EP1937316A2

EP1937316A2 - Radioprotective agents

Info

Publication number: EP1937316A2
Application number: EP06788493A
Authority: EP
Inventors: P. Fink Mitchell; Joel S. Greenberger; Michael W. Epperly
Original assignee: University of Pittsburgh
Current assignee: University of Pittsburgh
Priority date: 2005-07-26
Filing date: 2006-07-26
Publication date: 2008-07-02
Also published as: WO2007014237A2; WO2007014237A3; AU2006272596A1; CA2616380A1

Abstract

Methods and compositions are provided for treating or protecting a subject against the toxicities associated with ionizing radiation. The methods include administering to the subject a radioprotective amount of a radioprotective agent. The radioprotective agent includes an alpha-ketoalkanoic acid or a derivative thereof. For example, an ester of an alpha-ketoalkanoic acid, such as ethyl pyruvate, is used. The radioprotective agent may also comprise a superoxide dismutase, for example, EUK-134.

Description

RADIOPROTECTIVE AGENTS INVENTORS

1. Field of the invention

Methods and compositions are provided for protecting a subject against ionizing radiation using radioprotective agents.

2. Background

Exposure to ionizing irradiation may cause an increase in reactive oxygen species as well as an increase in the inflammatory cytokine response in the cells and tissues of an irradiated subject. Although methods maybe used to limit the number of cells directly exposed, it has been shown that non-irradiated cells may also suffer from radiation-induced effects through the "bystander effect." (Shao C. et al, Proc. Natl. Acad. Sci. USA 101:13495, 2004). Several mechanisms have been proposed to explain the bystander effect. One entails cell-cell communication via gap junctions. Another proposes that irradiated cells secrete cytokines, such as TGF-β or IL-8, or other factors, most notably NO, O₂*^", or H₂O₂, that act to promote oxidant stress in unirradiated cells. Shao C, Cancer Res. 63:8437, 2003. A third proposes that ionizing radiation recruits inflammatory cells (e.g., neutrophils and macrophages) that are capable of injuring tissues by releasing reactive oxygen species, cytokines or tissue-destructive enzymes. Lorimore S.A. et al, Oncogene 7058, 2005. Thus, inflammation may be a potentially important mechanism underlying damage to irradiated cells, as well as cells affected by the bystander effect.

Currently, Amifostine is the only radioprotective agent approved by the U.S. Food and Drug Administration. However, Amifostine has an unfavorable side-effect profile (e.g., administration may cause hypotension) and provides only minimal protection from cellular damage caused by ionizing radiation. Therefore, there is a need in the art for better, less toxic radioprotective agents.

Recognition that pyruvate is an effective reactive oxygen species scavenger prompted investigators to use this compound as a therapeutic agent for the treatment of various pathological conditions that are thought to be mediated, at least in part, by redox-dependent phenomena. One of the earliest efforts in this regard was carried out by Salahudeen et al, who showed that infusing a solution of sodium pyruvate (NaPyr) preserves kidney function in rat models of reactive oxygen species -mediated acute renal failure. Salahudeen AK et al, J.Clin.Invest. 88:1886, 1991. Other investigators reported that treatment with NaPyr ameliorates organ injury or dysfunction in a variety of other animal models of redox stress, such as myocardial (See Bunger R. et al, Eur.J. Biochem. 180:221, 1989), intestinal (See Cicalese L. et al, AmJ. Surg. 171:97, 1999), or hepatic ischemia-reperfusion-induced injury (See Sileri P et al, Transplantation 72:27, 2001). NaPyr-containing solutions also have been shown to have salutary effects in animal models of galactose- or diabetes-induced cataract formation (See Gupta SK, et al, Ophthalmic Res. 34:23, 2002; Zhao W et al, Diabetes Obes. Metab. 2:165, 2000), stroke (See Lee J.Y. et al, J. Neurosci. 21:1, 2001) and hemorrhagic shock (HS) (See Slovin P.N. et al, Resuscitation 50:109, 2001) and in vitro models of damage to the lens of the eye caused by exposure to galactose (See Vaπna S.D. et al, Free Rad. Res. 30:253, 1999), fructose (See Zhao W et al, Free Rad.Res. 33:23, 2000) or oxidants (See Varma S.D. et al, Free Rad. Res. 28:131, 1998).

