Compositions containing Purine Nucleosides and Manganese and their Uses

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application 60/935,494 (filed on August 16, 2007) which is incorporated by reference in its entirety.

GOVERNMENT SUPPORT

The present invention arose m part from research funded by grant DE-FG02- 04ER63918 from the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Remediation Research (BER), Environmental Remediation Sciences Program and by grant FA9559-07-1-0128 from the Air Force Office of Scientific Research. The Government has certain rights m the invention.

BACKGROUND OF THE INVENTION

The extremely radiation-resistant family Deinococcaceae is compπsed of greater than twenty distinct species that can survive acute exposures to ionizing radiation (IR) (10 IcGy), ultraviolet light (UV) (1 kJ/m ² ), and desiccation (years); and can grow under chronic IR (60 Gy/hour). In particular, Deinococcus radiodurans is an extremely ionizing radiation (IR) resistant bacterium that can survive exposures to gamma-radiation that exceed by a factor of one thousand the doses which are cytotoxic and lethal to mammalian cells

For extremely resistant bacteria, such as e.g., D. radiodurans, survival following high- doses of IR has been attributed to protection of proteins from oxidation during irradiation, with the result that enzymic repair systems survive and function with far greater efficiency duπng recovery than in sensitive bacteria, where cellular proteins are highly susceptible to carbonylation. In a report published m Science magazine (Daly et al. (2004), Accumulation of Mn(II) in Deinococcus radiodurans facilitates gamma-radiation resistance, Science 306: 925- 1084), intracellular manganese(II) was implicated in facilitating radiation resistance by protecting proteins, but not DNA, duπng exposure to ionizing radiation; and m a second report published m PLoS Biology (Daly et al. (2007) Protein oxidation implicated as the primary determinant of bacterial radioresistance, PLoS Biology 5(4) e92), radiation resistance was positively correlated to protein protection duπng irradiation, mediated by a non-enzymic mechanism. Unlike D. radiodurans, most proteins are not radiation-resistant. Similarly, most cells, whether in eukaryotes, prokaryotes or mammals (e g humans) are also not radiation resistant. As such, exposure to radiation is quite damaging to protein structure and/or function. For

example, ionizing radiation has been shown to induce (cause) cancer m man}* different species of animals and in almost all parts of the human body.

In humans, significant overexposure to radiation can result m radiation poisoning, also called "radiation sickness" or a "creeping dose". The term is generally used to refer to acute problems caused by a large dosage of radiation in a short peπod, though this also has occurred with long term exposure to low level radiation. The clinical name for "radiation sickness" is acute radiation syndrome as descπbed by the CDC. A chrome radiation syndrome does exist but is very uncommon: this has been observed among workers m early radium source production sites and in the early days of the Soviet nuclear program. A short exposure can result in acute radiation syndrome; chrome radiation syndrome requires a prolonged high level of exposure.

Humans routinely encounter radiation m daily life, including radiation from electronic equipment and cell phones as well as natural background radiation. Individuals that are m close proximity of radioactive elements such as e g. employees at a nuclear plant or members of the armed forces are particularly likely to encounter higher doses of radiation. Additionally, radiation is used m diagnostic tests such as X-rays and radiation therapy to treat cancers.

There are currently very few radioprotectors suitable for treating humans, and those which exist (e g , amifostme) are cytotoxic and have seπous side effects (e g., loss of consciousness, fast or irregular breathing, itching, nausea and vomiting).

Given the great exposure to radiation, there is a significant need for radioprotectors that are non-toxic, preserve protein function, and in particular are suitable for human use.

SUMMARY OF THE INVENTION

This invention provides for methods of radioprotection using compositions containing one or more puπne nucleosides and one or more antioxidant. These methods are suitable to protect proteins in vitro and in vivo from the damaging effects of radiation. One embodiment of the invention is a method of treating and/or preventing a side effect of radiation exposure in a subject in need thereof comprising administration of a pharmaceutically effective amount of a composition comprising one or more puπne nucleosides and one or more antioxidants to a subject in need thereof. The method is suitable for treating and/or preventing a side effect of exposure to many kinds of radiation. In one embodiment, radiation is selected from the group consisting of UV light, alpha radiation, beta radiation, gamma radiation, X-ray radiation and neutron radiation. The puπne nucleoside may be adenosine, uridine, β-pseudouπdme, mosme, and mixtures thereof. The antioxidant may be manganese, MnCl ₂ , manganous phosphate and Vitamin E and'or mixtures thereof In one

embodiment of the invention, the antioxidant is manganous phosphate. In another embodiment, the antioxidant is MnCl ₂ with phosphate added separately. In an alternate embodiment, the composition further composes an ammo acid, preferably any one of alanine, valine, and'or leucine. The composition may be a D radiodurans extract. In another embodiment, the composition prevents one or more side effects of radiotherapy. The composition may contain about 1 mM to about 15 mM of adenosine and'or undine. The composition may also contain about 1 mM to about 12.5 mM of manganese.

