a biological composition involves contacting the composition with an effective concentration of the selectively inactivating ethyleneimine oligomer.

Based on the method of the invention, vaccines containing killed microorganisms can be prepared by contacting purified or non-purified virus-containing compositions with an ethyleneimine selectively inactivating oligomer under virus inactivation conditions.

The vaccine so produced contains killed microorganisms or an effective amount of inactivated viruses, i.e., an amount sufficient to impart the desired degree of immunity to the body, and a pharmaceutically acceptable carrier. which produces inactivated viruses by incubating viruses with inactivating viruses and ethyleneimine oligomers under virus inactivating conditions that effectively reduce measurable infectivity by at least about 6.0 orders of magnitude (or to a desired degree by at least about 20 orders of magnitude). The vaccine may be administered to a patient or animal for therapeutic purposes. The patient is immunized against the virus by administering to the patient a vaccine containing such killed microorganisms.

Blood collection devices include a container for collecting blood or a fraction thereof, containing an ethyleneimine inactivating agent in an amount of effectively inactivating the virus in the blood or its fraction withdrawn into the container, and diagnostic reagents and diagnostic samples include viruses that have been treated with virus inactivating agents in a method according to the method of inactivation invention.

Figure 1. Structure of monomer (I), dimer (II), trimer (III), linear and branched tetramer (IV and V, respectively) inactivating the ethyleneimine oligomer.

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Figure 2. Potentiometric titration curves for ethyleneimine and its oligomers. The Roman numbers correspond to the structures present in Fig. 1.

Figure 3. Survival curves of MS2 phage under the action of ethyleneimine (0.025 M, curve 1), its dimer (0.007 M, curve 2), trimer (0.003 M, curve 3), and an equimolar mixture of linear and branched tetramer (0.0015 M, curve 4) in 0.15 M NaCl, pH 7.5, 20 ° C.

Figure 4. Survival curves of MS2 phage under the action of 0.007 M ethyleneimine in 0.15 M NaCl (20 ° C) at pH 6.5, 6.9, 7.5, and 8.5 (curves 1, 2, 3, and 4, respectively).

1. definitions:

"Selective inactivating agents" refers to ethyleneimine oligomers containing an aziridine moiety and having specific affinity for polyanions, e.g., polynucleotides, compared to other biological molecules. The selective inactivating agents of the invention include a class of relatively weakly toxic compounds that selectively bind to nucleic acids (single-stranded DNA, double-stranded DNA, or RNA) comprising genetic viral material and irreversibly modifying functional nucleic acids resulting in inactive viruses when used in inactivated conditions.

"Ethyleneimine oligomer" refers to ethyleneimine oligomers having a terminal aziridine group and optionally substituted. Preferred ethyleneimine oligomers have at least three ethyleneimine units and include, e.g., trimer or tetramer, linear or branched. The synthesis of ethyleneimine oligomers is carried out using synthetic schemes well known to those skilled in the art. See e.g., Kostyanovskii, RG et al. (translated from Izvestiya Academy of Sciences of the SSSR, Seriya Khimicheskaya, 11: 2566-2577, 1988). Representative ethyleneimine oligomers are shown in Figure 1. In the process of the invention, ethyleneimine oligomers having less than 10 units are preferred and ethyleneimine oligomers having about 3 or 4 units are more preferred.

Ethyleneimine oligomers can also be substituted so long as this does not eliminate the essential property of ethyleneimine. In one embodiment, the ethyleneimine oligomers are halogen-substituted and have the general formula (-Hal- (CH2-CH2-NH)_nH. Such compounds, often referred to as nitrogen mustards, are synthesized by the quantitative conversion of hydrogen chloride or hydrogen bromide of ethyleneimine or its oligomers into β-halo- mono or oligoethylamine. Nitrogen mustards are strong electrophiles and alkylate the nucleophilic groups of nucleic bases directly or by converting the intermediate to the corresponding aziridine. As ethyleneimine oligomers, P-halooligoethylamine has high affinity for polyanions. Thus, these ethyleneimine oligomers have high selectivity for nucleic acids, however the modification kinetics will need to be adjusted.

The inactivating agent exhibits "selectivity" for nucleic acids or "selectively" reacts with nucleic acids if the comparative reaction rate of the inactivating agent with nucleic acids is greater than the reaction rate with other biological molecules, e.g., proteins, carbohydrates or lipids. The level of selectivity of the inactivating ethyleneimine oligomer to nucleic acids compared to proteins is unexpected due to the ethyleneimine monomer, which is similarly selective for nucleic acids and other alkylating agents.

"Nucleic acid" refers to single- and double-stranded DNA and RNA.

"Biological composition" means a composition containing cells or biopolymers. Cell-containing compositions including, e.g., whole blood, red cell concentrates, platelet concentrates, leukocyte concentrates, blood cell proteins, blood plasma protein fractions, purified blood proteins, serum, semen, colostrum and mammalian milk, placental extracts, products fermentation, ascites fluid, and products made in cell culture from normal or transformed cells (e.g., by recombinant DNA or monoclonal antibody technique). "Biopolymer" or "biological molecule" means any class of organic molecule commonly found in living organisms, including, e.g., nucleic acids, polypeptides, post-translational modified proteins (e.g. glycoproteins), polysaccharides and lipids. Biopolymer containing compositions include, e.g., blood cell proteins,

187 759 blood plasma, blood plasma fractionation sediment, blood plasma fractionation supernatant, cryoprecipitate, cryosupematant, or part or derivative thereof, or a serum or non-blood product made from normal or transformed cells (e.g. by recombinant DNA techniques). Biological compositions may not contain cells.

"Functional nucleic acid" means a nucleic acid having sequence elements that serve as a pattern in the replication, transcription, translation or other activity of a nucleic acid molecule. Such elements include, e.g., sequences encoding the origin of replication of the nucleic acid molecule, transcriptional elements such as promoters / enhancers, transcriptional terminators and other regulatory elements; translational elements such as ribosome binding sites, translation start codons, coding sequences and phase stop codons; and sequences that confer RNA catalytic activity.

"Inhibit biopolymer activity" means measurably weaken the function or activity of the biopolymer. Impairment of function or activity can be determined in any standard test used to measure the activity of a particular biopolymer. For example, inhibition of enzyme (protein) or antigen activity can be determined by measuring changes in enzymatic process speed or immune response to the antigen using conventional assays. Another example of such inhibition is inhibition of RNA translation, which can be determined by measuring the amount of RNA encoded protein produced in a suitable in vitro or in vivo translation system.

"Inactivating", "inactivating" or "inactivating" with respect to functional nucleic acids means substantially eliminating DNA or RNA activity, e.g., damaging the ability to replicate, transcribe or translate a message. For example, inhibition of RNA translation can be determined by measuring the amount of protein encoded by a finite amount of RNA produced in a suitable in vitro or in vivo translation system. For viruses, the term means the reduction or elimination of infectious viral particles measured as the decrease in infectivity titre or number of infectious viral particles per ml. Such a decrease in the number of infectious virus particles is determined in tests well known to those of ordinary skill in the art. Lennette, EH and Schmidt, NJ (ed.) (1985) Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections, ed. 62, American Publisher's Assn., Washington. DC

Experimentally, the decrease in infectivity can be measured to at least about "six orders of magnitude" in a composition comprising a cell or biopolymer in which the virus is inactivated to the extent determined by infectivity studies, wherein the virus is present in untreated serum at a concentration such that even after dilution⁶ times viral activity can be measured. When a specific virus with a titer of 10 cannot be produced⁶, inactivation is quantified by measuring up to the titer of virus formed. Alternatively, such a decrease in the number of infectious virus particles is determined by calculating the method described herein to a range of at least about "20 orders" based on the kinetic description of the inactivation process based on accurate experimental determination of the infectivity of the virus suspension during inactivation, taking into account chemical, physical and biological factors affecting kinetics inactivation.