Despite these promising findings, the usefulness of pyruvate as a therapeutic agent is limited by its poor stability in solution. Aqueous solutions of NaPyr rapidly undergo an aldol-like condensation reaction to form parapyruvate (2-hydroxy-2-methyl-4-ketoglutarate), a potentially toxic inhibitor of a critical step in the mitochondrial Tricarboxylic acid cycle. Aqueous solutions of NaPyr also spontaneously undergo hydration to form pyruvate hydrate (2,2-dihydroxyproprionate). Neither parapyruvate nor pyruvate hydrate are capable of scavenging reactive oxygen species.

Ethyl pyruvate, the ester formed from pyruvic acid and ethanol, has been shown to be more effective than NaPyr as a cytoprotective or anti-inflammatory agent. Sappington P. L. et al, Shock 20:521, 2003; Sappington PX. et al, J. Pharmacol. Exp. Ther. 304:464, 2003. Given their close chemical similarity, it is remarkable that different pharmacological effects have been observed when ethyl pyruvate and pyruvate have been subjected to comparisons in models of inflammation or redox stress. For example, studies in the regulation of insulin secretion by pancreatic islet cells provide support that the pharmacological actions of pyruvate esters are quite distinct from those of pyruvate anion. It has been speculated that esterification renders pyruvate more membrane-permeable and thereby allows higher levels of the compound to accumulate in mitochondria. Zawalich WS et al, J. Biol. Chem. 272:3527, 1997. In many of the models of acute critical illness, treatment with ethyl pyruvate down- regulates the expression of various pro-inflammatory genes, including iNOS, TNF, cyclooxygenase-2, and interleukin IL-6. Similarly, ethyl pyruvate inhibits IL-6 and iNOS expression, NO production, and/or secretion of the pro-inflammatory protein, HGMBl, by immunostimulated Caco-2 human enterocyte-like cells or lipopolysaccharide-stimulated RAW 264.7 murine macrophage-like cells. Therefore, ethyl pyruvate has been shown to be an effective anti-inflammatory agent in a variety of in vitro and in vivo model systems.

Ethyl pyruvate also has been shown to inhibit activation of NF-κB (a family of transcription factors formed by the hetero- or homodimerization of proteins) in a variety of in vitro and in vivo systems. Varma SD, et al, Free Rad. Res. 28:131, 1998; Tawadrous Z.S. et al, Shock 17:473, 2002; Tsung A et al, Transplantation 27:196, 2005. NF-κB has been shown to regulate the transcription of approximately 200 genes {e.g., TNF, IL-6, IL-8, cyclooxygenase (COX)-2, and inducible nitric oxide synthase), many of which are involved in inflammatory response. Others, such as cellular inhibitors of apoptosis (cIAP-1 and cIAP- 2) and several members of the Bcl-2 gene family {e.g., Al/Bfl-1, BCI-X_L and Nrl3) are involved in regulating programmed cell death. Sonis S.T., Crit. Rev. Oral Biol. Med. 13:380, 2002.

Activation of NF-κB has also been implicated as being a factor modulating cell death secondary to ionizing radiation. The dose of ionizing radiation that is required to activate NF-κB-dependent signaling varies greatly, depending upon the cell type or system under study. Moreover, NF-κB appears to be pro-apoptotic in some situations and anti-apoptotic in others. Sonis S.T, Crit. Rev. Oral Biol. Med. 13:380, 2002. Since both inflammation and apoptosis appear to be important components of the response to ionizing radiation, NF -KB is generally regarded as an important target for radioprotection (and radiosensitization) strategies.

SUMMARY

In some embodiments, a method of treating or protecting a subject against toxicities associated with ionizing radiation is provided comprising administering to the subject a radioprotective amount of a radioprotective agent comprising an alpha-ketoalkanoic acid or a derivative thereof. In a further embodiment, the radioprotective agent is an ester of an alpha- ketoalkanoic acid. In yet a further embodiment, the ester of an alpha-ketoalkanoic acid is ethyl pyruvate. In other embodiments, a method of treating or protecting a subject against the toxicities associated with ionizing radiation is provided comprising administering to the subject a radioprotective amount of a radioprotective agent comprising a superoxide dismutase. In certain embodiments, the superoxide dismutase is EUK- 134.

In further embodiments, a radioprotective agent is provided for treating or protecting a subject against toxicities associated with ionizing radiation comprising an alpha- ketoalkanoic acid or a derivative thereof.

In still further embodiments, a radioprotective agent is provided for treating or protecting a subject against the toxicities associated with ionizing radiation comprising a superoxide dismutase.

