WO2003035238A2

WO2003035238A2 - Methods for combating bio-terrorism

Info

Publication number: WO2003035238A2
Application number: PCT/US2002/031584
Authority: WO
Inventors: David M. Mann; William N. Drohan; Glenn Calvert; Martin J. Macphee
Original assignee: Clearant, Inc.
Priority date: 2001-10-19
Filing date: 2002-10-21
Publication date: 2003-05-01
Also published as: AU2002356540A1; WO2003035238A3

Abstract

Methods are disclosed for combating bio-terrorism. More specifically, methods are disclosed for combating bio-terrorism by irradiating a material, such as a letter or package or currency, to reduce the level of one or more biological contaminants or pathogens that may be present on or in the material, such as viruses, bacteria (including inter- and intracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), yeasts, molds, fungi, single or multi-cellular parasites, and/or prions or similar agents responsible, alone or in combination, for TSEs.

Description

METHODS FOR COMBATING BIO-TERRORISM

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to methods for combating bio-terrorism. More specifically, the present invention relates to methods for combating bio-terrorism by irradiating a material, such as a letter or package or currency, to reduce the level of one or more biological contaminants or pathogens that may be present on or in the material, such as viruses, bacteria (including inter- and intracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), yeasts, molds, fungi, single or multi- cellular parasites, and/or prions or similar agents responsible, alone or in combination, for TSEs.

Background of the Related Art

Bio-terrorism involves the use of biological contaminants or pathogens as weapons of terror, designed to spread fear throughout a given population. Moreover, bio- terrorism can result in political instability, particularly in view of the panic that many fast-moving plagues have historically engendered. The extreme potency and virulence of such biological contaminants and pathogens means that as little as a few kilograms can be devastatingly effective (for example, it takes as few as 8000 inhaled anthrax spores to have a lethal effect on a human). Moreover, relatively large amounts of many biological contaminants and pathogens can be prepared from a small freeze-dried seed culture in a period of days to weeks.

Bio-terrorism is a lot easier to conduct and is thought by many experts to be much more likely to occur in the next few years than a replay of the terrorist tragedies of September 11, 2001. A small cloud of bacteria or viruses could easily and silently infect tens of thousands of people, triggering fatal outbreaks of anthrax, smallpox, pneumonic plague or any of a dozen other deadly diseases. And victims infected with contagious ailments could pass the microbes to thousands of others before doctors even figured out what was going on, if ever given that there are still natural outbreaks of certain diseases such as plague.

This sort of bio-terrorism would require just a small private plane, not a hijacked commercial jetliner. A terrorist could simply dump a bag of powdery bacterial spores while in flight, rather than having to overpower a planeload of passengers and federal marshals. And the terrorist could land and be home in time for dinner, instead of ending his life in a suicidal inferno.

"The events in New York and Washington were tragedies beyond what anyone had previously imagined, but the potential of biological terrorism is far greater in terms of loss of life and disruption. It would be less graphic — no flames and explosions — but much more insidious. Anyone with a cough would be a weapon," according to Michael Osterholm, director of the University of Minnesota's Center for Infectious Disease Research and Policy.

In many respects, the United States is less prepared for bio-terrorism than for conventional acts of terrorism. A report from the General Accounting Office (GAO) in

1999 documented major gaps in the nation's system for protecting itself against biological attacks. Inspectors found shortages of vaccines and medicines, stockrooms filled with expired drugs, and lax security measures where crucial drugs were stored.

A report by the Centers for Disease Control and Prevention (CDC) in Atlanta in January 2001 concluded that the nation's public health infrastructure was "not adequate to detect and respond to a bio-terrorist event." And a GAO report in March 2001 noted that 20 percent of the nation's pharmaceutical and medical supplies held by the federal Office of Emergency Preparedness for a bio-terrorist attack were stored in a vault whose temperature was 95 degrees and that had no air-conditioning. The potency of these medicines, however, could be assured only if kept cooler than 86 degrees.

Some improvements have been implemented since then. Still, the nation and the world are largely unprepared to fight major outbreaks of deadly diseases like plague.

Bio-terrorism is not new. Fourteenth-century barbarians tossed plague-infected corpses over the walls of fortified cities to spread the deadly infection among their enemies. In 1763, the English at Fort Pitt, Pa., gave smallpox-laden blankets to Indians who had been loyal to the French. And, as recently as the mid-1990s, U.N. weapons inspectors discovered that Iraq had stockpiled warheads containing anthrax spores and the toxin that causes botulism.

Russian scientists have revealed that the former Soviet Union produced large volumes of weapons-grade anthrax spores. And Aum Shinrikyo, the Japanese religious cult that released sarin nerve gas in the Tokyo subway system in 1995, made several tentative efforts to release biological agents. Members even went to Zaire to learn more about the deadly Ebola virus. An international biological weapons convention signed by 143 nations has outlawed the development, production and stockpiling of biological weapons since 1975, but the absence of any formal verification regime to monitor compliance has limited the effectiveness of the convention, according to the United Nations. In any case, terrorists don't play by the rules. And at least five countries known to sponsor international terrorism have acquired the capacity to produce biological weapons, according to U.S. Army experts.

Despite those capabilities, preparedness in the United States has lagged, in part because bio-terrorism has been deemed so unlikely. "Who would do such a thing?" skeptics asked. The attacks in New York and Washington in September 2001, however, seriously undermined such rational assurances.

Biological attacks can be far more difficult to respond to than conventional terrorist attacks. For one thing, they tend to be covert rather than overt; and so for days, no one might know that such an attack had even occurred. That's a huge problem for a disease like anthrax. Up to 80 percent of people infected by inhaled spores die within days if untreated. By the time symptoms appear — fever, rash and congested lungs — it's generally too late.

Another problem is that the proper first-line defenders against a biological attack would not be police and fire officials, who are specially trained for public safety emergencies. Rather, they should be local doctors and hospital staffers, most of who have received little training in the art and science of being able to recognize and respond to unusual outbreaks quickly.

And contagious diseases — unlike explosions — keep spreading long after an initial attack. Smallpox, for example, is easily spread by coughing and sneezing. The disease was declared eradicated in 1980, but vials of the virus were saved and the whereabouts of some are uncertain. Because regular vaccinations are no longer occurs, an entire generation is now susceptible to attack. And very few doses of the old vaccine remain in storage.

In a federal exercise in the summer of 2001, 24 simulated cases of smallpox were "discovered" in U.S. hospitals as part of an assessment of U.S. bio-terrorism preparedness. Less than two weeks after those cases popped up, computer models indicated that — if the exercise had been real — 15,000 people would have contracted the disease and 1,000 would have died. The "epidemic" was still raging when the exercise ended, and, the computer models predicted, rioting and looting would have broken out as vaccine supplies ran out.

A Clinton administration bio-terrorism initiative, administered jointly by the CDC and the National Institutes of Health, is speeding development of protective technologies, including portable DNA diagnostic devices that may someday help identify mystery microbes raining from the sky. But the initiative's $300 million budget is probably only a fraction of what will be needed to protect the nation in years to come.

Meanwhile, just in case, the CDC has contracted with two biotech companies to make and stockpile 40 million doses of smallpox vaccine. The first batches that could be used by civilians are not expected to be ready, however, until some time in 2004.

In view of the difficulties discussed above, there is a need for methods of combating bio-terrorism that are effective for reducing the level of at least one active biological contaminants or pathogens that may be present in or on a material.

Although there exist a number of methods for reducing the level of active biological contaminants or pathogens that may be present in or on a material, these methods are not suitable, for example, for treating letters and packages sent through the mail or for treating currency. Because such letters and packages and currency can serve as highly effective distribution systems for biological contaminants or pathogens, however, a suitable method of reducing the level of active biological contaminants or pathogens that may be present in or on a letter or package or currency is needed.

For example, autoclaving or heating of a material will frequently reduce the level of active biological contaminants or pathogens that may be present in or on that material.

Such treatment, however, is not suitable for use with letters and packages or currency because it will cause an unacceptable level of damage to the letters and packages or currency.