"Viral inactivation conditions" refer to conditions under which viral particles are incubated with selectively inactivating ethyleneimine oligomers of the invention, including, e.g., processing time, pH, temperature, salt composition and concentration of the selectively inactivating agent to inactivate the viral genome in desired degree. The conditions for virus inactivation are selected from the conditions described below by selective modification of nucleic acids.

"Virus" means DNA and RNA of viruses. Viruses include encapsulated and uncovered viruses, e.g., poxviruses, herpes viruses, adenoviruses, papy viruses, parvoviruses, reoviruses, orbiviruses, picomavirus, rotaviruses, alphaviruses, rubiviruses, influenza virus, type A and B, flaviviruses, coronaviruses, paravirus , pneumoviruses, rhabdoviruses, lissaviruses, orthomixoviruses, bunya viruses, phleboviruses, nairoviruses, hepadnaviruses, arenaviruses, retroviruses, enteroviruses, rhinoviruses and filoviruses.

"Vaccine" is used in its usual sense to designate an agent that effectively gives an appropriate level of immunity to the body, causing only a small amount

187 759 morbidity or mortality. Methods for making vaccines are, of course, useful in studying the immune system and preventing human and animal diseases.

"Pharmaceutically acceptable" means relatively non-toxic to the animal being treated with the compound. A "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, buffers and excipients such as phosphate buffered saline solution, water and emulsions such as oil / water or water / oil emulsions, and various types of wetting agents and / or adjuvants.

The invention is based on the unexpected and surprising discovery that, compared to many nucleic acid modifying agents, in particular the ethyleneimine (aziridine) monomer, ethyleneimine oligomers such as trimer and tetramer are much more selective in nucleic acid modification as opposed to other biopolymers such as proteins. Ethyleneimine oligomers are more selective than ethyleneimine monomer with many orders of magnitude, and in some cases, six orders of magnitude. The use of selectively inactivating ethyleneimine oligomers in the method of the invention is particularly effective for compositions comprising nucleic acids closely associated with proteins, such as nucleoproteins or viruses.

The present invention relates to a process for the selective aminoalkylation of nucleic acid molecules in a composition comprising a cell or biopolymer, comprising contacting the composition with an ethyleneimine inactivating oligomer. As a result of this process, the nucleic acids in the composition are chemically modified much faster than other biological molecules. This method is useful in any process in which the user wants to modify the nucleic acids, but leave other biological molecules relatively unchanged. For example, the method of the invention is useful in preferential labeling of nucleic acids in a nucleoprotein (e.g., chromatin or ribosomes) with an ethyleneimine oligomer carrying a detectable label (radioactive, fluorescent, enzymatic, etc.), e.g. for mapping or inactivating the genome of a virus or other living organism.

Inactivating ethyleneimine oligomers in the method of the invention modify nucleic acids preferably by reacting a protonated aziridine group with nucleic bases in polynucleotides. The ability of the virus inactivating agent to bind to polyanion depends, in part, on the number of protonable groups per molecule and on the total degree of protonation under given conditions. The modification occurs by binding the oligo-cationic virus inactivating agent to the polyanionic nucleic acid. The degree of protonation depends, in part, on the pH. As described herein, the pH of the aziridine group decreases with the length of the polymer. However, the ability of ethyleneimine inactivating oligomers to selectively associate with nucleic acids increases significantly with the length of the ethyleneimine inactivating oligomer. Accordingly, the practitioner can efficiently and selectively alkylate nucleic acids at physiological pH.

One variation of the method of selective amino-alkylation of nucleic acids includes the step of contacting the nucleic acids with about 0.0001 M to about 0.015 M selectively inactivating oligomer in ethyleneimine at a pH of about 6.5 to about 8.5 and preferably about 7.0 to about 8 , 0, and most preferably, a pH of about 7.5. The concentration of the inactivating ethyleneimine oligomer depends in part on the number of protonable groups in the inactivating ethyleneimine oligomer, and the choice of pH depends on the virion stability. The selectively inactivating ethyleneimine oligomer is preferably a trimer, linear tetramer or branched ethyleneimine tetramer. In another embodiment, the method comprises the step of exposing or contacting the nucleic acids with a selectively inactivating ethyleneimine oligomer in a solution with an ionic strength of about 0.1 M to about 0.5 M and preferably, about 0.15 M. The salts may be any of those normally used in biochemistry , including sodium, potassium, phosphates or acetates, and so on. The practitioner can change the pH of the solution using any buffers commonly used for biopolymers or cells, such as phosphate, acetate, borate, Tris, HEpES, MOPS and so on. MOPS and Tris are preferred buffers. High phosphate concentrations in buffers are not preferred. The practitioner can adjust the reaction conditions to obtain the desired pH and ionic strength. The practitioner may also adjust other factors such as reagent concentration, temperature, incubation time depending on reaction conditions and the desired degree of inactivation of infectivity.

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As, described here below; using an kinetic approach, the inactivation endpoint should be determined, as well as the conditions outlined herein, for aminoalkylation of nucleic acids to inactivate functional nucleic acids or viruses to a certain extent.

The practitioner can determine the degree of viral nucleic acid alkylation by the degree of inactivation of viral infectivity, using various assays known to those skilled in the art, such as determining cytopathic effects (CPE) in tissue cultures using serial dilutions of virus-containing mixtures introduced into susceptible cells, followed by incubation at 37 ° C. Modification of proteins, polysaccharides and glycoproteins with ethyleneimine oligomers will lead to the introduction of additional positive charges.

The degree of this biopolymer modification can be determined by known methods, including, e.g., isoelectric focusing, autoradiography, polyacrylamide gel electrophoresis, HPLC and other forms of chromatography.

Compositions for the selective modification, aminoalkylation, nucleic acids in a composition comprising a cell or biopolymer include about 0.0001 M to about 0.015 M of the inactivating ethyleneimine oligomer; with an ionic strength of about 0.1 M.

Compositions for the selective modification, aminoalkylation, nucleic acids in a composition comprising a cell or biopolymer include about 0.0001 M to about 0.015 M of the inactivating ethyleneimine oligomer; with an ionic strength of about 0.1 M to about 0.2 M and, preferably, about 0.15; and pH buffered about 6.5 to about 8.5 and preferably about 7.0 to about 8.0, and most preferably about 7.5. The inactivating ethyleneimine oligomer is preferably a linear or branched trimer or tetramer. Salts and buffers may be any of those described above. In one embodiment, the composition is ethyleneimine oligomer content, ionic strength and pH effective for aminoalkylation of a functional nucleic acid. In another embodiment, the composition has ethyleneimine oligomer concentrations, ionic strength, and pH effective to activate viruses. Such compositions are useful as disinfectants or as viricides, and in all of the methods of the invention described herein.