Other and further features and advantages of the present invention will be readily apparent to those skilled in the art upon a reading of the description of preferred embodiments which follows. For a better understanding of the present invention, together with other and further objects, reference is made to the following description, taken in conjunction with the accompanying drawings, and its scope will be pointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the survival curve of 32D cl 3 cells incubated with 100 μM EUK-134 or Tempol for 1 hour and then irradiated with doses from 0 to 800 cGy;

Figure 2 shows the survival curve of 32D cl 3 cells incubated in 20 mM ethyl pyruvate: 1 hour before irradiation, 1 hour before irradiation and also with ethyl pyruvate added to the methylcellulose following irradiation, and irradiated followed by the addition of ethyl pyruvate;

Figure 3 shows the percent of apoptotic cells twenty-four hours after irradiation in 32D cl 3 cells incubated in the presence of 20 mM ethyl pyruvate for 1 hour before irradiation to 10 Gy;

Figure 4 shows the survival curve of 32D cl 3 cells incubated for 1 hour with control medium, 5 mM ethyl pyruvate or 100 μM tempol prior to irradiation to doses ranging from 0 to 800 cGy;

Figure 5 shows the survival fraction of mice treated with ethyl pyruvate before and after irradiation or just after irradiation;

Figure 6 is a graph showing that ethyl pyruvate inhibits transcriptional activity of β- Catenin/TCF4 in 293T cells; Figure 7 shows inhibition of cyclin Dl expression by ethyl pyruvate;

Figure 8 shows irradiation induction of cyclin Dl; and

Figure 9 shows inhibition of cyclin Dl expression following irradiation of MCF7 cells incubated in the presence of EP.

DETAILED DESCRIPTION

The present invention relates to methods and compositions for protecting against ionizing radiation using radioprotective agents.

The compositions and methods described herein have many applications, for example, prevention and treatment of damage to normal tissues in patients exposed to ionizing radiation in therapeutic applications (e.g., radiation therapy for cancer), prevention and treatment of morbidity or mortality in victims exposed to ionizing radiation from a terrorist event or during military combat, or prevention and treatment of morbidity or mortality in victims exposed to ionizing radiation due to an industrial accident. Therefore, the compounds and methods described herein may be used to provide protection against cell death induced by all types of ionizing radiation including, for example, x-rays and gamma rays.

In some embodiments of the methods described herein, a method is provided of treating or protecting a subject against ionizing radiation comprising administering to the subject a radioprotective amount of an agent comprising an alpha-ketoalkanoic acid or a derivative thereof.

In one aspect, the alpha-ketoalkanoic acid is an ester of an alpha-ketoalkanoic acid, for example, a C₃-C₈ straight-chained or branched alpha-ketoalkanoic acid. Examples include alkyl, aryl, arylalkyl, alkoxyalkyl, carbalkoxyalkyl or acetoxyalkyl esters. hi further embodiments, the ester of an alpha-ketoalkanoic acid may include ethyl alpha-keto-butyrate, ethyl alpha-keto-pentanoate, ethyl alpha-keto-3-methyl-butyrate, ethyl alpha-keto-4-methyl-pentanoate, and ethyl alpha-keto-hexanoate. Specific examples include ethyl, propyl, butyl, carbmethoxymethyl (-CH₂COOCH₃), carbethoxymethyl (-CH ₂COOCH₂CH₃), acetoxymethyl (-CH₂OC(O)CH₃), carbmethoxyethyl (-CH₂CH₂COOCH₃), carbethoxyethyl (-CH₂CH₂COOCH₂CH₃), methoxymethyl (-CH₂OCH₃) and ethoxymethyl (- CH₂OCH₂CH₃). Ethyl esters are preferred.

In other embodiments, the ester of an alpha-ketoalkanoic acid may include an alpha- ketoalkanoic acid of the formula R¹C(O)C(O)OR² wherein R¹ and R² independently comprise an alkyl, aryl, arylalkyl, alkoxyalkyl, alcohol, amine, amide, aromatic, aliphatic, heterocyclic, carbalkoxyalkyl, acetoxyalkyl or combinations thereof. Thus, in some embodiments, R₁ and R₂ may independently include one or more combinations of functional groups.

In still other embodiments, the ester of an alpha-ketoalkanoic acid may comprise a glyceryl ester. As used herein, glycerol esters include glycerol esters of fatty acids, e.g., esters of fatty acids and glycerol or polyglycerol and their derivatives.