Another embodiment of the invention is a method for preserving a function of a protein comprising contacting a protein with a composition compnsmg one or more puπne nucleosides and one or more antioxidants. The one or more puπne nucleoside may be adenosine, uridine, β-pseudouπdme, mosine, and mixtures thereof. In one embodiment, purine nucleoside is adenosine and/or uridine. In another embodiment, the composition contains about 1 to about 15 mM of adenosine and/or uridine. The antioxidant may be manganese, MnCl ₂ , manganous phosphate and vitamin E. In one embodiment of the invention, the antioxidant is manganous phosphate. In another embodiment, the antioxidant is MnCl ₂ with phosphate added separately. In one embodiment, the composition contains about 0.01 mM to about 12.5 mM of the antioxidant (such as e g , manganese). In another embodiment, the composition contains adenosine, uridine, leucine, adenine, and manganese. In yet another embodiment, composition contains about 1 to about 15 mM adenosine and about 1 mM to about 12.5 mM MnCl ₂ . The composition may also contain an ammo acid such as e g , leucine, valine, and alanine. The method preserves the function of a protein (such as e g , an enzyme) during desiccation or when the protein is exposed to radiation (such as e g , UV light, alpha radiation, beta radiation, gamma radiation, X-ray radiation and neutron radiation).

Another embodiment of the invention is a method for stonng a protein compnsmg contacting a protein with a composition comprising one or more punne nucleosides and one or more antioxidants.

Yet another embodiment of the invention is a method of treating and/ or preventing a side effect of radiation exposure m a subject m need thereof compnsmg administration of a pharmaceutically effective amount of a D radiodurans extract compnsmg one or more punne nucleosides and one or more antioxidants to a subject in need thereof. The method is suitable for treating radiation exposure from a variety of sources. In one embodiment, the radiation is UV light, alpha radiation, beta radiation, gamma radiation, X-ray radiation and neutron radiation. The one or more punne nucleoside may be adenosine, uridine, β-pseudouπdme, mosine. and mixtures thereof. The one or more oxidants may be manganese, MnCl ₂ , manganous

phosphate and Vitamin E. In one embodiment of the invention, the antioxidant is manganous phosphate. In another embodiment, the antioxidant is MnCl ₂ with phosphate added separately. The D. radiodurans extract may also contain an ammo acid, preferably alanine, valine, leucine, or mixtures thereof. In one embodiment, the method prevents one or more side effects of radiotherapy. The D. radiodurans extract may contain about 1 mM to about 15 mM of adenosine and/or undine. The extract may also contain about 1 mM to about 12.5 mM of manganese.

The D radiodurans extract may be produced by harvesting a D. radiodurans culture by centnfugation, lying the D. radiodurans culture to create a D radiodurans lysate, washing the D. radiodurans lysate, centnfuging the D. radiodurans lysate for a time and under conditions sufficient to create a supernatant, passing the supernatant through a less than 3 kiloDalton filter; and boiling the supernatant for about 15 to about 45 minutes. The extract is soluble in butane, resistant to boiling and cell-free.

BRIEF DESCRIPTION OF THE DRAWINGS The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended figures. For the purpose of illustrating the invention, shown in the figures are embodiments of the present invention. It should be understood, however, that the invention is not limited to the precise arrangements, examples, and instrumentalities shown. Figure 1 shows that compounds in D. radiodurans ultrafiltrates protect proteins but compounds ultrafiltrates from m Pseudonomas putida (PP), Escherichia coli (EC), and Thermus therrnophilus (TT) do not. Protem-free, ultra-filtrated D. radiodurans (DR) cell extract prevents ionizing radiation (IR)-mduced protein oxidation in vitro, but extracts from the radiation sensitive bactena Pseudotnonas putida (PP), Escherichia coli (EC), and Thermus thermophilics (TT) did not. Punfied E. coli proteins were incubated in PP-, EC-, TT-, or DR- ultra-filtrated extract dunng irradiation, and subjected to a protein carbonyl assay. Coomassie-stamed polyacrylarmde denatunng gel; Carbonyl Western blot, revealing protein oxidation and protection (no signal).