Methods for selectively inhibiting the activity of functional nucleic acids in a biological sample include contacting the sample with an effective amount of a selectively inactivating ethylene oligomer. These methods have many uses. When the functional nucleic acid is an unprotected nucleic acid, such as a plasmid or DNA segment, inhibition is useful for reducing the ability of the molecule to transform the cell into which it is introduced, e.g., by transfection. In a cell-free translation system, inhibition is useful for reducing RNA translation. When the functional nucleic acid is a catalytic nucleic acid such as ribozyme, the methods are useful for inhibiting the action of a nucleic acid at its target site.

When the functional nucleic acid is a viral genome that is part of an infectious virus, the methods are useful for disinfecting the area, inactivating or eliminating viruses from the cell or biopolymer containing compositions such as whole blood, blood products or biological products such as proteins produced in cell culture, and for we deactivate virus infectivity to the desired degree (e.g., at least about 20 orders of magnitude by the calculation method described here). The biopolymer-containing composition may include, e.g., proteins purified from whole blood; blood products (such as, e.g., clotting factors such as factor VIII, hormones such as erythrypyetha and so on); cell culture products such as cell extracts, growth medium enriched with biological molecules (e.g., secymbination proteins); protein-containing compositions processed with blood products (e.g. inZubywahe compositions with calf serum) and plant products. These methods are useful for ensuring the purity and safety of these products for use in the laboratory and in therapy, while maintaining the critical biological properties of the biological product in the composition.

Devices for collecting blood, for collecting and / or processing blood samples and for inactivating viruses therein, include a container for collecting blood or a fraction thereof with an ethyleneimine inactivating agent in an amount effectively deactivating the virus in the blood or its fraction. One example of this variety includes corked vacuum tubes that contain 12

187 759 have an inactivating ethyleneimine oligomer. When the blood sample is assembled into a tube, it contacts the inactivating ethyleneimine oligomer. Another example is a blood bag used, e.g., in blood donation. Blood bags according to the invention contain an inactivating ethyleneimine oligomer in contact with the blood filling the bag.

Similarly, if the functional nucleic acid is bacterial or is part of the genome of another organism, methods are useful in disinfecting or eliminating such a bacterium or other organism.

Modification of the viral genome by the methods of the invention excludes the reproduction of viruses and thus excludes the infectivity of the vaccines comprising the killed microorganisms of the invention. In addition, since the virion coating proteins are not modified to the same extent, the vaccine retains significant immunogenicity. Since ethyleneimine oligomers are significantly more selective in modifying nucleic acids compared to other selective inactivating agents, compositions of the invention containing ethyleneimine oligomers have significant advantages over other less selective inactivating agents currently in use.

Vaccines containing killed microorganisms can be made based on the method of the invention including contacting the virus with a selectively inactivating ethyleneimine oligomer under virus inactivating conditions. The conditions for virus inactivation are selected from the methods described above for amino-alkylation and inactivation of viral, bacterial or other nucleic acids. Generally, a virus with a high titer of about 107 to 10⁸ units per ml are incubated with the ethyleneimine oligomer; at a pH of about 6.5 to about 8.5, in a solution with an ionic strength below 0.50 M at a temperature of about 4 ° C to about 40 ° C. The duration of treatment (i.e., endpoint of inactivation) depends on the structure and composition of the particular virus, incubation temperature, ionic strength and number of protonable groups in the ethyleneimine oligomers. However, kinetic studies indicate that, depending on the pH and inactivated virus, the incubation time may be as short as a few seconds, and may also be about 1 hour, 5 hours, 50 hours, 100 hours, 300 hours or 500 hours. Preferably, the selectively inactivating ethyleneimine oligomer is a trimer, linear tetramer or branched tetramer. Methods for making vaccines are well known and can be found, e.g., in Vaccines (ed. Slorein G., Martance E.). 2, 1994, Saunders, Harcourt-Brace, Phil, Toronto.

When used as a vaccine, the dead virus can be used directly in the vaccine composition, or lyophilized in single or multi-dose containers for further mixing with a pharmaceutically acceptable carrier. Lyophilized killed viruses are usually kept at 4 ° C.

The vaccine may also be administered in an adjuvant, i.e., a substance that enhances the immune response when used in combination with an antigen. The vaccine may be administered at an immunizing dose. The immunizing dose is the amount of antigen or immunogen necessary to generate or enhance an immune response. This amount varies depending on the presence and effectiveness of various adjuvants. The amount will vary depending on the animal and the immunogen or antigen or adjuvant, but will generally be less than about 100 mg per dose. The immunizing dose can be easily determined by methods well known to those skilled in the art by conducting statistically significant studies on immunization and challenge with an animal host. See, e.g., Manual of Clinical Immunology, HR Rose iH. Friedman, American Society for Microbiology, Washington, DC (1980).

Methods of treating a composition comprising cells or a biopolymer or producing vaccines containing killed microorganisms are particularly useful in inactivating viruses already known from irreversible inactivation with other alkylating agents such as ethyleneimine monomer and β-propiolactone. Thus, although the agents of the invention are more widely used due to their selectivity, in the selection of viruses for the production of vaccines or biological products for decontamination practices rely, in part, experience in the field of other inactivating agents.

A vaccine containing killed microorganisms may be composed of an effective amount of viruses inactivated by the method of the invention and a pharmaceutically acceptable carrier, wherein inactivated viruses are produced by treating viruses under virus inactivation conditions that effectively reduce infectivity to the desired range (by at least about 6 orders of magnitude in direct measurement or at least about 20 orders of magnitude in the calculation described here). Such vaccines are useful for preventing human and animal diseases. Vaccines capable of giving the desired degree of resistance obviously contain an amount of inactivated virus effective to elicit an immune response. The viral genome in these vaccines is alkylated on the endocyclic nitrogen nucleic base compared to other vaccines, but the significant difference is that inactivation is much more effective because the relative rate of modification of nucleic acids compared to other biopolymers in virion is significantly higher than in the case of any other selective inactivating agents. In the production of vaccines containing killed microbes, the virus sample is incubated with a selective inactivating agent in quantities and conditions to inactivate the virus while maintaining immunogenicity.

Suitable pharmaceutical carriers and compositions are described by Martin, Remington Pharmaceutical Sciences, ed. 19 (Mack Publishing Co., Easton 1995). Such compositions will generally contain an effective amount of the compound along with a sufficient amount of carrier to form the correct dosage form for appropriate administration to the patient.

The therapeutic application of the method of the invention are methods of immunizing a patient against a virus by administering to the patient a vaccine comprising killed microorganisms. The patient may be a human or an animal. In therapeutic practice, an effective amount of a compound described above is administered by any of the usual and acceptable methods known in the art, alone or in combination with other compounds of the invention.

In practice, the specific dose of vaccine administered to a patient will depend on a number of factors, including the nature of the virus, the administration regime, the age and physical characteristics of the patient, and so on. Appropriate doses can be determined using a clinical approach known in medicine.

Without wishing to be bound by theory, the increased specificity of selectively inactivating ethyleneimine oligomers for nucleic acids obviously includes the following factors that can be manipulated in the practice of the invention. The ethyleneimine monomer and ethyleneimine oligomers contain a single aziridine group (Figure 1). The reactivity of aziridines as electrophilic agents increases sharply with the protonation of aziridine nitrogen (Van Etten, RL and Dolhun, JJ (1968) J. Org. Chem. 33: 3904-3913), but only reacts poorly to its alkylation (Earley, JE et al. (1958) J. Am. Chem. Soc. 80: 3458-3462). Thus, the form of these compounds protonated on the aziridine group is probably the only reactive form. The rates of ordinary electrophilic aziridine reactions should be proportional to the concentration of their protonated forms in solution.