In other embodiments, the ester of an alpha-ketoalkanoic acid may comprise dihydroxyacetone esters of the formula R¹OCH₂C(O)CH₂OR² wherein R¹ and R² independently comprise an alkyl, aryl, arylalkyl, alkoxyalkyl, alcohol, amine, amide, aromatic, aliphatic, heterocyclic, carbalkoxyalkyl, acetoxyalkyl or combinations thereof. Thus, in some embodiments, R₁ and R₂ may independently include one or more combinations of functional groups.

In yet other embodiments, the ester of an alpha-ketoalkanoic acid may comprise a thiolester.

Specific example of alpha-ketoalkanoic esters suitable for use in the disclosed methods include ethyl pyruvate, propyl pyruvate, carbmethoxymethyl pyruvate, acetoxymethyl pyruvate, carbethoxymethymethyl pyruvate, ethoxymethyl pyruvate, ethyl alpha-keto-butyrate, ethyl alpha-keto-pentanoate, ethyl alpha-keto-3-methyl-butyrate, ethyl alpha-keto-4-methyl-pentanoate, or ethyl alpha-keto-hexanoate. In preferred embodiments the ester of an alpha-ketoalkanoic acid is ethyl pyruvate.

The methods and compositions described herein may optionally employ or include an enolization agent. An "enolization agent" is a chemical agent, which induces and stabilizes the enol resonance form of an alpha-keto ester and may be present in an amount induce to enolization of the alpha-keto functionality, e.g., from 0.0 to 4.0 molar equivalents relative to the ester. Enolization agents include a cationic material, preferably a divalent cation such as calcium or magnesium or, for example, a cationic amino acid such ornithine or lysine.

In other embodiments according to the present invention, the radioprotective agent includes an amide of an alpha-ketoalkanoic acid.

In other embodiments, the radioprotective agent is an amide of an alpha-ketoalkanoic acid of the formula R¹C(O)C(O)N(R²)R³, wherein R¹, R² and R³ independently comprise an alkyl, aryl, arylalkyl, alkoxyalkyl, alcohol, amine, amide, aromatic, aliphatic, heterocyclic, carbalkoxyalkyl, acetoxyalkyl or combinations thereof. Thus, in some embodiments, R¹, R² and R³ may independently include one or more combinations of functional groups. In yet other embodiments, the radioprotective agent includes a superoxide dismutase (SOD), i.e., a metal-containing antioxidant enzyme. It has previously been demonstrated that over expression of manganese containing SOD (MnSOD) protects cells from irradiation by down regulation of reactive oxygen species, as well as decreases the inflammatory cytokine response. The MnSOD mimetic, EUK-134 is a synthetic manganese-porphyrin complex that acts as a scavenger of oxidative species. As described below, EUK-134 has been shown to protect cells from irradiation damage and is a preferred superoxide dismutase according to the present invention.

The precise dose to be employed in the formulation of a therapeutic agent will depend on the route of administration, and the seriousness of the conditions, and should be decided according to the judgment of a practitioner and each subject's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems and will include an amount sufficient for treating (therapeutically or prophylactically), preventing, ameliorating or slowing effects from exposure to ionizing radiation.

An appropriate composition comprising the radioprotective agent to be administered can be prepared in any physiologically or pharmaceutically acceptable vehicle or carrier. For example, acceptable carriers include, e.g., a buffer solution for infusion or bolus injection, a tablet for oral administration or in gel, micelle or liposome form for on-site delivery. A preferred buffer solution is water or isotonic or hypertonic saline buffered with bicarbonate, phosphate, lactate or citrate. Alternatively, the therapeutic agent may be administered in a plasma extender, microcolloid or microcrystalline solution. One useful carrier is Ringer's isotonic saline solution. The solution may comprise from about 105 mM to 110 mM NaCl, from about 3.8 mM to about 4.2 mM KCl, and from about 2.5 to 2.9 mM CaCl₂. More preferably, the carrier is Ringer's Lactate solution comprising from about 105 mM to 110 mM NaCl, from about 3.8 mM to about 4.2 mM KCl, and from about 2.5 to 2.9 mM CaCl₂.

Because ethyl pyruvate hydrolyzes in aqueous solutions it is preferable that ethyl pyruvate solutions are prepared immediately prior to infusion. In addition, it may be preferable to administer ethyl pyruvate intravenously through a central venous catheter to avoid thrombophlebitis.