Similarly, ethylene oxide treatment is not suitable for use with letters and packages or currency because it leaves a toxic residue on the treated material. Ethylene oxide treatment also requires that the letters and packages or currency be gas permeable and stable at temperatures above 100°C. Moreover, many potential treatments for reducing the level of active biological contaminants or pathogens can be easily defeated by bio-terrorists. For example, treatment with steam and/or ethylene oxide can all be rendered ineffective by enclosing the active biological contaminant or pathogen in a foil pouch (that could be opened or punctured when the letter or package was opened) or simply using an envelope that is not porous. Treatment with steam may also have a deleterious effect on inks. Gas plasma sterilizers can be defeated by the presence of paper or other forms of cellulose.

Although radiation has been used to reduce the level of active biological contaminants or pathogens, most packaging materials are composed of large polymers, such as plastics or celluloses, which can be adversely affected by radiation. For example, radiation can cause excess polymerization of plastics, polymer breakdown and/or oxidation. The results of this damage can include the stiffening or embrittlement of plastic or paper, loss of adhesion by glues (resulting in the unintended opening of a letter or package) and/or discoloration. Accordingly, there remains a need for methods of combating bio-terrorism that are effective for reducing the level of at least one active biological contaminants or pathogens that may be present in or on a material, without an adverse effect on the material being treated.

The above references are incorporated by reference herein where appropriate for appropriate teachings of additional or alternative details, features and/or technical background.

SUMMARY OF THE INVENTION

An object of the invention is to solve at least the related art problems and disadvantages, and to provide at least the advantages described hereinafter.

Accordingly, it is an object of the present invention to provide methods of combating bio-terrorism by reducing the level of active biological contaminants or pathogens that may be present in a material, such as a letter or package or currency, without adversely effecting the material. Other objects, features and advantages of the present invention will be set forth in the detailed description of preferred embodiments that follows, and in part will be apparent from the description or may be learned by practice of the invention. These objects and advantages of the invention will be realized and attained by the compositions and methods particularly pointed out in the written description and claims hereof. In accordance with these and other objects, a first embodiment of the present invention is directed to a method for combating bio-terrorism involving the presence of active biological contaminants or pathogens in or on a material, said method comprising irradiating said material with radiation for a time and at a rate effective to reduce the level of active biological contaminants or pathogens in or on said material. Another embodiment of the present invention is directed to a method for combating bio-terrorism involving the presence of active biological contaminants or pathogens in or on a material, said method comprising: (i) performing at least one stabilizing process on said material, said stabilizing process selected from the group consisting of: (a) applying to said material at least one stabilizer in an amount effective to protect said material from said radiation; (b) placing said material in a container and reducing the moisture content of the atmosphere inside said container to a level effective to protect said material from said radiation; (c) reducing the temperature of said material to a level effective to protect said material from said radiation; (d) placing said material in a container and reducing the oxygen content of the atmosphere inside said container to a level effective to protect said material from said radiation; and (e) applying to said material at least one solvent in an amount effective to protect said material from said radiation; and (ii) iπadiating said material with a suitable radiation for a time and at a rate effective to reduce the level of active biological contaminants or pathogens in or on said material.

Another embodiment of the present invention is directed to a method for combating bio-terrorism involving the presence of active biological contaminants or pathogens in or on material, said method comprising: (i) performing at least two stabilizing process on said material, said stabilizing processes selected from the group consisting of: (a) applying to said material at least one stabilizer; (b) placing said material in a container and reducing the moisture content of the atmosphere inside said container; (c) reducing the temperature of said material; (d) placing said material in a container and reducing the oxygen content of the atmosphere inside said container; and (e) applying to said material at least one solvent; and (ii) irradiating said material with a suitable radiation for a time and at a rate effective to reduce the level of active biological contaminants or pathogens in or on said material, wherein said at least two stabilizing processes are together effective to protect said material from said radiation and further wherein said at least two stabilizing processes may be performed in any order.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objects and advantages of the invention may be realized and attained as particularly pointed out in the appended claims. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the relevant art.

As used herein, the singular forms "a," "an," and "the" include the plural reference unless the context clearly and unequivocally dictates otherwise.

As used herein, the term "biological contaminant or pathogen" is intended to mean a contaminant or pathogen that, upon direct or indirect contact with a mammal, particularly a human, may have a deleterious effect on that mammal. Such biological contaminants or pathogens include the various viruses, bacteria (including inter- and intracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), yeasts, molds, fungi, single or multi-cellular parasites, and/or prions or similar agents responsible, alone or in combination, for TSEs, all of which are known to those of skill in the art to be potentially employed as agents of bio-terrorism.

Examples of such biological contaminants or pathogens include, but are not limited to, the following: viruses, such as human immunodeficiency viruses and other retroviruses, herpes viruses, filoviruses, circoviruses, paramyxoviruses, cytomegaloviruses, hepatitis viruses (including hepatitis A, B and C and variants thereof), pox viruses, toga viruses, Epstein-Barr viruses, parvoviruses, Ebola virus, smallpox virus, Venezuelan equine encephalitis virus, brucellosis and viruses causing Viral Haemorrhagic Fever; bacteria (including mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias, including those responsible for Q fever and typhus), such as Escherichia, Bacillus, including B. anthracis, Yersinia, including Y. pestis, Campylobacter, Streptococcus and Staphylococcus; parasites, such as Trypanosoma and malarial parasites, including Plasmodium species; yeasts; molds; tularemia; and prions, or similar agents, responsible alone or in combination for TSE (transmissible spongiform encephalopathies), such as scrapie, kuru, BSE (bovine spongiform encephalopathy), CJD (Creutzfeldt-Jakob disease), Gerstmann-Straeussler-Scheinkler syndrome, and fatal familial insomnia.

As used herein, the term "active biological contaminant or pathogen" is intended to mean a biological contaminant or pathogen that is capable of causing a deleterious effect, either alone or in combination with another factor, such as a second biological contaminant or pathogen or a native protein (wild-type or mutant) or antibody, in a imal. The term "active biological contaminant or patnogen is micπucu ιυ un- ut biological contaminants or pathogens that can adopt or may be present in a dormant state, such as spores or the like, but are capable of causing a deleterious effect either in that state or in another state. An example of such an active biological contaminant or pathogen is Bacillus anthracis, which is often employed as a weapon of bio-terrorism in the form of spores. The term "active biological contaminant or pathogen" is also intended to include the products of certain biological contaminants or pathogens that are capable of causing a deleterious effect, either alone or in combination with another factor, in a mammal. An example such a product is botulism toxin, which is produced by Clostridium botulinum.

As used herein, the term "stabilizer" is intended to mean a compound or substance that, alone and/or in combination, reduces damage to the material being irradiated to an acceptable level. Illustrative examples of stabilizers that are suitable for such use include, but are not limited to, the following, including structural analogs and derivatives thereof: antioxidants; free radical scavengers, including spin traps, such as tert-butyl- nitrosobutane (tNB), a-phenyl-tert-butylnitrone (PBN), 5,5-dimethylpyrroline-N-oxide (DMPO), tert-butylnitrosobenzene (BNB), alpha-(4-pyridyl-l-oxide)-N-tert-butylnitrone (4-POBN) and 3,5-dibromo-4-nitroso-benzenesulphonic acid (DBNBS); combination stabilizers, i.e., stabilizers which are effective at quenching both Type I and Type II photodynamic reactions; and ligands, ligand analogs, substrates, substrate analogs, modulators, modulator analogs, stereoisomers, inhibitors, and inhibitor analogs, such as heparin, that stabilize the molecule(s) to which they bind.