Compounds I to V in Figure I differ significantly in pKa of their aziridine groups and the number, mutual arrangement and pKa of amino groups (Table 1). Thus, the proportion of the reactive form of these compounds in which the aziridine group is protonated, and their combined average positive charge significantly depends on the pH (Table 2).

Table 1

PKa values ​​of protonable oligoethyleneimine groups

<td></td><td>aziridino</td><td>amino primary</td><td>amino secondary</td><td>amino tertiary</td>

<td>monomer (I)</td><td> 8,10</td><td> -</td><td> -</td><td> -</td>

<td>trimer (III)</td><td> 4,10</td><td> 10,0</td><td> 7,35</td><td> -</td>

<td>tetramer (IV) (linear)</td><td> 3,3</td><td> 10,0</td><td> 7,05*</td><td> -</td>

<td>tetramer (V) (branched)</td><td> 3,0</td><td> 10,0</td><td> -</td><td> 6,4</td>

*) average value for two secondary amine groups **) average value for two primary amine groups

187 759

The action of aziridines on proteins and polynucleotides leads to amino-alkylation of nucleophilic groups in amino acid residues and nucleic bases (Dermer, OC and Ham, GE (1969) Ethyleneimine And Other Aziridines, Acad. Press, NY - London 52: 249-285). Aziridines, like many electrophilic agents, modify nucleic acids preferably with N7, N3 and Nl purines and to a lesser extent with N3 pyrimidines (Hemminki, K. and Ludlum, DB (1981) J. Natl. Cancer Inst. 73: 1021-1027; Musser , SM et al. (1992) Chem. Res. Toxicol. 5: 95-99; Singer, B. and Grunberger, D. (1983) Molecular Biology of Mutagens and Carcinogens, Plenum Press, New York-London; Loveless, A. (1966) Genetic and Allied Effects of Alkylating Agents. Butterworths, London; and Kochetkov, NK and Budowsky, EI ed. (1972) Organic Chemistry of Nucleic Acids, Part B, Plenum Press, London-New York). Reference synthesis is stopped by alkylating agents mainly due to the relatively slow opening of the imidazole ring of the alkylated N7 purines, mainly guanine (O'Connor, TR et al (1988) Nucl. Acids Res. 16: 5879-93; Hemminki, K. (1984) Chem .-Biol. Interactions, 48: 249260). For example, ethyleneimine modifies guanosine to form N7 (aminoethyl) guanosine showing a much faster imidazole ring opening rate than with N7-alkylguanosine (Hemminki, K. (1984) Chem. Biol. Interactions, 48: 249-260; Hemminki, K. et al. (1989) Chem. Biol. Interactions, 70: 289-303).

Table 2

Total average positive oligoethyleneimine charge (A) and degree of protonation of their aziridine group (B)

<td rowspan="2">pH values</td><td colspan="3">AND</td><td colspan="3">B</td>

<td> 6,5</td><td> 7,5</td><td> 8,5</td><td> 6,5</td><td> 7,5</td><td> 8,5</td>

<td>monomer (I)</td><td> 0,96</td><td> 0,80</td><td> 0,28</td><td> 0,98</td><td> 0,80</td><td> 0,28</td>

<td>trimer (III)</td><td> 1,88</td><td> 1,41</td><td> 1,04</td><td> 4,0-10-³</td><td> 4,0-10-⁴</td><td> 4,0-105</td>

<td>tetramer (IV) (linear)</td><td> 2,67</td><td> 1,41</td><td> 1,01</td><td> 6,3 ·10’⁴</td><td> 6,3-10-⁵</td><td> 6,3-10'⁶</td>

<td>tetramer (V) (branched)</td><td> 2,44</td><td> 2,07</td><td> 1,98</td><td> 3,2· 10*</td><td> 3,2 ·10’5</td><td> 3,2-10‘⁶</td>

The transition of monomer (I) into tetramers (IV and V) at pH 7.5 results in an increase of more than two orders of magnitude in the effective rate of inactivation of phage infectivity (k) calculated on the basis of the mean concentration of the agent in solution (Table 3). This transition leads to a decrease of 5 orders of magnitude by the pKa value of the aziridine group. Correspondingly, a fraction of the reactive form B of the agent decreases by about 4 orders of magnitude (Table 2). Thus, the transition from monomer to tetramers at pH 7.5 leads to an increase of about 6 orders of magnitude by a constant rate calculated based on the average concentration of the reactive form of the agent in the solution (s).

Table 3

Constant inactivation rates of phage MS2 infectivity (M'1-min'1) under the action of oligoethyleneimines in 0.15 M NaCl at 20 ° C calculated based on the total average concentration of the reactive form (k, in brackets)

<td>pH values</td><td> 6,5</td><td> . 6,9</td><td> 7,5</td><td> 8,5</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td>

<td>monomer (I)</td><td> -</td><td> -</td><td> 1,5±0,07 (1,25)</td><td> -</td>

<td>dimer (II)</td><td> 102±3*</td><td> 35±2,3</td><td> 13±1,4</td><td> 1,7±0,05</td>

<td></td><td> (2,4-10³)</td><td> (2,0-103)</td><td> (2,9-103)</td><td> (3,8-103)</td>

<td>trimer (III)</td><td> -</td><td> -</td><td> 47±2,7 (1,5-106)</td><td> -</td>

187 759 cont. table 3

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td>

<td>tetramer **</td><td> -</td><td> -</td><td> 150±12,5 (3,6-10⁶)</td><td> -</td>

* the rate constant was calculated using the initial part of the survival curve. ** equimolar mixture of linear and branched tetramer.

As oligocations, ethyleneimine oligomers have high affinity for polynucleotides, reflected by their association constant. This association constant, changed by electrostatic interaction, is proportional to the volumetric density density of the oligocation and polyanion, and therefore increases with the total average positive oligocation charge. The transition from the ethyleneimine monomer to its oligomers gives a significant increase in the total average positive particle charge (Table 2). In addition, the distance between protonable groups in ethyleneimine oligomers is comparable to the distance between internucleotide phosphate groups in polynucleotides. The increase in the number of protonable groups in oligoethyleneimines from monomer to tetramer should therefore lead to an increase in their association with polynucleotides, e.g. viral RNA (Manning, GS (1978) Q. Rev. Biophys. 2: 179-246; Thomas, TJ and Bloomfield, VA (1984) Biopolymers 23: 1295-1306; and Stevens, LS (1967) Biochem. J. 103: 811-815). Many of the alkylating agents used for nucleic acid modification have no clear affinity for polynucleotides. A comparison of the dependence on the pH of polynucleotide modifications with the pK values ​​of nucleic bases shows that in the pH range 6.0-8.0 mainly neutral nucleobases are subject to alkylation (Budowsky, EI and Zalesskaya, MA (1991) Vaccine, 9: 319-325; Singer, B. and Grunberger, D. (1983) Molecular Biology of Mutagens and Carcinogens, Plenum Press, New York-London; Loveless, A. (1966) Genetic and Allied Effects of Alkylating Agents, Butterworths, London; and Kochetkov, NK and Budowsky, EI ed. (1972) Organic Chemistry of Nucleic Acids, Part B, Plenum Press, London-New York) 48, 50, 51). A further increase in pH should lead to an increase in the fraction of more reactive protonated forms of guanosine and uridine, and thus to an increase in phage inactivation rate. However, the rate of inactivation of phage infectivity at pH 8.5 is lower than at pH 7.5 (Table 3). Thus, at least in this pH range, the effect of the proportion of the reactive agent on the inactivation rate is greater than that of nucleic bases. So when calculating the constant rate of inactivation of infectivity in the pH range of 6.0-8.5, we assume that the share of nucleic bases in their reactive (deprotonated) form is essentially constant. For oligoethyleneimine inactivation of MS2 phage, the dependence on the pH constant of the inactivation rate is therefore mainly caused by the participation of the reactive form and the total average positive charge of the inactivating agent.