To facilitate a better understanding of the present invention, the following examples of some of the preferred embodiments are given. In no way should such examples be read to limit, or define, the scope of the invention. Examples

32D cl 3 cells were incubated: 1) in the absence of any added compounds; 2) in the presence of EUK- 134 or ethyl pyruvate for 1 hour before irradiation; 3) in the presence of ethyl pyruvate in methyl cellulose following irradiation; or 4) in the presence of ethyl pyruvate for 1 hour before irradiation and also in the presence of ethyl pyruvate in methylcellulose following irradiation. For EUK-134 (Eukarion, Inc., Bedford, MA), 32D cl 3 cells were incubated for 1 hour in the presence of 100 μM concentration of EUK-134, or 100 μM tempol as a control, and plated in methylcellulose. In the case of ethyl pyruvate, cells were incubated for 1 hour in the presence of 20 mM ethyl pyruvate. In all groups, cells were irradiated to doses ranging from 0 to 800 cGy and incubated at 37⁰C for 7 days at which time colonies of >50 cells were counted and the data analyzed by linear quadratic or single-hit, multi-target models.

Cells incubated in EUK-134 had an increased shoulder on the survival curve with an n {i.e., the width of the shoulder region representing the quasi-threshold dose, or the point at which death becomes exponential) of 5.38 compared to an n of 1.43 for 32D cl 3 cells, as shown in Figure 1.

32D cl 3 cells incubated in the presence of ethyl pyruvate 1 hour before irradiation or plated in methylcellulose containing ethyl pyruvate had an increased D₀ (i.e., the dose required to reduce the surviving fraction to 37% in the exponential portion of the survival curve), compared to 32D cells alone (2.20 ± 0.25, 2.21 ± 0.15, and 1.84 ± 0.25 Gy, respectively) but no change in n (2.00 ± 1.01, 1.11 + 0.12, or 1.70 ± 0.37, respectively). The combination of incubating 32D cl 3 cells in ethyl pyruvate both before irradiation and in methylcellulose media after irradiation resulted in optimal radioprotection as shown by a significantly increased shoulder on the survival curve compared to untreated-irradiated 32D cl 3 cells (n = 4.14 ± 1.59 and 1.70 ± 0.6, respectively, p = 0.0485), as shown in Figure 2. Thus, ethyl pyruvate administered before and after irradiation resulted in increased radiation resistance and cells incubated in ethyl pyruvate either before or after irradiation also had an increased radiation resistance.

To determine whether ethyl pyruvate prevents irradiation-induced apoptosis, 32D cl 3 cells were incubated in the presence of 20 mM ethyl pyruvate 1 hour before irradiation to 10 Gy. The cells were then placed in tissue culture media and incubated for 24 hours at 37⁰C. The apoptotic cells were labeled with FITC-UTP using a Promega Dead End Fluorometric Tunel Assay. The cells were observed under a fluorescent microscope and the percent of apoptotic cells determined. As shown in Figure 3, ethyl pyruvate decreased the percent of 32D cl 3 cells undergoing irradiation apoptosis. Twenty-four hours after 10 Gy irradiation, cells incubated for 1 hour in ethyl pyruvate had 2.8 ± 0.7% (p<0.0001) apoptotic cells (1.2 ± 0.5% for non-irradiated cells) compared to 23.7 ± 1.8% for irradiated cells. Thus, incubation of cells in the presence of ethyl pyruvate before irradiation resulted in decreased percent of apoptotic cells.

32D cl 3 cells were also incubated for 1 hour with 5 mM ethyl pyruvate or 100 μM tempol, as a control medium. The cells were then irradiated to doses ranging from 0 to 800 cGy and plated in methylcellulose. After 7 days, colonies of greater than 50 cells were counted. The Do of cells incubated with ethyl pyruvate was 1.466 Gy compared to 0.797 Gy for control 32D cl 3 cells (Figure 4).

To determine whether ethyl pyruvate protects cells in vivo, C57BL/6NHsd female mice at 7-8 weeks of age were divided into five groups (10 mice per group) which received: 1) 9 Gy whole body irradiation only; 2) intravenous (IV) injections of MnSOD-PL (200 μg plasmid DNA) 24 hours before irradiation of 9 Gy whole body; 3) ethyl pyruvate (70 mg/kg) 30 minutes before 9 Gy whole body irradiation; 4) 9 Gy whole body irradiation followed 10 minutes later with an intraperitoneal injection of ethyl pyruvate (70 mg/kg) followed by additional injections of ethyl pyruvate at 24, 48, 72 or 96 hours; or 5) ethyl pyruvate 10 minutes before irradiation of 9 Gy and following irradiation as described for group 4 above. Mice treated with ethyl pyruvate had improved survival relative to controls as shown in Figure 5. Thus, treatment with ethyl pyruvate results in increased radiation resistance following whole body irradiation in mice. Similarly, administration of MnSOD-PL as an IV injection 24 hours before irradiation also resulted in increased radiation resistance.