Figure 2 shows that Adenosine and Uridine Prevent Gamma-Radiation-Induced Protein Breaks and Carbonylation (Oxidation). Adenosine and undine prevent ionizing radiation (IR)- mduced protein oxidation in vitro. Punfied E. coli proteins were incubated with the indicated agents. Coomassie-stamed polyacrylarmde denatunng gel; Carbonyl Western blot, revealing protein oxidation (black) and protection (no signal). For example, when E. coli proteins were

irradiated m potassium phosphate buffer (PPB 25 mM) + 15 mM adenosine (Ad), the proteins were highly protected from oxidation duπng irradiation compared to potassium phosphate buffer alone

Figure 3 shows the radioprotective properties for a composition containing Adenosine and Manganese, i e a radioprotective composition based on D radiodurans Post-irradiation functionality of restriction enzyme BamHl after indicated treatment and incubation with 1-DNA For example, when BamHl is irradiated m 25 mM potassium phosphate buffer (PPB) + 3 mM adenosine (Ad) +1 mM MnCl?, the enzyme remains functional after exposure to 17,500 Gy, but not when incubated in potassium phosphate buffer alone, adenosine alone or MnCl ₂ alone. Figure 4 shows radioprotection by D radiodurans protem-free cell extracts of human T- cells compared to well-established radioprotective compounds. D radiodurans extract (4 x concentrated, 100 μl/ml), Amifostme (200 μg/ml), or 5 -AED (10 mg/ml) were added to human Jurkat T cells 24 hr before exposure to 8 Gy (0.6 Gy/mm). Viability of cells 24 (Fig. 4A), 48 (Fig. 4B), and 72 hours (Fig. 4C) after irradiation was measured by trypan blue exclusion. *P<0.05 vs. Veh, DRE, and DRE+IR, determined by Student t- test. CON: non-irradiated, IR; gamma radiation; VEH: water as vehicle; DRE: D. radiodurans extract, AMF: Amifostme; 5- AED: 5-androstenediol.

Figure 5 shows in vivo desiceation-mduced oxidative protein damage. The proteins were isolated from the indicated strains after 6 days of desiccation. 20-mg samples were subjected to polyacrylamie denature gel electrophoreses and stained with Coomassie Blue (Fig. 5A). A duplicate unstained gel was subjected to assay for carbonyl groups. Abbreviations: O = oxidized protein standards; S = protem-size standards. Deinococcus species are m bold: Rl (type-stram); IAl; 1A6, 3Bl; 5A4 (surface isolates) and 7b- 1. Non-Demococcus species: 4A4 Methylobacteπum; 4A6, Chelatococcous. So, S oneidensis Figure 5C shows the corresponding values for intracellular Mn/Fe contractions, 10% IR survival (DlO) and survival following 1, 2, and 3 weeks (w) of desiccation are tabulated. Figure 5 D shows in vitro protection of restriction enzyme from drymg-mduced deactivation by D radiodurans protem-free cell extract

DETAILED DESCRIPTION

General Description The inventors have studied the radio-resistance of D radiodurans and prepared ultra- purified, protein free-cell extracts that exhibit radioprotective properties. Thus, the invention is based in part on the discovery of radioprotective components of D radiodurans cell free extract and artificial compositions containing such components

In particular, applicants have shown that D radiodurans ultra-purified and protem-free cell extracts are extremely radioprotective of proteins exposed to gamma-radiation Adenosine and uridine are accumulated m D radiodurans, but these nucleosides were undetectable m radiation sensitive bacteria In vitro, at doses >10,000 Gy, nucleosides were shown to be highly protective of proteins, preventing ionizing radiation (IR)-mduced protein carbonylation and preserve the function enzymes m the presence of Mn(II) A radioprotective composition of adenosine, manganese and phosphate was been developed Surprisingly, D radiodurans extracts have been shown to be potent radioprotectors for cultured human T-cells with greater potency than other well-established radioprotective compounds The present invention provides for radioprotective compositions either synthetic or deπved from D radiodurans and methods of uses of these compositions to protect proteins and/or cells from radiation damage These compositions are useful to prevent radiation damages m compositions as well as in subjects such as humans In particular, this invention provides for radioprotective compositions that contain one or more puπne nucleoside and one or more antioxidant The radioprotective compositions may furthei contain leucine, alanine, and/or valine Leucine is strongly implicated m scavenging hydrogen peroxide m the presence of Mn(II), and may be components of larger intracellular complexes that include uridine and adenosine Strong in vitro evidence indicates a synergistic effect between adenosine and manganese The stoichiometry of adenosine and manganese may be optimized for an apoptosis assay

Applicants have shown that adenosine alone and Mn(II) alone are radioprotective in vivo for a mammalian cell line

Although not being bound by any particular theory, it is believed that compositions comprising puπne nucleosides (e g adenosine) and an antioxidant (e g manganese) act as radioprotectants by shielding a proteins' active site The puπne nucleoside e g adenosine (and optionally with uπdme) mediates its radioprotective effects upon accumulation withm a cell, which inhibits radiation-mduced protein oxidation, and m the presence of Mn(II) preserves enzyme function Adenosine is thought to protect proteins, and therefore scavenge a subset of ROS Furthermore, without being bound by any particular theory, it is believed that under aerobic or anaerobic irradiation conditions, superoxide can build up m cells duπng irradiation because superoxide does not readily cross membranes Although superoxide does not react with DNA, superoxide will damage and inactivate enzymes with exposed 2Fe-2S or 4Fe-4S clusters, releasing Fe(II) The problem with iron m a cell, when it is unbound and "free", is that it causes