Preferred examples of stabilizers include, but are not limited to, the following: fatty acids, including 6,8-dimercapto-octanoic acid (lipoic acid) and its derivatives and analogues (alpha, beta, dihydro, bisno and tetranor lipoic acid), thioctic acid, 6,8- dimercapto-octanoic acid, dihydrolopoate (DL-6,8-dithioloctanoic acid methyl ester), lipoamide, bisonor methyl ester and tetranor-dihydrolipoic acid, omega-3 fatty acids, omega-6 fatty acids, omega-9 fatty acids, furan fatty acids, oleic, linoleic, linolenic, arachidonic, eicosapentaenoic (EPA), docosahexaenoic (DHA), and palmitic acids and their salts and derivatives; carotenes, including alpha-, beta-, and gamma-carotenes; Co- Q10; xanthophylls; sucrose, polyhydric alcohols, such as glycerol, mannitol, inositol, and sorbitol; sugars, including derivatives and stereoisomers thereof, such as xylose, glucose, ribose, mannose, fructose, erythrose, threose, idose, arabinose, lyxose, galactose, allose, altrose, gulose, talose, and trehalose; amino acids and derivatives thereof, including both D- and L-forms and mixtures thereof, such as arginine, lysine, alanine, valine, leucine, isoleucine, proline, phenylalanine, glycine, serine, threonine, tyrosine, asparagine, glutamine, aspartic acid, histidine, N-acetylcysteine (NAC), glutamic acid, tryptophan, sodium capryl N-acetyl tryptophan, and methionine; azides, such as sodium- azide; enzymes, such as Superoxide Dismutase (SOD), Catalase, and Δ4, Δ5 and Δ6 desaturases; uric acid and its derivatives, such as 1,3-dimethyluric acid and dimethylthiourea; allopurinol; thiols, such as glutathione and reduced glutathione and cysteine; trace elements, such as selenium, chromium, and boron; vitamins, including their precursors and derivatives, such as vitamin A, vitamin C (including its derivatives and salts such as sodium ascorbate and palmitoyl ascorbic acid) and vitamin E (and its derivatives and salts such as alpha-, beta-, gamma-, delta-, epsilon-, zeta-, and eta-tocopherols, tocopherol acetate and alpha-tocotrienol); chromanol-alpha-C6; 6-hydroxy-2,5,7,8- tetramethylchroma-2 carboxylic acid (Trolox) and derivatives; extraneous proteins, such as gelatin and albumin; tris-3-methyl-l-phenyl-2-pyrazolin-5-one (MCI-186); citiolone; puercetin; chrysin; dimethyl sulfoxide (DMSO); piperazine diethanesulfonic acid (PIPES); imidazole; methoxypsoralen (MOPS); l,2-dithiane-4,5-diol; reducing substances, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT); cholesterol, including derivatives and its various oxidized and reduced forms thereof, such as low density lipoprotein (LDL), high density lipoprotein (HDL), and very low density lipoprotein (VLDL); probucol; indole derivatives; thimerosal; lazaroid and tirilazad mesylate; proanthenols; proanthocyanidins; ammonium sulfate; Pegorgotein (PEG-SOD); N-tert-butyl-alpha-phenylnitrone (PBN); 4-hydroxy-2,2,6,6- tetramethylpiperidin-1-oxyl (Tempol); mixtures of ascorbate, urate and Trolox C (Asc/urate/Trolox C); proteins, such as albumin, and peptides of two or more amino acids, any of which may be either naturally occurring amino acids, i.e., L-amino acids, or non-naturally occurring amino acids, i.e., D-amino acids, and mixtures, derivatives, and analogs thereof, including, but not limited to, arginine, lysine, alanine, valine, leucine, isoleucine, proline, phenylalanine, glycine, histidine, glutamic acid, tryptophan (Trp), serine, threonine, tyrosine, asparagine, glutamine, aspartic acid, cysteine, methionine, and derivatives thereof, such as N-acetylcysteine (NAC) and sodium capryl N-acetyl tryptophan, as well as homologous dipeptide stabilizers (composed of two identical amino acids), including such naturally occurring amino acids, as Gly-Gly (glycylglycine) and Trp-Trp, and heterologous dipeptide stabilizers (composed of different amino acids), such as carnosine (β-alanyl-histidine), anserine (β-alanyl-methylhistidine), and Gly-T ; and flavonoids/flavonols, such as diosmin, quercetin, rutin, silybin, silidianin, silicristin, silymarin, apigenin, apiin, chrysin, morin, isoflavone, flavoxate, gossypetin, myricetin, biacalein, kaempferol, curcumin, proanthocyanidin B2-3-O-gallate, epicatechin gallate, epigallocatechin gallate, epigallocatechin, gallic acid, epicatechin, dihydroquercetin, quercetin chalcone, 4,4'-dihydroxy-chalcone, isoliquiritigenin, phloretin, coumestrol, 4',7- dihydroxy-flavanone, 4',5-dihydroxy-flavone, 4',6-dihydroxy-flavone, luteolin, galangin, equol, biochanin A, daidzein, formononetin, genistein, amentoflavone, bilobetin, taxifolin, delphinidin, malvidin, petunidin, pelargonidin, malonylapiin, pinosylvin, 3- methoxyapigenin, leucodelphinidin, dihydrokaempferol, apigenin 7-O-glucoside, pycnogenol, aminoflavone, purpurogallin fisetin, 2',3'-dihydroxyflavone, 3- hydroxyflavone, 3',4'-dihydroxyflavone, catechin, 7-flavonoxyacetic acid ethyl ester, catechin, hesperidin, and naringin. Particularly preferred examples include single stabilizers or combinations of stabilizers that are effective at quenching both Type I and Type II photodynamic reactions, and volatile stabilizers, which can be applied as a gas and/or easily removed by evaporation, low pressure, and similar methods.

As used herein, the term "moisture content" is intended to mean the amount or proportion of water in a particular atmosphere, such as that within a container. The moisture contents referenced herein refer to levels determined by the FDA approved, modified Karl Fischer method (Meyer and Boyd, Analytical Chem., 31:215-219, 1959; May, et al., J. Biol. Standardization, 10:249-259, 1982; Centers for Biologies Evaluation and Research, FDA, Docket No. 89D-0140, 83-93; 1990) or by near infrared spectroscopy.

As used herein, the term "sensitizer" is intended to mean a substance that selectively targets viruses, bacteria (including inter- and mtracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), yeasts, molds, fungi, single or multi-cellular parasites, and/or prions or similar agents responsible, alone or in combination, for TSEs, rendering them more sensitive to inactivation by radiation, therefore permitting the use of a lower rate or dose of radiation and/or a shorter time of irradiation than in the absence of the sensitizer.

Illustrative examples of suitable sensitizers include, but are not limited to, the following: psoralen and its derivatives and analogs (including 3-carboethoxy psoralens); inactines and their derivatives and analogs; angelicins, khellins and coumarins which

tθ contain a halogen substituent and a water solubilization moiety, such as quaternary ammonium ion or phosphonium ion; nucleic acid binding compounds; brominated hematoporphyrin; phthalocyanines; purpurins; porphyrins; halogenated or metal atom- substituted derivatives of dihematoporphyrin esters, hematoporphyrin derivatives, benzoporphyrinderivatives,hydrodibenzopoφhyrin dimaleimade, hydrodibenzopoφhyrin, dicyano disulfone, tetracarbethoxy hydrodibenzoporphyrin, and tetracarbethoxy hydrodibenzoporphyrin dipropionamide; doxorubicin and daunomycin, which may be modified with halogens or metal atoms; netropsin; BD peptide, S2 peptide; S-303 (ALE compound); dyes, such as hypericin, methylene blue, eosin, fluoresceins (and their derivatives), flavins, merocyanine 540; photoactive compounds, such as bergapten; SE peptide; and reactive gases, such as oxygen, nitrogen, chlorine, ozone and peroxides. In addition, atoms which bind to prions, and thereby increase their sensitivity to inactivation by radiation, may also be used. An illustrative example of such an atom would be the Copper ion, which binds to the prion protein and, with a Z number higher than the other atoms in the protein, increases the probability that the prion protein will absorb energy during irradiation, particularly gamma irradiation.

As used herein, the term "radiation" is intended to mean radiation of sufficient energy to reduce the level of at least one active biological contaminant or pathogen that may be present in or on a material. Types of radiation include, but are not limited to, the following: (i) corpuscular (streams of subatomic particles such as neutrons, electrons, and/or protons); (ii) electromagnetic (originating in a varying electromagnetic field, such as radio waves, visible (both mono and polychromatic) and invisible light, infrared, ultraviolet radiation, x-radiation, and gamma rays and mixtures thereof); and (iii) sound and pressure waves. Such radiation is often described as either ionizing (capable of producing ions in irradiated materials) radiation, such as gamma rays, and non-ionizing radiation, such as visible light.