There is a 60-fold increase in the effective rate of inactivation due to a decrease in pH from 8.5 to 6.5. This correlates with the increase in the reactive proportion of this agent in the solution (Tables 2 and 3). Thus, in this case, the effect of pH on the inactivation rate is mainly determined by changing the concentration of the reactive form in the solution. It should be emphasized, however, that the dependence on the pH of the total average positive charge for the trimer and tetramers is more strongly emphasized than for the ethyleneimine monomer (Table I), which leads to greater fluctuations in the rate of modification of polynucleotides for trimer and tetramers compared to monomers and dimers.

The rate of modification of any virion components with traditional inactivating agents is usually considered a function of the mean concentration in the agent solution. If the low molecular weight agent, however, has a specific affinity for a certain polymer, the local concentration of the agent near that polymer is higher than the average concentration in the agent solution and exponentially decreases with increasing distance from the polymer (Dolar, D. and Peterlin, A., (1969 ) J. Chem. Phys., 50: 3011-3015). The selectivity of inactivating the viral genome should be proportional to the difference in local concentration of the agent near these biopolymers.

Thus, even a local increase in oligoethyleneimine concentration near the genome should favorably increase its rate of modification. However, as considered above, it creates16

187 759 complexes between ethyleneimine oligomers and polynucleotides should increase the protonation range of the aziridine group, and thus the rate constant (s) of polynucleotide modification. Due to the exponential decrease in the concentration of the agent at a distance, at a distance of 1-2 nm from the polymer, the local concentration of the agent is practically identical to the average concentration in solution (Dolar, D. and Peterlin, A., (1969) J. Chem. Phys., 50: 3011-3015). Of course, the fraction of the reactive form of the reagent at this distance should be the same as in the free (unassociated) state in solution. Thus, the increase in the concentration of the inactivating agent in the vicinity of the polynucleotide, as well as the association of the inactivating agent with the polynucleotide, should not affect the rate of modification of the capsid component, especially its antigen-bearing regions on the virion surface. The selectivity of viral genome inactivation is improved by the use of selectively inactivating ethyleneimine oligomers having specific affinity for nucleic acids compared to traditional inactivating agents.

All these data and considerations allow the selection of an ethyleneimine trimer or tetramer, with a corresponding increase in polynucleotide affinity, with an increase in reaction rate and selectivity of viral genome modification. As shown above, this transition leads to an increase in selectivity of at least six orders of magnitude. Thus, even if the selectivity of the ethyleneimine monomer is not better than the selectivity of other agents currently used to make whole virion vaccines containing killed microbes, a significant increase in trimer and tetramer selectivity allows to ignore the effect of modification of the virion component on immunogenicity, stability and other properties of the virion.

The use of selectively inactivating ethyleneimine oligomers with selectivity for polynucleotide modification creates new possibilities for the production of safe and effective antiviral vaccines containing killed microorganisms. The same approach can also be used to selectively inactivate viruses, which are the most harmful impurities in donor's blood and serum, as well as in medical and veterinary preparations secreted from animal tissues and cell cultures. The same approach can be used to selectively inactivate biologically active (e.g. transforming) DNA fragments that can contaminate compositions containing cells or biopolymers.

The following examples are illustrative of the invention.

Example 1

Inactivation of bacteriophage Kinetic determination

MS2 bacteriophage was prepared according to a conventional procedure. Purification was performed by resedimentation on polyethylene glycol (PEG 6000, Serva) or by chromatography on DEAE-Sephadex A25 with a linear NaCl gradient (0.02-1.0 M, 20 mM Tris HCl, pH 7.4). The purified phage was suspended in 0.15 M NaCl solution (2-10 mg / ml) and stored at + 4 ° C. The infectivity of the virus suspension was determined by a conventional two-layer technique on peptone and Escherichia coli CA 180 meat agar.

Ethyleneimine (I), its trimer (III), and linear (IV) and branched (V) tetramer were prepared according to the method of Kostyanovsky et al. (1988) Izv. Akad. Sciences SSSR 11: 25662575. PMR data indicated that the purity of these compounds exceeded 95%. Ethyleneimine solutions were prepared immediately before use by adding the calculated volume of the compound (densities at 20 ° C are 0.836, 0.945, and 0.968 g · em³ for compounds I, III, and IV (V) respectively) to 0.15 M NaCl solution. The pKa values ​​of the protonable ethyleneimine groups in 0.15 M aqueous NaCl solution at 25 ° C were calculated based on the results of potentiometric titration (Fig. 2) HCl using the TTT-60 titrator (Radiometer) with a thermostated cell chamber. The accuracy of determining the pKa value is not less than 0.05. The proportion of reactive (protonated on aziridine) form of agent (Q) was calculated from the following equation:

l + 10^pH_pK' (1)

187 759 where pKj is the pKa value of the aziridine group in the corresponding compound.

The total average positive particle charge (p) was calculated using the following equation:

p = Jewish (H ".Aⁿ*) (2) where d- (H_n-ANDⁿ+) means the proportion of agent having a positive charge n at a given pH value.

Before mixing, the phage-containing composition and the freshly prepared solution of the selectively inactivating ethyleneimine oligomer were kept at 20 ° C and the desired pH was obtained by adding diluted NaOH or HCl solutions. Samples of the reaction mixture incubated at 20 ° C (final concentration of inactivating agent are given in the brief description of Figures 3 and 4) were taken at defined intervals and after immediate dilution 100-fold used to determine infectivity (titers).

Effective infectivity inactivation rate constants (k) were calculated from the following equation:

k- ^ ioSSo / s, (3)

A -t where A is the concentration (total or reactive part) of the agent; So and St indicate the infectivity (titer) of the suspension before and minutes after the start of inactivation.

The well-defined shapes of the potentiometric titration curves (Fig. 2) reflect the changes in the ionic state of the ethyleneimine inactivating oligomer. In the case of monomer, a single plateau corresponds to the protonation of an aziridine group. The calculated pKa value (Table 1) is quite consistent with the literature data (O'Rourke, CE, et al., (1956) J. Am. Chem. Soc., 78: 2159-2160. For other plateau compounds they correspond to the protonation of amino groups and aziridine (table 1). The acid consumption of oligomers II to V indicates that each has a single zpKa group 5.15-3.0, which corresponds to the presence of a single aziridine group in these molecules. Molecules II to V exhibit increased aziridine pKa relative to ethyleneimine, because a higher number of strongly basic amino groups increases the total positive charge of the molecule, thus reducing the protonation of aziridine groups. The less emphasized pKa difference between the IV and V molecules probably comes from the difference in the arrangement of the aziridine and amino groups in these molecules. One equivalent of acid is required to proton the most basic amino groups for compounds II to IV, while two are necessary for compound V.

