Abstract:
This invention is directed to the use of novel methods and nitroxide compositions having high redox activity to treat diseases and conditions related to free radicals. The compositions and methods described include pro-drug forms of nitroxide that enhance the therapeutic effect through their ability to penetrate readily into skin cells and remain sequestered within the cells. The formulations and methods described herein result in a significant accumulation of nitroxides in the skin cells, thus achieving a targeted delivery of a therapeutic or diagnostic dose. Applications using the novel compositions and methods described herein include use as improved imaging agents and as improved topical agents for treating conditions of the skin, including aging of the skin, wrinkles caused by sun-exposure, skin cancer, burns, psoriasis and alopecia, among others.

Description:
FIELD OF THE INVENTION  
         [0001]    The present invention generally relates to compositions of nitroxides and the therapeutic and diagnostic uses of specially formulated nitroxide compositions compatible with pharmaceutical use. These formulations are specifically designed for increased diagnostic and therapeutic utility when applied in a biological context where intracellular retention is important and where hydrophobic barriers such as skin, stratum corneum, or other such membranes are selectively permeable. These compositions are particularly useful when delivered to esterase-containing cells. These compositions may be used to alleviate the toxic effects of free radicals in a living organism that result from exposure to chemical agents and toxins, as well as sun, UV, or other forms of ionizing radiation. Specialized methods and formulations also enable the ability to diagnose and treat a wide variety of physiological conditions, and to analyze in vivo reactions with imaging techniques that measure nitroxides and their reactivity. The invention also relates to novel nitroxide compositions that permit targeting or compartmentalization of a therapeutic dose of nitroxide within a localized area, particularly penetration and retention within an intra-dermal interface in the skin.  
         BACKGROUND OF THE INVENTION  
         [0002]    Radiation and free-radical mechanisms are the source for a wide variety of diseases and physiological damage to the body. Reactive oxygen species such as the superoxide an ion (.O 2 ), the hydroxyl radical (.OH), hydrogen peroxide (H 2 O 2 ), and singlet oxygen ( 1 O 2 ) are usually formed by ultraviolet or ionizing radiation and react through oxidation mechanisms to damage DNA, proteins, and biologically important lipids in the body. The whole body effect of radiation and other such toxic reactive oxygen species is frequently manifested in damage to the skin. The acute effects include inflammation and the appearance of sun-damaged cells, principally as a result to overexposure to the sun. The exposure to reactive oxygen species also creates photosensitivity reactions and can lead to immunological suppression when the overall exposure is large in quantity or duration. The long-term effects include photoaging, typically manifested through the appearance of the skin as well as a tendency for certain physiological mechanisms that rejuvenate the skin and body to be compromised. Also, carcinogenesis is observed and the resulting skin cancers or keratoses can appear after years or even decades following a significant exposure incident.  
           [0003]    Skin may be viewed a major protective barrier that preferentially admits some compounds and yet excludes external agents that may be hazardous to a living organism, including toxic chemicals and free radical species. The skin features certain endogenous protective mechanisms against reactive oxygen species including the enzymes superoxide dismutase, catalase, goutathione peroxidase, and reductase. Each of these enzymes acts to convert free radical oxygen species to a compound that is less toxic to the body and can ultimately be metabolized through normal mechanisms. However, these enzymes are also involved in a cascade of free radical reactions that may inherently involve damage to the organism. Also, when an exposure to radiation or reactive oxygen species is particularly severe, these protective mechanisms are often overwhelmed by the free radical cascade and significant cell or tissue damage or injury results.  
           [0004]    Recognizing the potential damage from exposure to reactive oxygen species, several topical antioxidants have been developed that attempt to protect the skin from the majority of damage caused by reactive oxygen species. Examples of the non-enzymatic antioxidants that may be applied to the skin, and which exist in some quantities naturally, include vitamin C and vitamin E, para-amino benzoc acid (PABA) and others. These topical antioxidants may reduce the acute effects of exposure to radiation and may also, over time, reduce the chronic effects observed with long-term or high dose exposure to radiation and reactive oxygen species.  
           [0005]    The selective permeability creates unique problems and offers unique opportunities for drug administration. This environment of the dermal layers, as well as other membranes such as the enothelial layers of the vascular system and the blood/brain barriers, demonstrate a complex drug clearance and pharmacodynamic and pharmacokinetic parameters when exposure to ultraviolet and ionizing radiation causes changes to normal cell metabolism.  