The sources of such radiation may vary and, in general, the selection of a specific source of radiation is not critical provided that sufficient radiation is given in an appropriate time and at an appropriate rate to effect sterilization. In practice, gamma radiation is usually produced by isotopes of Cobalt or Cesium, while UN and X-rays are produced by machines that emit UV and X-radiation, respectively, and electrons are often used to sterilize materials in a method known as "E-beam" irradiation that involves their production via a machine. Visible light, both mono- and polychromatic, is produced by

tl machines and may, in practice, be combined with invisible light, such as infrared and UN, that is produced by the same machine or a different machine.

As used herein, the term "to protect" is intended to mean to reduce any damage to the material being irradiated, which would otherwise result from the irradiation of that material, to a level that is acceptable. In other words, a substance or process "protects" a material from radiation if the presence of that substance or carrying out that process results in less damage to the material from irradiation than in the absence of that substance or process.

As used herein, an "acceptable level" of damage may vary depending upon certain features of the particular method(s) of the present invention being employed, such as the nature and characteristics of the particular material being irradiated, and can be determined empirically by one skilled in the art. An "unacceptable level" of damage would therefore be a level of damage that would preclude the intended use of the material being irradiated. The particular level of damage in a given material may be determined using any of the methods and techniques known to one skilled in the art. For example, when the material is a letter or package delivered through the mail or similar commercial delivery service, unacceptable levels of damage include the following: paper that becomes brittle; discoloration of paper and/or inks; fading of inks; fading or discoloration of transparent windows for address(es); and stiffening of paper. As used herein, the term "D₁₀ dose" is intended to mean the dose of radiation necessary to reduce the level of at least one active biological contaminant or pathogen to 10% of its pre-irradiation level.

Particularly Preferred Embodiments A first embodiment of the present invention is directed to a method for combating bio-terrorism involving the presence of active biological contaminants or pathogens in or on a material, said method comprising irradiating said material with radiation for a time and at a rate effective to reduce the level of active biological contaminants or pathogens in or on said material. A second embodiment of the present invention is directed to a method for combating bio-terrorism involving the presence of active biological contaminants or pathogens in or on a material, said method comprising: (i) performing at least one stabilizing process on said material, said stabilizing process selected from the group consisting of: (a) applying to said material at least one stabilizer in an amount effective to protect said material from said radiation; (b) placing said material in a container and reducing the moisture content of the atmosphere inside said container to a level effective to protect said material from said radiation; (c) reducing the temperature of said material to a level effective to protect said material from said radiation; (d) placing said material in a container and reducing the oxygen content of the atmosphere inside said container to a level effective to protect said material from said radiation; and (e) applying to said material at least one solvent in an amount effective to protect said material from said radiation; and (ii) radiating said material with a suitable radiation for a time and at a rate effective to reduce the level of active biological contaminants or pathogens in or on said material.

A third embodiment of the present invention is directed to a method for combating bio-teπorism involving the presence of active biological contaminants or pathogens in or on material, said method comprising: (i) performing at least two stabilizing process on said material, said stabilizing processes selected from the group consisting of: (a) applying to said material at least one stabilizer; (b) placing said material in a container and reducing the moisture content of the atmosphere inside said container; (c) reducing the temperature of said material; (d) placing said material in a container and reducing the oxygen content of the atmosphere inside said container; and (e) applying to said material at least one solvent; and (ii) iπadiating said material with a suitable radiation for a time and at a rate effective to reduce the level of active biological contaminants or pathogens in or on said material, wherein said at least two stabilizing processes are together effective to protect said material from said radiation and further wherein said at least two stabilizing processes may be performed in any order.

The particular geometry of the material being irradiated, such as the thickness and distance from the source of radiation, may be determined empirically by one skilled in the art. A preferred embodiment is a geometry that provides for an even rate of iπadiation throughout the material. A particularly prefeπed embodiment is a geometry that results in a short path length for the radiation through the material, thus minimizing the differences in radiation dose between the front and back of the material or at its edges and center, if it or the radiation source is rotated. This may be further minimized in some preferred geometries, particularly those wherein the material has a constant radius about its axis that is perpendicular to the radiation source, by the utilization of a means of rotating the preparation about said axis. Similarly, there may be preferred geometries of the radiation source that may be determined empirically by one skilled in the art. Preferably, a plurality of dosimeters is employed during irradiation to ensure the effectiveness of the inventive methods. Proper placement of dosimeters, as well as the use of the correct number thereof, is necessary to ensure adequate irradiation of the entire material being treated according to the present invention According to certain methods of the present invention, a stabilizer, or mixture of stabilizers, is applied to the material prior to irradiation thereof with radiation. This stabilizer is preferably applied to the material in an amount that is effective to protect the material from the radiation. Suitable amounts of stabilizer may vary depending upon certain features of the particular method(s) of the present invention being employed, such as the particular stabilizer being used and/or the nature and characteristics of the material being irradiated, and can be determined empirically by one skilled in the art.

According to certain methods of the present invention, the material to be irradiated is placed within an effective container prior to irradiation. An "effective container" for containing a material during irradiation is one that is stable under the influence of irradiation, minimizes the interactions between the radiation and the material and isolates the material from the external environment. As used herein, the term "container" includes a facility, such as a sealed room, provided that it sufficiently isolates the material from the external environment.

Preferred containers both maintain an effective seal against the external environment pre-, during and post-iπadiation, and are not reactive with the material within, nor do they produce chemicals that may interact with the material within. Other preferred containers include means for modifying the atmosphere inside the container which maintaining an effective seal against the external environment. Illustrative examples include, but are not limited to, containers that comprise glasses stable when iπadiated, stoppered with stoppers made of rubber that is relatively stable during radiation and liberates a minimal amount of compounds from within, and sealed with metal crimp seals of aluminum or other suitable materials with relatively low Z numbers. Suitable containers can be determined by measuring their physical performance, and the amount and type of reactive leachable compounds post-iπadiation and by examining other characteristics known to be important to the containment of materials empirically by one skilled in the art.

When placed within a container, the moisture content of the atmosphere inside the container may be reduced prior to irradiation of the material with radiation. The moisture content of the atmosphere inside the container is preferably reduced to a level that is

M effective to protect the material from the radiation. Suitable levels of moisture content may vary depending upon certain features of the particular method(s) of the present invention being employed, such as the nature and characteristics of the particular material being iπadiated and/or its intended use, and can be determined empirically by one skilled in the art. There may be materials for which it is desirable to maintain the moisture content of the atmosphere inside the container to within a particular range, rather than a specific value.

In addition, the moisture content of the material itself may be reduced prior to iπadiation of the material with radiation. The moisture content of the material is preferably reduced to a level that is effective to protect the material from the radiation. Suitable levels of moisture content may vary depending upon certain features of the particular method(s) of the present invention being employed, such as the nature and characteristics of the particular material being iπadiated and/or its intended use, and can be determined empirically by one skilled in the art. There may be materials for which it is desirable to maintain the moisture content to within a particular range, rather than a specific value.

Preferably, the moisture content of the atmosphere inside the container and/or the material may be less than 80%, 60%, 40% or 20%, generally less than about 15%, typically less than about 10%, more typically less than about 9%, even more typically less than about 8%, usually less than about 5%, preferably less than about 3.0%, more preferably less than about 2.0%, even more preferably less than about 1.0%, still more preferably less than about 0.5%, still even more preferably less than about 0.2% and most preferably less than about 0.08%.

In certain embodiments of the present invention, the moisture content for a particular atmosphere and/or material may be found to lie within a range, rather than at a specific point. Such a range for the prefeπed moisture content may be determined empirically by one skilled in the art.

While not wishing to be bound by any theory of operability, it is believed that the reduction in moisture content may reduces the number of targets for free radical generation. Similar results might therefore be achieved by lowering the temperature of the material. These results may permit the use of a higher rate and/or dose of radiation than might otherwise be acceptable. Thus, the methods described herein may be performed at any temperature that doesn't result in unacceptable damage to the material, i.e., damage that would preclude the intended use of the material. Preferably, the

IS methods described herein are performed at ambient temperature or below ambient temperature.