Fenton reactions m the presence of hydrogen peroxide, generating hydroxyl radicals. Therefore, conditions which liberate bound Fe(II) are extremely dangerous, not only because of the generation of hydroxyl radicals, but because the loss of Fe from Fe-dependent enzymes leads to the failure of the biochemical pathways withm which they operate. The methods of the instant application optimally protect against these dangerous conditions.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and mateπals similar or equivalent to those described herein can be used m the practice or testing of the present invention, the preferred methods, and mateπals are descπbed.

As used herein, "a" or "an" means at least one, unless clearly indicated otherwise. The term "about," unless otherwise indicated, refers to a value that is no more than 10% above or below the value being modified by the term. For example, the term "about 5% (w/w)" means a range of from 4.5% (w/w) to 5.5% (w/w).

Methods of preserving protein function

This invention provides for methods of preserving protein function by contacting a protein with a composition comprising one or more purine nucleosides and one or more antioxidants. One embodiment of the invention is a method preserving protein function when the protein is exposed to the extreme conditions of radiation such as e.g. gamma radiation. In another embodiment of the invention, the method preserves protein function duπng desiccation.

The methods of preserving protein function provide radioprotection when the protein is exposed to high dose of radiation such as doses m excess of 10 kGy, e g., 17.5 kGy.

In another embodiment, the invention provides for methods of protecting protein function m a cell culture comprising one or more puπne nucleosides and one or more antioxidants. The cell culture may be prokaryotic or eukaryotic. In one embodiment, the cell culture is mammalian.

Any purine nucleoside may be used m the composition. Suitable puπne nucleosides include, but are not limited to, adenosine, uπdme, β-pseudouπdme, mosme, and mixtures thereof. Preferably the puπne nucleoside is adenosine or uπdme. In one embodiment, the composition contains adenosine. In other embodiment of the invention, the composition contains uπdme. The amount of puπne nucleoside in the composition vanes on its use. Those of skill m the art will be able to determine the suitable amount. In some embodiments of the invention, the amount of puπne nucleoside ranges from about 0.01 mM to about 15 mM, from

about 0.1 rnM to about 1 mM, from about 1 mM to about 10 mM, from about 1 mM about 15 mM. In one embodiment, the concentration of one or more puπne nucleosides composes about 1 mM to about 15 mM of adenosine and/or uridine.

A variety of antioxidants maybe used m the composition. Suitable antioxidants include manganese, vitamin E and manganous phosphate. In one embodiment of the invention, the antioxidant is manganese. In another embodiment, the antioxidant is MnCl ₂ . In yet another embodiment, the antioxidant is vitamin E. The amount of antioxidant m the composition vanes on its use. Those of skill in the art will be able to determine the suitable amount. In one embodiment, the composition contains about 0.01 mM to about 15 mM of the antioxidant. In another embodiment, the composition contains about 0.01 mM to about 12.5 mM.

In one embodiment of the invention, a cπtical antioxidant is manganous phosphate which may be provided at near-millimolar concentrations. In another embodiment, the antioxidant is MnCl ₂ , with phosphate added separately. The amount of antioxidant in the composition vanes on its use. Those of skill m the art will be able to determine the suitable amount. In one embodiment, the composition contains about 0.01 mM to about 15 mM of the manganous (Mn(II)) ions and 1 mM to about 25 mM phosphate buffer.

The compositions may further contain one or more ammo acids that exhibit cytoprotective properties. In one embodiment of the invention, composition further contains at least one or more ammo acid selected from the group consisting of leucine, valine, and alanine. In another embodiment, the ammo acid is leucine. In an alternate embodiment, the ammo acid is glycine.

In one embodiment, the composition comprises adenosine, uridine, leucine, adenine, and manganese. In an alternate embodiment, the composition comprises about 1 to about 15 mM adenosine and about 1 to about 12.5 mM MnCl ₂ . In another embodiment, the composition compnses a D. radiodurans extract containing one or more punne nucleosides and one or more antioxidants.

Any protein function may be preserved by use of the methods of this invention. In a preferred embodiment of the invention, the protein is an enzyme. The methods of the instant disclosure are particularly useful m preventing protein oxidation associated with ultraviolet radiation and aging. Furthermore, the methods also preserve protein functionality dunng desiccation and thus help increase the shelf life of desiccated blood products and enzyme-based drugs, which are stored dry.

The methods of the invention optimally preserve protein function (such as e g , enzymatic activity) dunng exposure to radiation. One embodiment of the invention is a method

of preservation compπsing contacting a protem (such as e g , an enzyme) with a composition compπsmg one or more purine nucleosides and one or more antioxidants.