           [0006]    The properties of an ideal topical antioxidant would include chemical stability, the ability to effectively penetrate both the epidermal and dermal layers of the skin, the maximum possible retention in the target cells to maintain protective levels as long as possible, and, of course, overall lack of toxicity. The formulation of an antioxidant having all of these properties, that is also suitable for topical administration, is difficult because free radical species are typically extremely unstable and highly reactive. The high reactivity of free radical special is particularly problematic for therapeutic drug design because the physiological damage is caused by chemical reactions between free radicals and body tissue that occur in very close proximity to the site where the free radical chemical species is generated. The effect of ultraviolet light upon molecular oxygen present in the dermal layers penetrated by UV light, for example, can produce high levels of free radicals in the skin that cause erythema and may ultimately lead to skin cancer and pre-mature skin aging (and wrinkling). Since molecular oxygen contains two unpaired electrons in its normal state, the effect of ultraviolet light upon triplet oxygen results in the creation of a more reactive oxygen species (i.e., singlet oxygen).  
           [0007]    In addition to the direct damage to biological tissue components such as structural proteins, superoxide radicals are extremely reactive with a variety of molecules naturally present in the body, including membrane lipids and nucleic acids. Detrimental free radical reactions result in the alteration or loss of tissue and cell function, increased permeability of skin cell membranes, cell death, and cancer. See e.g. Canavese, C. et al.,  Int. J. Artif. Organs  10(6): 379-89 (1987). Many studies have demonstrated that free radicals cause or aggravate a number of other pathologic states, including cancer, ulcers, cataracts, closed head injury, renal failure, injury to the nervous system, ischemia-reperfusion injury resulting from heart attack or stroke, shock, alopecia, sepsis, apoptosis, toxicity caused by certain drugs resulting from oxygen therapy in the treatment of pulmonary disease, psoriasis, the aging process, and many others. (See e.g. Dhalla, N. S.,  Can. J. Cardiol.  15(5):587-593 (1999); Basaga, H. S.,  Biochem. Cell Biol.  68(7-8): 989-98 (1990); Canavese, C. et. al.,  Int. J. Artif. Organs  10(6):379-89 (1987); Granger D. N. et. al.,  Gastroenterology  81:22 (1981); Babior, B. M. et. al,  J. Clin. Invest.  52:741 (1973)).  
           [0008]    A particularly important chemical reaction in the pathophysiology of skin damage occurs when nitric oxide and a superoxygen species react to form peroxynitrite. Peroxynitrite was recently discovered to pathologically activate epidermal growth factor receptor. These pathologies resulting in chronic lesioning of dermal tissue can eventually lead to DNA mutagenicity and cancer. For example, while its etiology is unknown, free radicals have been implicated in the etiology and/or symptoms of the chronic skin disorder, psoriasis. See e.g. Er-Raki A, et al.,  Skin Pharmacol.  6:253-258 (1993); Miyachi, Y. and Niwa, Y.,  Arch. Dematol. Res.  275:23-26 (1983). Psoriasis is accompanied by increased levels of the enzyme xanthin oxidase in the epidermis of the skin, which itself is capable of generating oxygen free radicals. See Hersh, U.S. Pat. No. 6,011,067. Further, psoriasis patients display an increase in the production of malondialdehyde, which is a reflector of free radical damage in the body. Corrocher,  Clin. Chim. ACTA  179:121 (1989). Additionally, the effect of ultraviolet radiation from exposure to the sun on human skin is a growing concern since the majority of changes associated with an aged appearance result from chronic sun-damage. Warren et al.,  J. Am. Acad. Dermatol.  25:751-760 (1991); Frances, C. and Robert L.,  Int. J. Dermatol.  23:166-179 (1984).  
           [0009]    Recently, topical retinoids became a popular treatment for sun-damaged skin, as well as for a wide variety of other skin disorders. See A. Haas et al., “Selected Therapeutic Applications of Topical Tretinoin”,  JAAD,  15:870 (1986). Among the topical retinoids, Tretinoin (Retin-A or Renova®) is one of the most widely used, most commonly as an anti-wrinkle agent for sun-damaged skin. Tretinoin, however, has a number of undesirable side effects including, ironically, heightened sensitivity to sun exposure. See U.S. Pat. No. 5,721,275, Bazzano (1998). Further, even the lowest dose formulations of tretinoin can be unacceptable to certain individuals with sensitive skin since local irritation is a common side effect of tretinoin therapy. See U.S. Pat. No. 4,021,573 Lee (1977); 4,214,000 Papa (1980). Alpha-hydroxy acids have also been used to treat various skin conditions, including sun damage. However, when used at high concentrations, the application of these formulations must be carefully monitored and can lead to inflammation, infection and scarring. See Morganti, P. et al.,  J. Applied Cosmetology  14(1):1-8 (1996); U.S. Pat. No. 4,247,547 Marks (1981). Even at lower concentrations, alpha hydroxy acids can cause skin sensitivity and irritation, and may require the user to use special cosmetic and shampoo formulations due to this sensitivity. See e.g. U.S. Pat. No. 6,019,967 Breton et al. (2000).  