The moisture content may be reduced by any of the methods and techniques known to those skilled in the art for reducing moisture in an atmosphere or a material without producing an unacceptable level of damage. When reducing the moisture content of the atmosphere inside the container, prefeπed examples of such methods include, but are not limited to, applying heat and/or reduced pressure to the atmosphere inside the container, introducing at least one hygroscopic material into the container and introducing dry gas into the atmosphere inside the container. When reducing the moisture content of the material, preferred examples of such methods include, but are not limited to, lyophilization, evaporation, concentration, centrifugal concentration, vitrification, spray- drying, distillation, freeze-distillation and partitioning during and/or following lyophilization.

The radiation employed in the methods of the present invention may be any radiation effective for reducing the level of one or more active biological contaminants or pathogens. The radiation may be coφuscular, including E-beam radiation. Preferably the radiation is electromagnetic radiation, including x-rays, infrared, visible light, UV light and mixtures of various wavelengths of electromagnetic radiation. A particularly prefeπed form of radiation is gamma radiation. According to the methods of the present invention, the material is iπadiated with the radiation at a rate effective for reducing the level of one or more active biological contaminants or pathogens, while not producing an unacceptable level of damage to the material being irradiated. Suitable rates of iπadiation may vary depending upon certain features of the methods of the present invention being employed, such as the nature and characteristics of the particular material being iπadiated, the particular form of radiation involved and/or the particular biological contaminants or pathogens being inactivated. Suitable rates of iπadiation can be determined empirically by one skilled in the art. Preferably, the rate of iπadiation is constant for the duration of the sterilization procedure. When this is impractical or otherwise not desired, a variable or discontinuous irradiation may be utilized.

According to the methods of the present invention, the rate of irradiation may be optimized to produce the most advantageous combination of protection to the material and time required to complete the operation. Both low (<3 kGy/hour) and high (>3 kGy/hour) rates may be utilized in the methods described herein to achieve such results. The rate of irradiation is preferably be selected to optimize the protection of the material while still reducing the level of one or more active biological contaminants or pathogens.

Although reducing the rate of iπadiation may serve to decrease damage to the material, it will also result in longer iπadiation times being required to achieve a particular desired total dose. A higher dose rate may therefore be preferred in certain circumstances, such as to minimize logistical issues and costs, and may be possible when used in accordance with the methods described herein for protecting a material from iπadiation.

As used herein, the "rate" of iπadiation, particularly as relates to E-beam radiation, means the total dose of radiation over the total time of iπadiation, or may be based on the total dose received from the time of first exposure to the time of last exposure or the total dose received during the time the machine is actually producing radiation.

According to a particularly prefeπed embodiment of the present invention, the rate of irradiation is not more than about 3.0 kGy/hour, more preferably between about 0.1 kGy/hr and 3.0 kGy/hr, even more preferably between about 0.25 kGy/hr and 2.0 kGy/hour, still even more preferably between about 0.5 kGy/hr and 1.5 kGy/hr and most preferably between about 0.5 kGy/hr and 1.0 kGy/hr.

According to another particularly preferred embodiment of the present invention, the rate of iπadiation is at least about 3.0 kGy/hr, more preferably at least about 6 kGy/hr, even more preferably at least about 16 kGy/hr, and even more preferably at least about 30 kGy/hr and most preferably at least about 45 kGy/hr or greater.

According to yet another particularly preferred embodiment of the present invention, such as when the radiation is E-beam, the rate of iπadiation is preferably at least in the range of 10,000 to 100,000 kGy/hr and more preferably at least about 1,000,000 kGy/hr.

According to the methods of the present invention, the material being iπadiated is irradiated with the radiation for a time effective for reducing the level of one or more active biological contaminants or pathogens that may be present in or on the material.

Combined with iπadiation rate, the appropriate iπadiation time results in the appropriate dose of iπadiation being applied to the material to achieve such a result. Suitable irradiation times may vary depending upon the particular form and rate of radiation involved and/or the nature and characteristics of the particular material being iπadiated.

Suitable iπadiation times can be determined empirically by one skilled in the art.

1:7 According to the methods of the present invention, the material being iπadiated is irradiated with radiation up to a total dose effective for reducing the level of one or more active biological contaminants or pathogens that may be present in or on the material, while not producing an unacceptable level of damage to that material. Suitable total doses of radiation may vary depending upon certain features of the methods of the present invention being employed, such as the nature and characteristics of the particular material being iπadiated, the particular form of radiation involved and or the particular biological contaminants or pathogens that may be present. Suitable total doses of radiation can be determined empirically by one skilled in the art. Preferably, the total dose of radiation is at least 25 kGy, more preferably at least 45 kGy, even more preferably at least 75 kGy, and still more preferably at least 100 kGy or greater, such as 150 kGy or 200 kGy or greater.

According to certain methods of the present invention, an effective amount of at least one sensitizing compound may optionally be applied to the material prior to iπadiation, for example to enhance the effect of the iπadiation on the biological contaminant(s) or pathogen(s) that may be present therein, while employing the methods described herein to minimize the deleterious effects of irradiation upon the material. Suitable sensitizers are known to those skilled in the art, and include psoralens and their derivatives and inactines and their derivatives. According to the methods of the present invention, the iπadiation of the material may occur at any temperature that is not deleterious to the material being iπadiated. According to one prefeπed embodiment, the material is iπadiated at ambient temperature. According to an alternate preferred embodiment, the material is iπadiated at reduced temperature, i.e. a temperature below ambient temperature or lower, such as 0°C, -20°C, - 40°C, -60°C, -78°C or even -196°C. According to another alternate prefeπed embodiment, the material is iπadiated at elevated temperature, i.e. a temperature above ambient temperature or higher, such as 37°C, 60°C, 72°C or 80°C. While not wishing to be bound by any theory, the use of elevated temperature may enhance the effect of irradiation on the biological contaminant(s) or pathogen(s) that may be present in or on the material and therefore allow the use of a lower total dose of radiation.

Most preferably, the irradiation of the material occurs at a temperature that protects the material from radiation. Suitable temperatures can be determined empirically by one skilled in the art.

t8 In certain embodiments of the present invention, the temperature at which irradiation is performed may be found to lie within a range, rather than at a specific point. Such a range for the preferred temperature for the iπadiation of a particular material may be determined empirically by one skilled in the art. According to another prefeπed embodiment, the material to be irradiated may be shielded from radiation other than that desired in order to minimize the deleterious effects upon the material and/or any added stabilizer(s) by undesired radiation.

According to the methods of the present invention, the irradiation of the material may occur at any pressure which is not deleterious to the material being iπadiated. According to one prefeπed embodiment, the material is iπadiated at elevated pressure. More preferably, the material is irradiated at elevated pressure due to the application of sound waves or the use of a gas or volatile solvent. While not wishing to be bound by any theory, the use of elevated pressure may enhance the effect of irradiation on the biological contaminant(s) or pathogen(s) that may be present in or on the material and/or enhance the protection afforded by one or more stabilizers, and therefore allow the use of a lower total dose of radiation. Suitable pressures can be determined empirically by one skilled in the art.

Similarly, according to the methods of the present invention, the iπadiation of the material may occur under any atmosphere that is not deleterious to the material being treated. According to one prefeπed embodiment, the material is held in a low oxygen atmosphere or an inert atmosphere. When an inert atmosphere is employed, the atmosphere is preferably composed of a noble gas, such as helium or argon, more preferably a higher molecular weight noble gas, and most preferably argon.

According to another prefeπed embodiment, the material is held under vacuum while being irradiated. According to a particularly prefeπed embodiment of the present invention, a material is stored under vacuum or an inert atmosphere (preferably a noble gas, such as helium or argon, more preferably a higher molecular weight noble gas, and most preferably argon) prior to iπadiation. According to an alternative prefeπed embodiment of the present invention, a material is held under low pressure, to decrease the amount of gas, particularly oxygen, prior to irradiation, either with or without a prior step of reducing the moisture content.

In another prefeπed embodiment, where the atmosphere inside the container contains oxygen, the amount thereof these may be reduced by any of the methods and techniques known and available to those skilled in the art, such as the controlled reduction of pressure within the container (rigid or flexible) holding the material to be iπadiated or by introducing an inert gas, such as a noble gas (e.g. helium or argon).