Another embodiment of the invention is a method of increasing the durability and longevity of microbial and enzyme-driven fuel cells compπsmg contacting the components of the fuel cell with a composition compπsmg one or more puπne nucleosides and one or more antioxidants.

This method may be suitable to preserve the function of many proteins including but not limited to proteins with Fe-S complexes (such as metabolic enzymes) and enzymatic repair functions that are dependent on redox-active (4Fe-4S) clusters. Exemplary proteins include protein groups associated with the production of reactive oxygen species (ROS), transport protein precursors which might reduce biosynthetic demands and suppress the production of ROS, proteins that defend against ROS, proteins that participate in repair of damaged molecules (non-DNA) and redox regulation as well as Mn and Fe-dependent systems. Other exemplary proteins are listed in Ghosal et al. (2005), FEMS Microbiology Reviews 29: 361-375, the disclosure of which is herein incorporated in its entirety.

Methods of treating or preventing the effects of radiation exposure

The invention also provides for methods of treating or preventing the effects of radiation exposure. The methods comprise treating or preventing the effects of radiation exposure with a therapeutic agent comprising one or more puπne nucleosides and one or more antioxidants. In one embodiment of the invention, the radiation exposure is due to UV exposure. In another embodiment of the invention, the radiation exposure is due to ionizing radiation. In another embodiment of the invention, the radiation exposure is chronic.

As used herein, the term "therapeutic agent" shall encompass compositions comprising one or more puπne nucleosides and one or more antioxidants as well as formulations containing other pharmaceutically acceptable components such as e.g pharmaceutically acceptable earners. As used herein, the term "radiation exposure" shall mean exposure to any radiation in a dose and for a period sufficient to cause damage. Radiation exposure includes but it is not limited to exposure to UV light, alpha radiation, beta radiation, gamma radiation, X-ray radiation and neutron radiation. In one embodiment, the invention provides for methods of treating or preventing the side effects of radiotherapy. As used herein, the term "radiotherapy" shall refer to the use of certain types of energy (such as e g , ionizing radiation) to kill cancer cells and shπnk tumors. The term "radiotherapy" includes all types of radiotherapy including but not limited to external

radiation therapy (such as e g , intraoperative radiotherapy and prophylactic cranial irradiation (PC)), internal radiation therapy (such as e g , interstitial radiation therapy, intracavitary or intraluminal radiation therapy), systemic radiation therapy, stereotactic (or stereotaxic) radiosurgery, three-dimensional (3-D) conformal radiation therapy, intensity-modulated radiation therapy (IMRT) Furthermore, the term "radiotherapy" also encompasses radiotherapy using a vaπety of sources of radiation including but not limited to X-rays, gamma rays, particle beams, proton beam therapy, and high-energy photon radiation Radiotherapy is used to treat a vaπety of cancers including solid tumors (such as e g , cancers of the bram, breast, cervix, larynx, lung, pancreas, prostate, skin, spme, stomach, uterus, or soft tissue sarcomas) Radiotherapy is also used to treat leukemia and lymphoma (z e , cancers of the blood-formmg cells and lymphatic system, respectively) as well as cancers of the skm, cervix, and thyroids

As used herein, the term "side effects of radiotherapy" shall be refer to any side effect experienced by a subject undergoing radiotherapy Such side effects include but are not limited to tiredness and skm reactions, anemia, increased πsk of bruising or bleeding, decreased fertility, dry mouth, loss of appetite and weight, hair loss etc

A "subject in need of treatment" is an animal with a bacteπal infection that is potentially life-threatening or that impairs health or shortens the lifespan of the animal The animal can be a fish, bird, or mammal Exemplary mammals include humans, domesticated animals (e g , cows, horses, sheep, pigs, dogs, and cats), and exhibition animals, e g , in a zoo In a preferred embodiment, the subject is human

The terms "treating", "treatment," and "therapy" as used herein refer to curative therapy, prophylactic therapy, and preventative therapy

As used herein, unless stated otherwise, the term composition is meant to encompass, and not limited to, pharmaceutical compositions and nutraceutical compositions containing one or more puπne nucleosides and one or more antioxidants The composition may also contain one or more "excipients" that are "inactive ingredients" or "compounds" devoid of pharmacological activity or other direct effect m the diagnosis, cure, mitigation, treatment, or prevention of disease or to affect the structure or any function of the human body

A "pharmaceutically acceptable" component is one that is suitable for use with humans, animals, and/or plants without undue ad\erse side effects (such as e g , toxicity, irritation, and allergic response) commensurate with a reasonable benefit/risk ratio

The therapeutic agent may contain any puπne nucleoside Suitable puπne nucleosides include but are not limited to adenosine, undine, β-pseudouπdme, mosme, and mixtures thereof Preferably the purine nucleoside is adenosine or uridine In one embodiment, the therapeutic

agent contains adenosine. In other embodiment of the invention, the therapeutic agent contains uridine.