Ethyl pyruvate (EP) has been shown to protect cells in vitro and in vivo from irradiation. Ethyl Pyruvate has also been shown to protect cells from the negative physiological consequences associated with ischemia injuries. Tsung A, Kaizu T, et al Transplantation 79 (2): 196-204, Fink, MP, Minerva Anestesiologica 70 (5):365-371, 2004, Uchiyama T, Delude RL, and Fink MP: Intensive Care Medicine 29 (11):2050-2058, 2003, and Sims CA, Wattanasirichaigoon S, et al. Critical Care Medicine. 29:(8):1513-1518, 2001. The mechanism of EP's protection is not clearly understood, but is thought to inhibit NF-Kβ in the ischemia models. One possible mechanism for radioprotection is by inhibition of the GADD45 (growth arrest and DNA damage-inducible protein 45) pathway. Irradiation stimulates GADD45 which results in a cell cycle arrest at G2/M phase allowing cells time to repair before proceeding through the cell cycle. This is thought to occur by the GADD45 protein binding to the transcription factor TCF4 protein, which binds to β-catenin, resulting in the stimulation of cyclin Dl. This mechanism prevents cells from arresting in Gl, allowing them to proceed through the cell cycle and undergo apoptosis. Cell cycle arrest at Gl is thought to be of increased significance because it occurs before DNA synthesis and is the stage of cell cycle arrest which is more growth factor dependent. Therefore blocking cyclin Dl production should result in a Gl phase arrest which would allow the cells time to repair the irradiation-induced damage.

Using a β-catenin/TCF4 luciferase assay in 293T cells, it is demonstrated that incubation of the cells in 20 mM EP inhibits β-catenin/TCF4 activity (Figure 6). Figure 6 shows that ethyl pyruvate inhibits transcriptional activity of β-Catenin/TCF4 in 293T cells as measured by luciferase activity. Incubation of 293T cells with EP blocked the luciferase activity from a β-Catenin/TCF4 luciferase reporter constrcut, demonstrating that EP inhibits the β-Catenin/TCF4 signal transduction pathway. This inhibition of luciferase activity demonstrates that EP is able to block GADD45 dependent activation of β-Catenin/TCF4.

Inhibition of GADD45 pathway is further demonstrated by expression of cyclin Dl. Expression of cyclin Dl which is the target gene for GADD45/β-catenin/TCF4 is inhibited by EP (Figure 7A). Cells from GADD45+/+ cells, GADD45-/- cells or HeLa cells were incubated in the presence of EP and cyclin Dl expression was determined by Western blot analysis. EP inhibited cyclin Dl expression in the GADD45 +/+ and HeLa cells (Figure 7A). The importance of the GADD45 protein is demonstrated in that GADD457- mice are more likely to exhibit radiation-induced carcinogenesis and have increased genomic instability (Sheikh, MS, MC Hollander and AJ Fornace, Biochemical Pharmacology, 59:43-45, 2000). Furthermore, no change in cyclin Dl expression was observed in GADD45-/- cells in the presence of EP. This suggests that the mechanism by which EP protects irradiated cells involves modulating the GADD45 pathway. Incubation of HeLa cells with ethyl lactate (EL, used as a negative control) had no effect on the expression of cyclin Dl (Figure 7B).

Irradiation has been shown to increase the expression of cyclin Dl in several cell types. Figure 8 shows irradiation induced increases in cyclin Dl at 24 hours after irradiation. Western blot analysis was used to demonstrate increased cyclin Dl expression in HCT 116 cells and MCF-7 cells at 24 hr following exposure to 20 Gy irradiation.

Figure 9 shows inhibition of cyclin Dl expression following irradiation of MCF7 cells incubated in the presence of EP. MCF-7 cells were incubated in the presence of 10 or 20 mM EL or EP and irradiated with 20 Gy (+ were irradiated (IR), - were not). Western blot analysis of cyclin Dl was performed 24 hr later. MCF-7 cells incubated in 20 mM EP demonstrated decreased expression of cyclin Dl following irradiation, while cells incubated in the presence of ethyl lactate demonstrated no inhibition of cyclin Dl expression.