In certain embodiments of the present invention, a stabilizer is applied to the material according to any of the methods and techniques known and available to one skilled in the art, including soaking the material in a solution containing the stabilizer, preferably under pressure, at elevated temperature and/or in the presence of a penetration enhancer, such as dimethylsulfoxide. Other methods of introducing the stabilizer(s) include, but are not limited to, applying a gas containing the stabilizer(s), preferably under pressure and/or at elevated temperature, placing the material under reduced pressure and then introducing a gas or solution containing the stabilizer(s), and combinations of two or more of these methods. One or more sensitizers may also be introduced into a tissue according to such methods.

It will be appreciated that the combination of one or more of the features described herein may be employed to further minimize undesirable effects upon the material caused by iπadiation, while maintaining adequate effectiveness of the iπadiation process on the biological contaminants) or pathogen(s). For example, in addition to the use of a stabilizer, a particular material may also be held at a reduced temperature and kept under vacuum prior to irradiation to further minimize undesirable effects.

According to a particularly prefeπed embodiment of the present invention, the material to be iπadiated is examined with a suitable detection technique, such as the use of X-rays or "sniffers". Such screening can, for example, ensure that no radiodense substance is present in or on the material that might reduce the effectiveness of the methods of the present invention. Any such materials should be destroyed or, in the alternative, the conditions of iπadiation may be varied so that the methods of the present invention are effective at reducing the level of at least active biological contaminant or pathogen in or on the material.

Similarly, such screening can be employed to determine the level of active biological contaminants or pathogens. According to such an embodiment, the conditions of irradiation may be varied so that the methods of the present invention are effective at reducing the level of at least active biological contaminant or pathogen in or on the material. Screening can also be used to identify suspect materials, i.e. materials which may have in or on them an active biological contaminant or pathogen, through characteristics such as excess postage, foreign postmarks, suspicious packaging or misspellings and incoπect or unknown return addresses. According to the methods of the present invention, the materials that can be iπadiated to reduce the level of at least one active biological contaminant or pathogen include, but are not limited to, letters and/or packages delivered through conventional mail or delivery services and cuπency. Preferably, such letters or packages or cuπency are contained within a material, such as an envelope or the like, that is resistant to the effects of radiation, particularly gamma radiation. Such materials are known to those skilled in the art.

Experiment 1:

Purpose: The puφose of this experiment was to determine the effects of gamma iπadiation on various paper samples such as stationery, envelopes, packages, and binding tape that might be sent through the postal system. Background: Various paper samples (paper, envelopes, packages, adhesive tapes, etc.) were gathered together and placed in a container for irradiation.

The various samples received modifications with laser jet printing, irikjet printing, ballpoint printing, tape, packaging tape, wettable stamps, and adhesive stamps as appropriate. All adhesives were applied or sealed using pressure or water to wet the adhesive.

Six identical groups were prepared as follows: Control group (no irradiation) Iπadiation with ambient temperature and ambient atmosphere

Iπadiation on dry ice and inert argon atmosphere Control group with dithiothreitol (DTT) (no irradiation) Iπadiation with ambient temperature and ambient atmosphere with DTT Iπadiation on dry ice and inert argon atmosphere with DTT

Samples gamma iπadiated at ambient temperature and atmosphere were iπadiated at dose rates of 6.42 - 6.68 kGy/hr to total doses of 46.3 kGy - 50.0 kGy. Samples gamma iπadiated on dry ice were irradiated at dose rates of 1.43-1.54 kGy/hr to total doses of 46.3-50.0 kGy.

Evaluation Procedures:

After irradiation, the containers were opened. The containers containing DTT were evaluated for odor by sniffing the container and pieces of paper for odor permeance.

The corresponding paper samples from each of the 6 groups were pooled and compared in a blinded manner by three people. Any changes in appearance such as color change of the paper, inks, and adhesives were noted. Adhesive function was evaluated manually. Any gross differences in the physical properties of the papers such as brittleness or changes in texture were also noted. Observations are set forth in Tables 1 and 2. The papers were stored in a cardbox with little exposure to light for one year. The papers were then re-examined by three people (the 3 who viewed them at time zero) in a blinded manner to determine if there were any changes upon long-term storage.

Observations:

The observations made by 2 observers for each paper sample are presented in Table 1.

In addition, selected samples were observed by a third observer. This third observer noted subtle changes in hue in the red ink of those samples irradiated under ambient conditions. Observations are set forth in tables 3-5. Table 1. Observations Made

L = Laserjet printing I = Inkjet printing B = Bic ballpoint ink printing

T = Scotch Magic tape P = Packaging tape W = Wettable stamp

A = Adhesive Stamp Results and Discussion:

2§

+ = Slight color change ++ = Moderate color change

+ = Slight color change ++ = Moderate color change

Table 5. Observations Made 1 Year After Irradiation (Samples that initially did not chan e that showed chan e after 1 ear

+ = Slight color change ++ = Moderate color change

Of those paper samples that showed changes that were visually apparent, two were subjected to further analysis. The analysis of the paper color was conducted by scanning the samples in transmission mode and analyzing the colour of the resulting image using Adobe Photoshop. The colour analysis consisted of determining the relative contribution of the following colours: red, green, blue. The values are given a number from 0 (darkest) to 255 (lightest) by the program. Four points on the paper were analyzed, and the mean + the standard deviation are presented below in Tables 6 and 7.

Table 7. Red, Green, Blue Color Analysis of Epson Matte Paper (Heavyweight) After 1 Year

Summary

Overall, the following observations were made immediately after irradiation:

1. Gamma irradiation caused some changes in paper color. There were no noted changes in the physical strength of the papers.

2. There was some slight yellowing of the packaging tape only on mailers from the US Post Office. Color change was not noted on any other paper sample. This could be due to the papers used by the Post office to produce the envelopes or the colored inks applied to them. Gamma irradiation did not affect the adhesive properties of the transparent Scotch tape or the packaging tape.

3. Gamma irradiation did not affect the appearance of any of the inks printed on the paper. This was true for the laser jet ink, ballpoint pen ink, and all of the colors from the ink jet ink. One observer was given a select group of samples. This person noticed a lightening of the red ink in the samples irradiated at ambient temperature.

4. Gamma irradiation did not affect the coloring or adhesive properties of the stamps whether they were wettable or self-adhesive.

After storing for 1 year, the following observations were made: 1. For the papers that changed color after 1 year, irradiation on dry ice produced markedly less change than irradiation at ambient temperature. 2. There was further yellowing of the packaging tape on mailers from the US Post

Office and on the Mail Away DVD mailer. Color change was not noted on any other paper sample. 3. There were no visible changes in the appearance of any of the inks printed on the paper. This was true for the laser jet ink, ballpoint pen ink, and all of the colors from the ink jet ink. One observer, however, noticed some lightening of the red inkjet ink. 4. Gamma irradiation had no noted changes in the physical strength of the papers. 5. There were no visible changes to the coloring or adhesive properties of the stamps whether they were wettable or self-adhesive.

Experiment 2:

Purpose: The purpose of this experiment was to determine the effects of gamma irradiation on various packaging & paper samples such as stationery, envelopes, and packaging tape that might be sent through the postal system. Methods: Various samples (paper, envelopes, labels, adhesive tapes, etc.) were gathered together and placed in a container for irradiation. The material samples are described in Table 8 below: Table 8. Material Sam les Used

Some samples received modifications with laser jet printing to determine the affects of irradiation on the ink. All adhesives applied to the samples were applied using hand pressure.

Where noted, the air inside the containers was replaced with dry Argon gas. As noted, DTT (Dithiothretol, 50 mg) was placed inside open eppendorf microtubes within each container and allowed to sublime prior to irradiation.

For each material sample, twelve identical groups were prepared as follows:

The 100 kGy samples first received a dose of 50 kGy at a dose rate of 4.45 kGy/hr. All groups were then gamma irradiated at ambient or dry ice temperatures at a rate of approx. 2.2 kGy/hr to a total of 50 kGy. Evaluation Procedures:

The corresponding paper samples from each of the 6 groups were pooled and compared in a blinded manner by three people. Any changes in appearance such as color change of the paper, inks, or adhesives were noted. Adhesive function was evaluated manually. Any gross differences in the physical properties of the papers such as brittleness or changes in texture were also noted.