The therapeutic agent may contain a variety of suitable antioxidants. Suitable antioxidants include but are not limited to manganese, vitamin E, and manganous phosphate. In one embodiment of the invention, the antioxidant is manganese. In another embodiment, the antioxidant is MnCl ₂ . In yet another embodiment, the antioxidant is vitamin E.

In one embodiment of the invention, a cπtical antioxidant is manganous phosphate, which may be provided at near-millimolar concentrations. In another embodiment, the antioxidant is MnCl ₂ , with phosphate added separately. The amount of antioxidant m the composition vanes on its use. Those of skill in the art will be able to determine the suitable amount. In one embodiment, the composition contains about 0.01 mM to about 15 mM of the manganous (Mn(II)) ions and 1 mM to about 25 mM phosphate buffer.

The amount of puπne nucleoside and antioxidant m the therapeutic agent vanes. Those of skill m the art will be able to determine the suitable amount depending on a vanety of factor such as the subject, the duration of the radiation exposure, the amount of the radiation exposure etc. In some embodiments of the invention, the amount of punne nucleoside ranges from about 0.01 mM to about 15 mM, from about 0.1 mM to about 1 mM, from about 1 mM to about 10 mM, from about 1 mM about 15 mM. In one embodiment, the concentration of one or more purine nucleosides comprises about 1 mM to about 15 mM of adenosine and/or undme. In another embodiment, the amount of antioxidant ranges from about 0.01 mM to about 15 mM. In another embodiment, the therapeutic agent contains about 0.01 mM to about 12.5 mM. The therapeutic agent may further contain one or more ammo acids that exhibit cytoprotective properties. In one embodiment of the invention, therapeutic agent further contains at least one or more ammo acid selected from the group consisting of leucine, valine, and alanine. In another embodiment, the ammo acid is leucine. In another embodiment, the ammo acid is glycine.

In one embodiment, the therapeutic agent compnses adenosine, undme, leucine, adenine, and manganese. In an alternate embodiment, the therapeutic agent compnses about 1 mM to about 15 mM adenosine and about 1 mM to about 12.5 mM MnCl ₂ . In another embodiment, the therapeutic agent comprises a D radiodurans extract containing one or more punne nucleosides and one or more antioxidants.

In yet another embodiment of the invention, the therapeutic agent is a composition suitable for human use compπsing one or more punne nucleoside (such as e g , adenosine, uridine, β-pseudoundme, mosme, and mixtures thereof), one or more antioxidant (such as e g ,

manganese, vitamin E) and optionally one or more ammo acid selected from the group consisting of leucine, valine, and alanine. In one embodiment, the composition suitable for human use compπses adenosine and manganese.

In an alternate embodiment of the invention, the therapeutic agent is a D radiodurans extract containing one or more puπne nucleosides and one or more antioxidants.

The methods for treating or preventing the effects of radiation exposure compπse administration of a therapeutic agent comprising one or more puπne nucleosides and one or more antioxidants to a subject m need thereof.

One embodiment is a method of preventing a side effect of radiotherapy, comprising administration of a -D radiodurans extract comprising one or more puπne nucleosides and one or more antioxidants to a subject in need thereof.

Another embodiment of the invention is a method of preventing a side effect of radiotherapy comprising administration of a composition compπsmg one or more puπne nucleosides, an antioxidant and optionally an ammo acid selected from the group consisting of alanine, valine and leucine to a subject in need thereof. Preferably the one or more puπne nucleoside is adenosine and/or uridine, which may be present in amounts from about 1 mM to about 15 mM of adenosine and/or uridine. The one or more puπne nucleosides may also selected from the group consisting of adenosine, uridine, β-pseudouπdme, inosine, and mixtures thereof. The antioxidant may be manganese (e g of about 1 mM to about 12.5 mM). In one embodiment, the antioxidant is MnCl ₂ . In another embodiment, the antioxidant is vitamin E. In another embodiment, the composition compπses adenosine, undine, leucine, adenine, and manganese.

The methods of the instant application are particularly advantageous. Compared to well- established radioprotectors (such e g amifostme), compositions compπsmg one or more puπne nucleosides and one or more antioxidants (e.g., adenosine, uridine and Mn) are relatively nontoxic.

The methods of the invention are particularly suitable for pre- and post-exposure treatments of military personnel and civilians accidentally or deliberately exposed to ionizing radiation. The methods may also used prophylactically for individuals exposed to significant chrome levels of radiation such as in nuclear power plants, dunng long-duration space flight, or on the international space station.