These data correlate with the presence of one primary amine group in each of the first three compounds, and two such groups in V. The consumption of one and two equivalents of acid, respectively, for the occurrence of one and two secondary amino groups, is necessary for ionic transformation of compounds III and IV at pH 7.35 and 7.05, respectively. The consumption of one equivalent of acid for the ionic transformation of compound V at pH 6.4 corresponds to the existence of one tertiary amine group. In these compounds, higher pKa values ​​of primary versus secondary and tertiary amine groups are probably caused by electronic effects as well as the structure of the polyamine molecule (see MD Bratek-Wiewiorowsak et al. (1986) Bull. Pol. Ac. Sci., Chem. 34: 229-249). These data were used to calculate the total average positive charge and protonation range of the aziridine group at any pH value for compounds I to V (Table 2).

Exponential decrease in MS2 phage infectivity under the action of compounds I to V at pH

7.5 (Figure 3) indicates that the concentration of these compounds remains constant under the conditions used. This allows the calculation of effective rate constants (k) to inactivate infectivity based on the data received. As shown in Table 3, the transition of the ethyleneimine monomer to its tetramer leads to an increase of two orders of magnitude by this rate constant.

A decrease in pH from 8.5 to 6.5 causes a 60-fold increase in the rate of inactivation of phage infectivity by ethyleneimine (Figure 4, Table 3). The use of this compound at pH 6.5 leads to a significant deviation from the exponential curve shape (Fig. 4). This change indicates a rapid decrease in the concentration of compound II due to its cationic polymerization at this pH (cf. Gembitsky, PA, et al. (1791) Nauka, Moscow). The inactivation rate constant for compound II at pH 6.5 was therefore calculated using only the initial part of the curve

187 759 survivability. The rate constant for consumption of compound II at pH 6.5 is about 0.02 min¹, as calculated using a rate constant in the early inactivation period and the final range of inactivation of infectivity (cf. Budowski, EI and Zalesskaya, MA, (1991) Vaccine, 9: 319-325).

Example 2

Calculation of the inactivation endpoint

The extent of reduction of infectivity of virus-containing compositions can be experimentally controlled by at least about six orders of magnitude, even when the sample volume increases (within reasonable limits) and a series of successive passes is used. However, the production of safe antiviral vaccines containing killed microorganisms requires that the infectivity of the original virus-containing composition is reduced by at least about 20 orders of magnitude. Thus, in general, the safety of antiviral vaccines containing killed microorganisms cannot be determined only experimentally and must rely on the method described herein and can be evaluated more significantly by calculation using a kinetic approach. This requires an accurate kinetic description of the inactivation conditions for the virus, taking into account the characteristics of the selectively inactivating agent of the invention. Data can be obtained from the early (experimentally controlled) part of the survival curve.

Accurate kinetic description should be based on accurate determination of viral infectivity of the suspension during inactivation. The skilled person should make sure that the determination of infectivity (titres) is accurate to ensure that a constant inactivation rate and minimum duration of action of the selectively inactivating agent (ti) are determined. A certain ti value can be obtained using the kinetic approach described above, especially when the survival curves give a good kinetic description to a certain degree of infectivity reduction. Assuming that the modification of the viral genome is not distorted by biological factors (such as DNA repair) and assuming that the formation of the first inactivating damage, regardless of its location in the viral nucleic acid, blocks full genome replication, survival curves for the virus during the action of the selectively inactivating agent according to the invention satisfies the following equation:

S = So exp (-Akt) where So and S are titers of the virus-containing composition before and during t after the start of the activity of the selectively inactivating agent, A is the concentration of the selectively inactivating agent and k is the inactivation rate constant, i.e. modification of the nucleotide residue on the genome.

Assuming that A and k values ​​are constant during inactivation, survival curves are exponential. In this case, the inactivation time required to reduce the infectivity to a given can be calculated from the following equation:

Tj = [2,3 / (Ak) log (S / Sc)

Using the inactivation rate constant obtained in Example 1 given in Table 3, the inactivation time, assuming 10 'ethyleneimine oligomer concentration³ M (for tetramer) and inactivation rate constant, k = 150, taken from Table 3 (pH 7.5) and log (S / So) is the desired degree of inactivation, in this case at least about 20, infectivity reduction time in Example 1 is 15.3 minutes at 20 ° C.

According to the invention, the inactivation endpoint for any virus and any selectively inactivating agent of the invention can be determined under inactivation conditions based on data obtained from the initial part of the survival curve.

Example 3

Pikomawirus. Hepatitis A virus (a type of enterovirus) and mouth and hoof virus (aftovirus) containing single-stranded RNA, positive chain, with capsid containing only virus protein were tested. Viruses are produced according to conventional procedures involving purification and determination of infectivity and stability. The ethyleneimine selectively inactivating oligomer of the invention is prepared as described above. PK values ​​are determined_and ethyleneimine in a specific salt solution and at a specific temperature. virus

187 759 and the virus inactivant selected, i.e., the ethyleneimine oligomer, are mixed at a particular pH, temperature and concentration using the conditions for each of the viruses as given in Table 4.

Table 4

Time to inactivate the infectivity of some animal viruses (15-20 orders of magnitude) under the action of ethyleneimine tetramer at 20 ° C.

<td rowspan="3">Virus</td><td rowspan="3">Durability of treatment in conditions</td><td colspan="4">Incubation time in hours * The concentration of the agent</td>

<td colspan="2">1 mM</td><td colspan="2">15 mM</td>

<td>pH 7.0</td><td>pH 8.0</td><td>pH 7.2</td><td>pH 8.0</td>

<td>Foot-and-mouth disease virus</td><td>moderate</td><td> 120</td><td> 1200</td><td></td><td></td>

<td>Group A virus</td><td>moderate</td><td> 1-50</td><td> 10-300</td><td> 0,07-0,2</td><td> 0,7-2</td>

<td>Hepatitis virus</td><td>Okay</td><td> 1-30</td><td> 10-300</td><td> 0,15-0,3</td><td> 1,5-3</td>

<td>Face and hoof disease virus</td><td>moderate</td><td> 2-5</td><td> 20-500</td><td> 0,15-0,3</td><td> 1,5-3</td>

ł) depends on the type and strain of virus, the method of virus purification and the composition of the reaction mixture

Samples of the reaction mixture taken at appropriate intervals were supplemented with thiosulfate (0.1 M final concentration) for 30 minutes to flood the excess inactivating agent and the infectivity of the reaction mixture was measured as described above. The time ti of reducing the viral infectivity by 20 orders of magnitude is calculated using the equation described above. For example, using a constant inactivation rate of about 500 (known to the skilled person and based on comparing the genome length of hepatitis A virus compared to MS2 bacteriophage as described in Example 1), the inactivation time, assuming 10 'ethyleneimine oligomer concentration⁴ M (for tetramer) and constant inactivation rate, k = 500 (pH 7.5) and log (S / So) is the desired degree of inactivation, in this case at least about 20, the infectivity reduction time in this example is 30 minutes at 20 ° C.

Example 4

Rhabdoviruses. Inactivation of the shampoo virus (VSV) containing the single-stranded virus RNA and the lipid-shielded nucleocapsid were tested.