The papers were then scanned on a flatbed scanner in transmission mode. The images were analyzed for color composition (red, green, and blue) using commercial imaging software (Adobe Photoshop, Version 5.0). For samples #1-4, one point on the paper and each of the color dots were analyzed. For samples #5-7, one point from the paper and one point from the tape were analyzed. No inks were put on these papers.

Results: Observations:

The smell of sulfur similar to rotten eggs was detected in the vials containing DTT, indicating that DTT had permeated the vial.

The observations made by 3 observers for each paper sample are presented in Tables 10-16. In addition, selected samples were observed by a fourth observer.

Table 10. Observations Made Immediately After Irradiation for Material Sample

#1

Table 11. Observations Made Immediately After Irradiation for Material Sample

#2

Table 12. Observations Made Immediately After Irradiation for Material Sample

#3

Table 13. Observations Made Immediately After Irradiation for Material Sample

#4

Table 14. Observations Made Immediately After Irradiation for Material Sample

#5

Table 15. Observations Made Immediately After Irradiation for Material Sample

#6

Table 16. Observations Made Immediately After Irradiation for Material Sample

#7

Affect of Irradiation on the Color of the Paper Samples

After visual inspection, the papers were scanned using an Epson Expression 1600 using the reflective mode. The images were analyzed for color composition (red, green, and blue) Adobe Photoshop. Representative samples of these data are presented below.

Visually, the samples that showed color changes were those irradiated at ambient temperature, irregardless of other modifications. Representative color analyses are presented below in Table 17.

Table 17. Color Analysis Results: Paper

Affect of Irradiation on the Color of the Paper Samples

Visually, the samples that showed visible color changes were those irradiated at ambient temperature. Representative color analyses are presented below in Tables 18-21.

Table 18. Color Analysis Results: Red Ink

In comparison to the unirradiated control, the papers above showed a reduction in the Red value when irradiated at ambient temperature. Consequently, the Blue and Green values are higher, making the red color appear less intense. Irradiation at dry ice temperature did not have this effect and was comparable to the control. Table 19. Color Analysis Results: Blue Ink

In comparison to the unirradiated control, the papers above showed an increase in the Red and Green values when irradiated at ambient temperature, making the blue color appear less intense. Irradiation at dry ice temperature did not have this effect and was comparable to the control. When extended to 100 kGy, this effect is not as pronounced with the paper irradiated on dry ice as it is with the paper irradiated at ambient conditions.

Table 20. Color Analysis Results: Green Ink

In comparison to the unirradiated control, the papers above showed an increase in the Red and Blue values when irradiated at ambient temperature, making the green color appear less intense. Irradiation at dry ice temperature did not have this effect and was comparable to the control. When extended to 100 kGy, this effect is not as pronounced with the paper irradiated on dry ice as it is with the paper irradiated at ambient conditions.

Table 21. Color Analysis Results: Yellow Ink

In comparison to the unirradiated control, the papers above showed an increase in the Red and Green values when irradiated at ambient temperature, making the yellow color appear less intense. Irradiation at dry ice temperature did not have this effect and was comparable to the control.

Affect of Irradiation on the Color of the Packaging Tape

Visually, the samples that showed visible color changes were those irradiated at ambient temperature. Due to the reflectivity of the tape, color analysis was not possible.

Summary

Overall, the following observations were made immediately after irradiation: 1. Gamma irradiation caused some changes in paper color. There were no noted changes in the physical strength of the papers.

2. There was some slight yellowing of the packaging tape on mailers from the US Post Office. Color change was not noted on any other paper sample. This could be

! due to the papers used by the Post office to produce the envelopes or the colored inks applied to them. Gamma irradiation did not affect the adhesive properties of the packaging tape.

3. Gamma irradiation did affect the appearance of the ink jet inks printed on the paper. This was more profound for the samples irradiated at ambient temperature.

Having now fully described this invention, it will be understood to those of ordinary skill in the art that the methods of the present invention can be carried out with a wide and equivalent range of conditions, formulations, and other parameters without departing from the scope of the invention or any embodiments thereof.

All patents and publications cited herein are hereby fully incorporated by reference in their entirety. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that such publication is prior art or that the present invention is not entitled to antedate such publication by virtue of prior invention.

The foregoing embodiments and advantages are merely exemplary and are not to be construed as limiting the present invention. The present teachings can be readily applied to other types of apparatuses. The description of the present invention is intended to be illustrative, and not to limit the scope of the claims. Many alternatives, modifications, and variations will be apparent to those skilled in the art.

Claims

WHAT IS CLAIMED IS

1. A method for combating bio-terrorism involving the presence of active biological contaminants or pathogens in or on a material, said method comprising irradiating said material with radiation for a time and at a rate effective to reduce the level of active biological contaminants or pathogens in or on said material.

2. A method for combating bio-terrorism involving the presence of active biological contaminants or pathogens in or on a material, said method comprising:

(i) performing at least one stabilizing process on said material, said stabilizing process selected from the group consisting of:

(a) applying to said material at least one stabilizer in an amount effective to protect said material from said radiation; (b) placing said material in a container and reducing the moisture content of the atmosphere inside said container to a level effective to protect said material from said radiation;

(c) reducing the temperature of said material to a level effective to protect said material from said radiation; (d) placing said material in a container and reducing the oxygen and/or nitrogen content of the atmosphere inside said container to a level effective to protect said material from said radiation;

(e) applying to said material at least one solvent in an amount effective to protect said material from said radiation; (f) reducing the moisture content of the material to a level effective to protect said material from said radiation; and

(ii) irradiating said material with a suitable radiation for a time and at a rate effective to reduce the level of active biological contaminants or pathogens in or on said material.

3. A method for combating bio-terrorism involving the presence of active biological contaminants or pathogens in or on a material, said method comprising:

(i) performing at least two stabilizing process on said material, said stabilizing processes selected from the group consisting of:

(a) applying to said material at least one stabilizer;

(b) placing said material in a container and reducing the moisture content of the atmosphere inside said container;

(c) reducing the temperature of said material; (d) placing said material in a container and reducing the oxygen and/or nitrogen content of the atmosphere inside said container;

(e) applying to said material at least one solvent; and

(f) reducing the moisture content of the material; and

(ii) irradiating said material with a suitable radiation for a time and at a rate effective to reduce the level of active biological contaminants or pathogens in or on said material, wherein said at least two stabilizing processes are together effective to protect said material from said radiation and further wherein said at least two stabilizing processes may be performed in any order.

4. The method according to claim 1, 2 or 3, wherein said effective rate is not more than about 3.0 kGy/hour.

5. The method according to claim 1, 2 or 3, wherein said effective rate is not more than about 2.0 kGy/hr.

6. The method according to claim 1, 2 or 3, wherein said effective rate is not more than about 1.0 kGy/hr.

7. The method according to claim 1, 2 or 3, wherein said effective rate is not more than about 0.3 kGy/hr.

8. The method according to claim 1, 2 or 3, wherein said effective rate is more than about 3.0 kGy/hour.

9. The method according to claim 1, 2 or 3, wherein said effective rate is at least about 6.0 kGy/hour.

10. The method according to claim 1, 2 or 3, wherein said effective rate is at least about 18.0 kGy/hour.

11. The method according to claim 1, 2 or 3, wherein said effective rate is at least about 30.0 kGy/hour.

12. The method according to claim 1, 2 or 3, wherein said effective rate is at least about 45 kGy/hour.

13. The method according to claim 1, 2 or 3, wherein said material is maintained in a low oxygen atmosphere.

14. The method according to claim 1, 2 or 3, wherein said material is maintained in an atmosphere comprising at least one noble gas or nitrogen.

15. The method according to claim 14, wherein said noble gas is argon.

16. The method according to claim 1, 2 or 3, wherein said material is maintained in a vacuum.

17. The method according to claim 2 or 3, wherein said moisture content is reduced by a method selected from the group consisting of lyophilization, drying and evaporation.