A "safe and effective amount" refers to a quantity of a component that is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity,

irritation, or allergic response) commensurate with a reasonable benefit risk ratio when used m the manner of this invention. By "therapeutically effective amount" is meant an amount of a component effective to yield a desired therapeutic response, e g , an amount effective to slow the rate of bacterial cell division, or to cause cessation of bacteπal cell division, or to cause death or decrease rate of population growth of the bacteria. The specific safe and effective amount or therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the subject, the type of subject being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its deπvatives. Means of application include, but are not limited to direct, indirect, earner and special means or any combination of means. Direct application of the phage may be by nasal sprays, nasal drops, nasal ointments, nasal washes, nasal injections, nasal packings, bronchial sprays and inhalers, or indirectly through use of throat lozenges, or through use of mouthwashes or gargles, or through the use of ointments applied to the nasal nares, the bridge of the nose, or the face or any combination of these and similar methods of application. The forms m which the phage may be administered include but are not limited to lozenges, troches, candies, mjectants, chewing gums, tablets, powders, sprays, liquids, ointments, and aerosols.

The therapeutic agent may also be placed in a nasal spray, wherein the nasal spray is the carrier. The nasal spray can be a long acting or timed release spray, and can be manufactured by means well known m the art. An inhalant may also be used, so that the therapeutic agent may reach further down into the bronchial tract, including into the lungs.

The therapeutic agent may be added to these substances in a liquid form or in a lyophihzed state, whereupon it will be solubihzed when it meets body fluids such as saliva. The enzyme may also be in a micelle or liposome. While these methods may be used m any mammalian species such as farm animals including, but not limited to, horses, sheep, pigs, chicken, and cows, the preferred use of compositions is for a human.

The effective dosage rates or amounts of the compositions will depend m part on whether the composition will be used therapeutically or prophylactically, the duration of exposure of the recipient to radiation, the type of radiation, the size, and weight of the individual, etc. The duration for use of the composition also depends on whether the use is for prophylactic purposes, wherein the use may be hourly, daily or weekly, for a short time peπod, or whether the use will be for therapeutic purposes wherein a more intensive regimen of the use of the composition may be needed, such that usage may last for hours, days or weeks, and/or on

a daily basis, or at timed intervals during the day. Any dosage form employed should provide for a minimum number of units for a minimum amount of time. The concentration of the active units of phage believed to for an effective amount or dosage of phage may be m the range of about 100 units/ml to about 100,000 units 'ml of fluid m the wet or damp environment of the nasal and oral passages, and possibly m the range of about 100 units/ml to about 10,000 umts/ml. More specifically, time exposure to the radiation may influence the desired concentration of active radioprotective composition units per ml. It should be noted that earners that are classified as "long" or "slow" release earners (such as, for example, certain nasal sprays or lozenges) could possess or provide a lower concentration of the composition per ml, but over a longer peπod of time, whereas a "short" or "fast" release earner (such as, for example, a gargle) could possess or provide a high concentration of composition per ml, but over a shorter period of time. It will furthermore be appreciated that a therapeutically effective amount of a particular composition can be determined by those of ordinary skill m the art with due consideration of the factors pertinent to the subject. Selection of the preferred effective dose can be determined (e.g., via clinical trials) by a skilled artisan based upon the consideration of several factors which will be known to one of ordinary skill m the art. Such factors include the disease to be treated or prevented, the symptoms involved, the patient's body mass, the patient's immune status and other factors known by the skilled artisan to reflect the accuracy of administered pharmaceutical compositions.

The precise dose to be employed in the formulation will also depend on the route of administration and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves denved from in vitro or animal model test systems. For the prophylactic and therapeutic treatment and/or prevention of the effects of radiation exposure, the compositions comprising purine nucleosides and antioxidants may also be applied by direct, indirect, earners and special means or any combination of means. Direct application of the phage may be by nasal sprays, nasal drops, nasal ointments, nasal washes, nasal injections, nasal packings, bronchial sprays and inhalers, or indirectly through use of throat lozenges, or through use of mouthwashes or gargles, or through the use of ointments applied to the nasal nares. the bndge of the nose, or the face or any combination of these and similar methods of application. The forms m which the phage may be administered include but are not limited to lozenges, troches, candies, mjectants, chewing gums, tablets, powders, sprays, liquids, ointments, and aerosols. For the therapeutic treatment of anthrax, the bronchial sprays and

aerosols are most beneficial, as these earners, or means of distributing the composition, allow the phage to reach the bronchial tubes and the lungs.