These results demonstrate that EP 's ability to protect irradiated cells involves the GADD45 pathway. By blocking this pathway the cells are able to arrest in the Gl phase of the cell cycle where cellular repair may happen before proceeding through the cell cycle, thus avoiding apoptotic cell death.

While the invention has been depicted and described by reference to exemplary embodiments of the invention, such a reference does not imply a limitation on the invention, and no such limitation is to be inferred. The invention is capable of considerable modification, alteration, and equivalents in form and function, as will occur to those ordinarily skilled in the pertinent arts having the benefit of this disclosure. The depicted and described embodiments of the invention are exemplary only, and are not exhaustive of the scope of the invention. Consequently, the invention is intended to be limited only by the spirit and scope of the appended claims, giving full cognizance to equivalence in all respects. All references cited herein are hereby incorporated by reference in their entirety.

Claims

We claim:

1. A method of treating or protecting a subject against toxicities associated with ionizing radiation comprising administering to the subject a radioprotective amount of a radioprotective agent comprising an alpha-ketoalkanoic acid or a derivative thereof.

2. The method of claim 1 , wherein the radioprotective agent is an ester of an alpha- ketoalkanoic acid.

3. The method of claim 2, wherein the ester of an alpha-ketoalkanoic acid is a C₃-C₈ straight-chained or branched alpha-ketoalkanoic acid.

4. The method of claim 2, wherein the ester of an alpha-ketoalkanoic acid is an alkyl, aryl, arylalkyl, alkoxyalkyl, carbalkoxyalkyl or acetoxyalkyl ester.

5. The method of claim 2, wherein the ester of an alpha-ketoalkanoic acid is selected from the group consisting of ethyl pyruvate, propyl pyruvate, carboxymethyl pyruvate, acetoxymethyl pyruvate, carbethoxymethymethyl pyruvate, and ethoxymethyl pyruvate.

6. The method of claim 2, wherein the ester of an alpha-ketoalkanoic acid is selected from the group consisting of ethyl alpha-keto-butyrate, ethyl alpha-keto-pentanoate, ethyl alpha-keto-3-methyl-butyrate, ethyl alpha-keto-4-methyl-pentanoate, and ethyl alpha-keto- hexanoate.

7. The method of claim 1 , wherein the radioprotective agent is an ester of an alpha- ketoalkanoic acid of the formula R¹C(O)C(O)OR² wherein R¹ and R² independently comprise an alkyl, aryl, arylalkyl, alkoxyalkyl, alcohol, amine, amide, aromatic, aliphatic, heterocyclic, carbalkoxyalkyl, acetoxyalkyl or combinations thereof.

8. The method of claim 2, wherein the ester of an alpha-ketoalkanoic acid comprise a glyceryl ester.

9. The method of claim 2, wherein the ester of an alpha-ketoalkanoic acid comprises dihydroxyacetone esters of the formula R¹OCH₂C(O)CH₂OR²WhCrCm R¹ and R² independently comprise an alkyl, aryl, arylalkyl, alkoxyalkyl, alcohol, amine, amide, aromatic, aliphatic, heterocyclic, carbalkoxyalkyl, acetoxyalkyl or combinations thereof.

10. The method of claim 2, wherein the ester of an alpha-ketoalkanoic acid is a thiolester.

11. The method of claim 2, wherein the ester of an alpha-ketoalkanoic acid is an ethyl ester.

12. The method of claim 2, wherein the ester of an alpha-ketoalkanoic acid is an ester of pyruvic acid.

13. The method of claim 2, wherein the ester of an alpha-ketoalkanoic acid is ethyl pyruvate.

14. The method of claim 2, wherein the ester of an alpha-ketoalkanoic acid is contained in Ringer's isotonic saline.

15. The method of claim 2, wherein the radioprotective agent comprises in Ringer's isotonic saline comprising potassium ions or sodium ions.

16. The method of claim 2, wherein the radioprotective agent further comprises an enolization agent.

17. The method of claim 1 , wherein the radioprotective agent is an amide of an alpha- ketoalkanoic acid.

18. The method of claim 1 , wherein the radioprotective agent is an amide of an alpha- ketoalkanoic acid of the formula R¹C(O)C(O)N(R²)R³, wherein R¹, R² and R³ independently comprise an alkyl, aryl, arylalkyl, alkoxyalkyl, alcohol, amine, amide, aromatic, aliphatic, heterocyclic, carbalkoxyalkyl, acetoxyalkyl or combinations thereof.