18. The method according to claim 2 or 3, wherein said moisture content is less than about 15%.

19. The method according to claim 2 or 3, wherein said moisture content is less than about 10%.

20. The method according to claim 2 or 3, wherein said moisture content is less than about 3%.

21. The method according to claim 2 or 3, wherein said moisture content is less than about 2%.

22. The method according to claim 2 or 3, wherein said moisture content is less than about 1%.

23. The method according to claim 2 or 3, wherein said moisture content is less than about 0.5%.

24.> The method according to claim 2 or 3, wherein said moisture content is less than about 0.08%.

25. The method according to claim 1, 2 or 3, wherein at least one sensitizer is applied to said material prior to said irradiation.

26. The method according to claim 1, 2 or 3, wherein said material contain at least one biological contaminant or pathogen selected from the group consisting of viruses, bacteria, yeasts, molds, fungi, parasites and prions or similar agents responsible, alone or in combination, for TSEs.

27. The method according to claim 2 or 3, wherein said at least one stabilizer is an antioxidant.

28. The method according to claim 2 or 3, wherein said at least one stabilizer is a free radical scavenger.

29. The method according to claim 2 or 3, wherein said at least one stabilizer is a combination stabilizer.

30. The method according to claim 2 or 3, wherein said at least one stabilizer reduces damage due to reactive oxygen species.

31. The method according to claim 2 or 3, wherein said at least one stabilizer is a gas.

32. The method according to claim 31, wherein said at least one stabilizer is selected from the group consisting of ethanol, acetone, beta-mercaptethanol, dithiothreitol, dimethylsulfoxide and mixtures thereof.

33. The method according to claim 1, 2 or 3, wherein said radiation is corpuscular radiation, electromagnetic radiation, or a mixture thereof.

34. The method according to claim 33, wherein said electromagnetic radiation is selected from the group consisting of radio waves, microwaves, visible and invisible light, ultraviolet light, x-ray radiation, gamma radiation and combinations thereof.

35. The method according to claim 1, 2 or 3, wherein said radiation is gamma radiation.

36. The method according to claim 1, 2 or 3, wherein said radiation is E-beam radiation.

37. The method according to claim 1, 2 or 3, wherein said radiation is visible light.

38. The method according to claim 1, 2 or 3, wherein said radiation is ultraviolet light.

39. The method according to claim 1, 2 or 3, wherein said radiation is x-ray radiation.

40. The method according to claim 1, 2 or 3, wherein said radiation is polychromatic visible light.

41. The method according to claim 1, 2 or 3, wherein said radiation is infrared.

42. The method according to claim 1, 2 or 3, wherein said radiation is a combination of one or more wavelengths of visible and ultraviolet light.

43. The method according to claim 1, 2 or 3, wherein said irradiation is conducted at ambient temperature.

44. The method according to claim 1, 2 or 3, wherein said irradiation is conducted at a temperature below ambient temperature.

45. The method according to claim 1, 2 or 3, wherein said irradiation is conducted at a temperature above ambient temperature.

46. The method according to claim 2 or 3, wherein said moisture content is about 0%.

47. The method according to claim 2 or 3, wherein said moisture content is about 10%.

48. The method according to claim 2 or 3, wherein said moisture content is about 20%.

49. The method according to claim 2 or 3, wherein said moisture content is about 33%.

50. The method according to claim 2 or 3, wherein said moisture content is less than about 33%.

51. The method according to claim 2 or 3, wherein said moisture content is reduced by the addition of at least one hygroscopic agent to said container.

52. The method according to claim 2 or 3, wherein said moisture content is reduced by the addition of at least one dry gas to said atmosphere.

53. The method according to claim 2 or 3, wherein said oxygen content is reduced by the application of a vacuum to said atmosphere.

54. The method according to claim 2 or 3, wherein said oxygen and/or nitrogen content is reduced by the addition of at least one noble gas to said atmosphere.

55. The method according to claim 2 or 3, wherein said oxygen and/or nitrogen content is reduced by the application of a vacuum followed by the addition of at least one noble gas to said atmosphere.

56. The method according to claim 1, 2 or 3, wherein the total dose of said radiation is at least about 2 times the D₁₀ dose for at least one predetermined biological contaminant or pathogen that may be present in or on the material.

57. The method according to claim 1, 2 or 3, wherein the total dose of said radiation is at least about 3 times the Dio dose for at least one predetermined biological contaminant or pathogen that may be present in or on the material.

58. The method according to claim 1 , 2 or 3, wherein the total dose of said radiation is at least about 4 times the D₁₀ dose for at least one predetermined biological contaminant or pathogen that may be present in or on the material.

59. The method according to claim 1 , 2 or 3, wherein the total dose of said radiation is at least about 5 times the D₁₀ dose for at least one predetermined biological contaminant or pathogen that may be present in or on the material.

60. The method according to claim 1 , 2 or 3, wherein the total dose of said radiation is at least about 6 times the D₁₀ dose for at least one predetermined biological contaminant or pathogen that may be present in or on the material.

61. The method according to claim 1 , 2 or 3 , wherein the total dose of said radiation is at least about 7 times the D₁₀ dose for at least one predetermined biological contaminant or pathogen that may be present in or on the material.

62. The method according to claim 1, 2 or 3, wherein the total dose of said radiation is at least about 8 times the Dio dose for at least one predetermined biological contaminant or pathogen that may be present in or on the material.

63. The method according to claim 1, 2 or 3, wherein the total dose of said radiation is at least about 9 times the D₁₀ dose for at least one predetermined biological contaminant or pathogen that may be present in or on the material.

64. The method according to claim 1 , 2 or 3, wherein the total dose of said radiation is greater than about 9 times the Dm dose for at least one predetermined biological contaminant or pathogen that may be present in or on the material.

65. The method according to claim 1, 2 or 3, wherein the total dose of said radiation is at least about 2 kGy.

66. The method according to claim 1 , 2 or 3, wherein the total dose of said radiation is at least about 5 kGy.

67. The method according to claim 1 , 2 or 3, wherein the total dose of said radiation is at least about 10 kGy.

68. The method according to claim 1, 2 or 3, wherein the total dose of said radiation is at least about 20 kGy.

69. The method according to claim 1 , 2 or 3, wherein the total dose of said radiation is at least about 25 kGy.

70. The method according to claim 1 , 2 or 3, wherein the total dose of said radiation is at least about 45 kGy.

71. The method according to claim 1, 2 or 3, wherein the total dose of said radiation is at least about 75 kGy.

72. The method according to claim 1, 2 or 3, wherein the total dose of said radiation is at least about 100 kGy.

73. The method according to claim 1, 2 or 3, wherein the total dose of said radiation is at least about 150 kGy.

74. The method according to claim 1, 2 or 3, wherein the total dose of said radiation is at least about 200 kGy.

75. The method according to claim 1, 2 or 3, wherein the total dose of said radiation is greater than about 200 kGy.

76. The method according to claim 2 or 3, wherein said solvent increases the permeability of said material to radiation.

77. The method according to claim 76, wherein said solvent is a non-aqueous solvent.

78. The method according to claim 76, wherein said solvent is an aqueous solvent.

79. The method according to claim 1, 2 or 3, further comprising the step of screening said material prior to said irradiating to determine the presence of at least one active biological contaminant or pathogen.

80. The method according to claim 1, 2 or 3, further comprising the step of screening said material prior to said irradiating to determine the possible presence of a radiodense substance.

81. The method according to claim 1, 2 or 3, wherein said effective rate is at least about 1,000,000 kGy/hour.

82. The method according to claim 2 or 3, wherein said moisture content is less than about 80%.

83. The method according to claim 2 or 3, wherein said moisture content is less than about 60%.

84. The method according to claim 2 or 3, wherein said moisture content is less than about 40%.

85. The method according to claim 1, 2 or 3, further comprising the step of screening said material prior to said irradiating to determine the optimal conditions for irradiation.

86. The method according to claim 2 or 3, wherein said container is stable under the influence of irradiation, minimizes the interactions between the radiation and the material and/or isolates the material from the external environment.

87. The method according to claim 1, 2 or 3, wherein said material is a letter or package.

88. The method according to claim 1, 2 or 3, wherein said material is currency.