The compositions of the present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. For example, an agent may be administered locally to a site of injury via micromfusion. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired In one embodiment of the invention, the method comprises administration of the therapeutic agent in a pharmaceutically acceptable earner. Suitable earners and their formulations are descnbed in Remington's Pharmaceutical Sciences, 2005, Mack Publishing Co. Typically, an appropnate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically acceptable earner include liquids such as salme, Ringer's solution, and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5 The formulation may also compnse a lyophihzed powder. Further carriers include sustained release preparations such as semi-permeable matnces of solid hydrophobic polymers, which matrices are in the form of shaped articles, e g , films, liposomes or microparticles It will be apparent to those persons skilled in the art that certain earners may be more preferable depending upon, for instance, the route of administration and concentration of proinflammatory cytokine inhibitor being administered.

The methods optimally provide therapeutics against numerous redox-related forms of cell injury mediated by protein damage, and facilitate wound healing.

Methods of preparing D. radiodurans extracts One embodiment of the invention is a method of preparing D radiodurans cell-free extracts that exhibit radio-protective properties. In one embodiment, the methods has the steps of harvesting a D radiodurans by e g , centnfugation, lysmg the D radiodurans culture to create a lysate, washing the D radiodurans lysate followed by centnfugmg the lysate for a time and under conditions sufficient to create a supernatant. After centnfugation, the supernatant is passed through a microfϊlter, preferably a 3 kiloDalton Microfilter, and boiled for a penod for a suitable amount of time In one embodiment, the supernatant is boiled for about 15 to about 45 minutes after filtration. The resulting D radiodurans extract contains one or more punne nucleosides and one or more antioxidants, is soluble in butanol, resistant to boiling, and cell-

free.

In one embodiment, the extract contains adenosine and manganese. In another embodiment, the extract contains adenosine and/or uridine manganese. The cell extracts may also further contain leucine, alanine, and/or valine. In one embodiment, the D. radiodurans extract contains at least adenosine, undine, leucine, adenine, and manganese.

Without further descπption, it is believed that one of ordinary skill m the art can, using the preceding descπption and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

Examples

Example 1

Preparation of protein-free extract from D. radiodurans. D. radiodurans (ATTC BAA- 816) was grown to OD600 0.9 in TGY, harvested by centrifugation, and lysed by French pressure treatment. The cells were washed and then lysed in double-distilled, de-iomzed stenle water (dH ₂ O). Pπor to lysis, cell density was adjusted with dH ₂ O to yield lysates representing approximately 50% intracellular concentration. Crude cell extracts were centnfuged for 20 hours at 175,000 x g. The supernatant was passed through a <3 kiloDalton Microcon centrifugal filter (Millrpore, USA) and boiled for 30 mm. The Coomassie (Bradford) protein assay was used to confirm the virtual absence of proteins in the ultra-punfied extracts, which were ahquoted and stored at -80° C.

Example 2

Analysis of protein-free extract from D. radiodurans. The D. radiodurans extracts were analyzed using TOF MS and chromatography. Using these techniques, the extracts were found to contain a variety of compounds including but not limited to leucine, adenine, uridine, and adenosine. Previous analysis showed that these extracts also contain manganese. The ammo acids leucine, alanine, and valine are very elevated m D. radiodurans and D. geothermalis compared to radiation sensitive bacteria (data not shown).

Example 3 Radioprotective effects in mammalian cells. Apoptosis of Human Fetal Osteoblastic

1.19 cell line from ATCC (hFOB) was determined by death and apoptotic markers (Annexm V and propidium iodide PI) using a flow cytometry assay. In brief, hFOB cells were cultured m

DMEM-F 12 with 10% fetal bovine serum, 2.5 mM L-glutamme and 1% antibiotic to full confluency followed by treatment for expeπmental groups with adenosine (10 mM) or MnCl2 (0.25 mM) or adenosine and MnCl ₂ for 6 days peπod starting 3 h before IR. Apoptotic cell death was measured by flow cytometry with Annexin V and PI staining 6 days after IR. hFOB cells also were plated (lxl03/well, 6-well plate) for clonogenic survival assay after IR. Colonies were counted 10 days later. The results of the assay are shown below in Table 1.

For the assays, adenosine +'- 0.25 mM Mn(II) was added 3 hours pπor to irradiation. Cells were assayed 6 days after irradiation by flow cytometry. Annexm-V conjugates allow for the identification of cell surface changes that occur early duπng the apoptotic process using flow cytometry. Early in the apoptotic process, phosphatidylseπne becomes exposed on the cell surface. Propidium Iodide (PI) in un-fixed cells discriminates between apoptosis and necrosis on the basis of dye exclusion. While necrotic cells lose membrane integrity early on m their death throws, apoptotic cells may have compromised membranes.

While the invention has been described and illustrated herein by references to vaπous specific materials, procedures and examples, it is understood that the invention is not restπcted to the particular combinations of mateπal and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art. It is intended that the specification and examples be considered as exemplary, only, with the true scope and spiπt of the invention being indicated by the following claims. All references, patents, and patent applications referred to m this application are herein incorporated by reference m their entirety.