VSV is cultured in human A549 cells. The EMC stock generated is either mouse L929 cells or human A459 cells. Culture and trial procedures are known to those skilled in the art. VSV infectivity is tested in final, 10-fold serial dilutions in DMEM culture with 10% fetal calf serum. Each dilution is used to inoculate eight replicate wells with human A549 cells in 96-well microtiter plates. Virus induced cytopathology is assessed after 72 hours of incubation at 37 ° C, in 5% CO₂. The virus titer reported is assessed using known methods.

Bound to VSV cells is produced by incubating the confluent monolayer of human A549 cells with 5 ml⁷ IDso / ml VSV in serum free DmEm for 1 hour at 37 ° C with 5% CO₂ in 150 cm tissue culture flasks². The multiple of infections under these conditions is about 2.1 TCIDsc / cell. To assess inactivation, selectively inactivating ethyleneimine oligomer is added to the cell-associated virus in DMEM in 3 ml aliquots in polystyrene tubes. Samples of the reaction mixture are taken at finite intervals and made up with thiosulfate (final concentration 0.1 M) for 30 minutes to flood the excess selectively inactivating agent. Cells are removed by centrifugation and the infectivity titers of the supematant and the redispersed papule are determined. The time ti of reducing the viral infectivity by 20 orders of magnitude is calculated using the equation described above.

Inactivation of VSV cell free added to whole blood was evaluated (5x10⁹red blood cells / ml) in the presence of a selectively inactivating ethyleneimine oligomer.

187 759

Virus infectivity is assessed as described herein. The time t) for reducing the infectivity of viruses by 20 orders of magnitude is calculated using the equation described above. The structure and function of red blood cells are assessed.

Example 5

OstymiOsywisusy. Inactivation of influenza A virus with a single cleaved RNA strand, negative chain, lipid-shielded virus capsid is assessed. The virus is produced according to conventional procedures involving purification and determination of infectivity and stability. The ethyleneimine selectively inactivating oligomer of the invention is prepared as described above. The pKa values ​​of the ethyleneimine oligomer are determined in a particular salt solution and a specific temperature. Influenza A virus and the selected virus inactivating agent, i.e. the ethyleneimine oligomer, are mixed at a particular pH, temperature and concentration using the virus conditions as given above. Virus infectivity is assessed as described herein. The time ti of reducing the viral infectivity by 20 orders of magnitude is calculated using the equation described above.

Example 6

Human immunodeficiency virus (two copies of single-stranded RNA genome, often ravaged capsid proteins) was tested. HIV in cell-free or intracellular form is added to whole blood or red cell concentrate in the tube. A selectively inactivating ethyleneimine oligomer is added and the antigen is measured after processing the HIV samples. Virus infectivity is assessed as described herein. The time ti of reducing the viral infectivity by 20 orders of magnitude is calculated using the equation described above.

It can be understood from the above description that the selectively activating ethyleneimine oligomers and methods of the invention can be used to inactivate blood-borne viruses, bacteria or parasites in compositions containing cells or biopolymer in various contexts, e.g., in a hospital, laboratory, as part of a kit . Because the cell compositions also contain various proteins, the method of virus inactivation described herein is also suitable for protein fractions, particularly blood plasma protein fractions or purified blood products, including, but not limited to, fraction containing fractions (such as factor VIII and factor IX) , serum albumin and / or gammaglobulin. Viral and bacterial inactivation can be performed by subjecting the protein or purified protein fraction to the selectively inactivating ethyleneimine oligomer described herein.

The method of the invention may be combined with other methods of virus inactivation. For example, certain processes used in the manufacture of medical products (e.g., buffer chromatography at low pH, or storage of red cells in acidic solutions containing calcium chelating agents) may have incidental viral macro-cultivation properties for selected sensitive viruses, usually shielded viruses. The use of ethylene will help such agents to inactivate such viruses.

Example 7

Independence of the reaction rate from the concentration of surrounding protein

Inactivation of MS2 phage was evaluated in the absence and presence of protein up to 3% (30 mg / ml) to assess whether high concentrations of protein in the medium would significantly affect the rate of reaction with the viral genome. Phage MS2 suspended in 0.15 M NaCl was added to equal volume of human serum albumin. Yligyethyleneimine trimer or tetramer was added to the phage suspensions to final concentrations of 0.4 mM or 0.2 mM, respectively, and inubstained at pH 7.0, 25 ° C. Periodically, samples were removed and analyzed for residual infectivity (phage titer). The calculated rate constants are shown in Table 5:

Table 5

Constant rates of activation of MS-2 phage infectivity in the presence of human serum albumin

<td colspan="2">Trigot inactivation with yligyetylehyimihy (0.4 mM)</td><td colspan="2">^ activation with yligyethylenimine trimer (0.2 mM)</td>

<td>Albumin concentration (mg / ml)</td><td>Speed ​​constant (mM ^ U + standard error</td><td>Albumin concentration (mg / ml)</td><td>Speed ​​constant (mM '¹ hj + standard error</td>

<td> 0</td><td> 10,8±1,1</td><td> 0</td><td> 22,5±4,2</td>

<td> 10</td><td> 9,8±0,3</td><td> 1</td><td> 19,9±2,2</td>

<td> 20</td><td> 10,4±0,3</td><td> 2</td><td> 18,6±1,8</td>

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The lack of a clear effect of high protein concentrations on the rate of virus inactivation confirms the high degree of selectivity of the reaction of ethyleneimines with the viral genome and confirms that modification of nucleic acid can be achieved in biological or other fluids containing high concentration protein or other biopolymers.

Example 8

Specificity of inactivation of phage infectivity (nucleic acid modification): no protein modification by ethyleneimine oligomers

Inactivation of MS2 phage infectivity by ethyleneimine monomer (25 mM), trimer (0.4 mM) and tetramer (0.2 mM) was performed in the presence of 0.9% human serum albumin to various degrees of inactivation in 0.02 M phosphate buffer, pH 7.0-7.2, at 25 ° C. After completion of the thiosulfate inactivation reaction (0.1 M final concentration), samples from each incubation were compared by 12.5% ​​reducing and non-reducing SDS-PAGE and isoelectric focusing (pH range 3 to 7).

As shown in Table 6, reduced and unreduced SDS-PAGE did not detect any difference in albumin after inactivation to 80 orders of magnitude, indicating no significant effect of inactivation on protein size.

Table 6

No protein modification by ethyleneimine oligomers, SDS-PAGE analysis

<td colspan="2">Inactivation with monomer (25 mM)</td><td colspan="2">Trimmer inactivation (0.4 mM)</td><td colspan="2">Tetramer inactivation (0.2 mM)</td>

<td>Degree inactivation (Logio)</td><td>SDS-PAGE</td><td>Degree inactivation (Log,)</td><td>SDS-PAGE</td><td>Degree inactivation (Logio)</td><td>SDS-PAGE</td>

<td> 0</td><td>baseline *</td><td> 0</td><td>baseline *</td><td> 0</td><td>baseline *</td>

<td> 11</td><td>no change **</td><td> 11</td><td>no change **</td><td> 11</td><td>no change **</td>

<td> 32</td><td>no change **</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 41</td><td>no change **</td><td> 40</td><td>no change **</td><td> 40</td><td>no change **</td>

<td> 83</td><td>no change **</td><td> 79</td><td>no change **</td><td> 81</td><td>no change **</td>

* for reduced and non-reduced SDS-PAGE, main band at about 68 kDa with about 5 weak bands with a higher molecular weight.