19. A method of treating or protecting a subject against toxicities associated with ionizing radiation comprising administering to the subject a radioprotective amount of a radioprotective agent comprising a superoxide dismutase.

20. The method of claim 19, wherein the superoxide dismutase is EUK-134.

21. A radioprotective agent for treating or protecting a subject against toxicities associated with ionizing radiation comprising an alpha-ketoalkanoic acid or a derivative thereof.

22. The radioprotective agent of claim 21 , wherein the radioprotective agent is an ester of an alpha-ketoalkanoic acid.

23. The radioprotective agent of claim 22, wherein the ester of an alpha-ketoalkanoic acid is a C₃-C₈ straight-chained or branched alpha-ketoalkanoic acid.

24. The radioprotective agent of claim 22, wherein the ester of an alpha-ketoalkanoic acid is an alkyl, aryl, arylalkyl, alkoxyalkyl, carbalkoxyalkyl or acetoxyalkyl ester.

25. The radioprotective agent of claim 22, wherein the ester of an alpha-ketoalkanoic acid is selected from the group consisting of ethyl pyruvate, propyl pyruvate, carboxymethyl pyruvate, acetoxymethyl pyruvate, carbethoxymethymethyl pyruvate, and ethoxymethyl pyruvate.

26. The radioprotective agent of claim 22, wherein the ester of an alpha-ketoalkanoic acid is selected from the group consisting of ethyl alpha-keto-butyrate, ethyl alpha-keto- pentanoate, ethyl alpha-keto-3-methyl-butyrate, ethyl alpha-keto-4-methyl-pentanoate, and ethyl alpha-keto-hexanoate.

27. The radioprotective agent of claim 21 , wherein the radioprotective agent is an ester of an alpha-ketoalkanoic acid of the formula R¹C(O)C(O)OR² wherein R¹ and R² independently comprise an alkyl, aryl, arylalkyl, alkoxyalkyl, alcohol, amine, amide, aromatic, aliphatic, heterocyclic, carbalkoxyalkyl, acetoxyalkyl or combinations thereof.

28. The radioprotective agent of claim 22, wherein the ester of an alpha-ketoalkanoic acid comprise a glyceryl ester.

29. The radioprotective agent of claim 22, wherein the ester of an alpha-ketoalkanoic acid comprises dihydroxyacetone esters of the formula R¹OCH₂C(O)CH₂OR²WhCrCm R¹ and R² independently comprise an alkyl, aryl, arylalkyl, alkoxyalkyl, alcohol, amine, amide, aromatic, aliphatic, heterocyclic, carbalkoxyalkyl, acetoxyalkyl or combinations thereof.

30. The radioprotective agent of claim 22, wherein the ester of an alpha-ketoalkanoic acid is a thiolester.

31. The radioprotective agent of claim 22, wherein the ester of an alpha-ketoalkanoic acid is an ethyl ester.

32. The radioprotective agent of claim 22, wherein the ester of an alpha-ketoalkanoic acid is an ester of pyruvic acid.

33. The radioprotective agent of claim 22, wherein the ester of an alpha-ketoalkanoic acid is ethyl pyruvate.

34. The radioprotective agent of claim 22, wherein the ester of an alpha-ketoalkanoic acid is contained in Ringer's isotonic saline.

35. The radioprotective agent of claim 22, wherein the radioprotective agent comprises in Ringer's isotonic saline comprising potassium ions or sodium ions.

36. The radioprotective agent of claim 22, wherein the radioprotective agent further comprises an enolization agent.

37. The radioprotective agent of claim 21 , wherein the radioprotective agent is an amide of an alpha-ketoalkanoic acid.

38. The radioprotective agent of claim 21 , wherein the radioprotective agent is an amide of an alpha-ketoalkanoic acid of the formula R¹C(O)C(O)N(R²)R³, wherein R¹ _, R² and R³ independently comprise an alkyl, aryl, arylalkyl, alkoxyalkyl, alcohol, amine, amide, aromatic, aliphatic, heterocyclic, carbalkoxyalkyl, acetoxyalkyl or combinations thereof.

39. A radioprotective agent for treating or protecting a subject against the toxicities associated with ionizing radiation comprising a superoxide dismutase.

40. The radioprotective agent of claim 39, wherein the superoxide dismutase is EUK-134.