** no change for reduced and unreduced SDS-PAGE

However, as shown in Table 7, isoelectric focusing (IEF) analysis of albumin samples showed that inactivation with ethyleneimine monomer, but not with trimer or tetramer, resulted in a significant increase in charge heterogeneity in albumin, increasing its alkalinity. This indicates that the ethyleneimine monomer modified albumin, changing its charge. In contrast, the ethyleneimine trimer and tetramer, with the same degree of inactivation, up to 81 orders of magnitude, had no detectable effect on albumin load. This indicates that the inactivation reaction with ethyleneimine oligomers is strongly selective for nucleic acid, while inactivation with ethyleneimine monomer under the same conditions modifies proteins as well as nucleic acids.

Table 7

No protein modification by ethyleneimine oligomers, isoelectric focusing analysis

<td colspan="2">Inactivation with monomer (25 mM)</td><td colspan="2">Trimmer inactivation (0.4 mM)</td><td colspan="2">Tetramer inactivation (0.2 mM)</td>

<td>Degree inactivation (Logio)</td><td>IEF template</td><td>Degree inactivation (Logio)</td><td>wzórIEF</td><td>Degree inactivation (Logio)</td><td>IEF template</td>

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td>

<td> 0</td><td>baseline *</td><td> 0</td><td>baseline *</td><td> 0</td><td>baseline *</td>

187 759 cont. table 7

<td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td>

<td> 11</td><td>extended band **</td><td> 11</td><td>no change **</td><td> 11</td><td>no change **</td>

<td> 32</td><td>extended band **</td><td> -</td><td> -</td><td> -</td><td> -</td>

<td> 41</td><td>extended band **</td><td> 40</td><td>no change **</td><td> 40</td><td>no change **</td>

<td> 83</td><td>extended band **</td><td> 79</td><td>no change **</td><td> 81</td><td>no change **</td>

* Single main band at pI about 5.5, ** Expanded and blurred main band, mainly towards increased positive charge (more basic).

Example 9

Inactivation of the enveloped animal virus To demonstrate the inactivation of the enveloped animal virus, Venezuelan Equine Encephalitis Virus ("VE") was incubated with ethyleneimine trimer (2 mM) or tetramer (0.5 mM) for up to 24 hours at 22 or 37 ° C in 0.02 M phosphate buffer containing 0.2% bovine serum albumin. Samples were periodically removed from the incubation mixture and titrated to residual virus concentration. Inactivation rates were determined from the data for the first 7 hours. The results are shown in Table 8 below:

Table 8

Constant rates of Venezuelan equine encephalitis virus inactivation by ethyleneimine oligomers

<td>Temperature (° C)</td><td>Inactivation rate constant for ethyleneimine trimer (mM''h '')</td><td>Rate of inactivation by the ethyleneimine tetramer (mM ^ h¹)</td>

<td> 25</td><td> 0,33</td><td> 1,64</td>

<td> 37</td><td> 1,25</td><td> 3,40</td>

Example 10

Behavior of antigenic epitopes during inactivation of enveloped animal virus

In order to demonstrate the behavior of antigenic epitopes during inactivation with ethyleneimine oligomers, Venezuelan equine encephalitis virus was inactivated for up to 7 hours with 2mM ethyleneimine trimer at 37 ° C. Virus antigenicity after 3 and 7 hours of inactivation was tested by ELISA using three different monoclonal antibodies. Endpoint titers were determined and compared with viral titers prior to inactivation. The monoclonal antibodies used were 1A1B-9, 7A1A-1 and 7A3A-4. Antibody 1A1B-9 recognizes the type-specific antigen, while antibodies 7A1A-1 and 7A3A-4 recognize the latent epitope that is presented after the development of the viral capsid antigen.

As shown in Table 9, no change in antigenic reactivity was detected after inactivation by more than 8 orders of magnitude (three hours of inactivation) or after an estimated 18.7 orders of magnitude (seven hours of inactivation).

Table 9

Behavior of antigenic epitopes during the inactivation of Venezuelan Equine Encephalitis virus with an ethyleneimine trimmer (2 Mm)

<td></td><td colspan="3">Incubation time (hours)</td>

<td></td><td> 0</td><td> 3</td><td> 7</td>

<td>Virus titer</td><td>2x10⁸</td><td> 0</td><td> 0</td>

<td>Endpoint, antibody 1A1B-9</td><td> 10’⁵</td><td> 10-5</td><td> 10'⁵</td>

<td>Endpoint, antibody 7A1A-1</td><td> 10-3</td><td> 10-3</td><td> 10-3</td>

<td>Endpoint, antibody 7A3A-4</td><td> 10-2</td><td> 10-2</td><td> 10-2</td>

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Example 11

Kinetics of phage inactivation of MS2 with bromide salts of ethyleneimine oligomers To assess kinetics of inactivation of phage MS2 with bromide salts of ethyleneimine oligomers, the phage were incubated at 25 ° C for various periods of up to 24 hours in one of several buffers at pH 7.0 together with ethyleneimine trimer tetramer . ethyleneimine trimer hydrobromide (β-bromoethyl diethylenetriamine tribromohydrate) or ethyleneimine tetramer hydrobromide (β-bromoethyltriethylenetetramine tetrabromohydrate). Samples were periodically removed to assess residual infectivity, and infectious inactivation rate constants were determined. The results shown in Table 10 indicate that the phage MS2 was inactivated upon incubation with bromide salts by conversion into the corresponding ethyleneimine trimer or tetramer. A slightly lower inactivation rate for bromide salts compared to inactivation in the presence of the corresponding ethyleneimine oligomers indicates that the conversion was only about 30-40%. However, the use of halogen salts may be beneficial due to their greater stability and convenience. The data also illustrate that the phosphate anion inhibits the reaction, probably because of its large negative charge interfering with the interaction of ethyleneimine oligomers with nucleic acid.

Table 10

Constant inactivation rates of phage MS2 with ethyleneimine trimer and tetramer and appropriate bromide salts

<td>Buffer</td><td colspan="4">Speed ​​constant (mM ^ h⁴)</td>

<td></td><td>trimer ethyleneimine</td><td>tetramer ethyleneimine</td><td>Tribromohydrat β-bromoetylodietylenotriaminy</td><td>Tetrabromohydrat β-bromoetylotrietylenotetraminy</td>

<td>AND</td><td> 7,4±0,6</td><td> -</td><td> -</td><td> -</td>

<td>B</td><td> 7,6±0,8</td><td> 18,2±1,6</td><td> 3,0±0,4</td><td> 6,3±0,9</td>

<td>C</td><td> 5,1±0,8</td><td> 8,7±0,9</td><td> -</td><td> -</td>

<td>D</td><td> 0,26±0,03</td><td> 0,32±0,04</td><td> 0,17±0,02</td><td> 0,29±0,04</td>

A: 0.15 M NaCl

B: 0.075 M NaCl, 0.2 M MOPS

C: 0.1 M NaCl, 0.025 M phosphate

D: 0.075 M NaCl, 0.2 M phosphate

Although the description refers to viruses, it should be understood that the methods of the present invention are generally also useful in deactivating any biological impurities found in stored blood or blood products, including bacteria and blood borne parasites.

Although the invention has been described in detail for the purposes of clarity, it will be apparent that some modifications may be made while remaining within the scope of the appended claims.

All publications and patent documents cited in this specification are attached as references in their entirety for all purposes as if each individual publication and patent document were described individually.

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187 759

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