           [0010]    However, clinical studies indicate that the ability of typical agents to provide protection is variable. See Miyachi Y.,  J. Dermatol. Sci.  1995, 9:79-86; Bissett et al.  Photodermatol. Photoimmunol. Photomed.  1990, 7:56-62. Moreover, the usefulness of many anti-oxidants is limited by short duration of action in vivo, toxicity at effective dosage levels, the inability of many compounds to cross cell membranes, and an inability to counter the effects of high levels of free radicals. For example, superoxide disuntates and catalase do not function effectively in the intracellular space, and procystein as a GSH precursor, vitamins and other antioxidants are unable to alleviate the effects of the high levels of free radicals encountered in injury and disease and are rapidly bioreduced. Nitroxides are a class of stable free radical compounds which have an unpaired electron maintained in an orbital configuration that yields both stability and unusual reactivity. Nitroxides exhibit antioxidant, enzyme-mimetic and catalytic activities in vitro and in vivo. (See Hsia U.S. Pat. Nos. 5,725,839; 5,741,893; 5,767,089; 5,804,561; 5,807,831; 5,817,632; 5,824,781; 5,840,701; 5,591,710; 5,789,376; 5,811,005; 6,048,967). The impact of small molecular weight nitroxides on a variety of reactive oxygen species (ROS)-induced diseases is also well documented in animal models. However, the intracellular and extracellular isotropical distribution and rapid bio-reduction of these nitroxides narrows their therapeutic index and application. Further, where high dose, continuous infusions have been employed, toxicity has been an issue that limits the therapeutic utility. Small molecule nitroxides have been discovered to protect cells against free radical-mediated damage, including inflammation and injury caused by ionizing radiation, Mitchell U.S. Pat. No. 5,462,946 (1991); Hahn (1992), and post-ischemic reperfusion injury, Gelvan (1991). Certain low molecular weight nitroxides have also been identified that mimic the activity of superoxide dismutase (SOD), (A. Samuni et. al.  J. Biol. Chem.  263:17921 (1988)) and catalase (R. J. Mehlhorn et. al.,  Free Rad. Res. Comm.,  17:157 (1992)). Studies also show that permeable nitroxides are capable of short-term protection of mammalian cells against cytotoxicity from superoxide anion generated by hypoxanthine/xanthine oxidase and from hydrogen peroxide exposure. See e.g. Noel-Hudson et al.,  Toxicol. Vitro.  3(2):103-109 (1989).  
           [0011]    Previously, small molecular weight nitroxides have been covalently attached to macromolecules, such as albumin, hemoglobin or starch, at low molar ratios for non-therapeutic applications. Therapeutic efficacy can be achieved by covalently attaching the nitroxides at comparatively higher molar ratios (See Hsia U.S. Pat. Nos. 5,725,839; 5,741,893; 5,767,089; 5,804,561; 5,807,831; 5,817,632; 5,824,781; 5,840,701; 5,591,710; 5,789,376; 5,811,005; 6,048,967). The resulting macromolecules have extended half-lives and demonstrate both reduced toxicity and enhanced therapeutic effect in animal models of vascular-related disease (e.g. stroke, gut ischemia) (21, 22) and have utility in imaging and diagnostic applications. Application of macromolecular nitroxides, in combination with an isotropically distributes nitroxide (e.g. TPL), or its hydroxylamine derivative (e.g. TPA), results in a superior therapeutic index. This formulation exhibits a spin-spin exchange between the macromolecular nitroxide and the cell permeable nitroxide that occurs with a “halo” surrounding the vessel. Within the “halo” TPH reoxidizes to TPL. The creation of such a TPL “halo” and the corresponding increase in therapeutic index occurs through the compartmentalization of cell-permeable and macromolecular nitroxides. However, unmodified membrane permeable nitroxides are rapidly reduced to an inactive form because a dynamic equilibrium between the nitroxide and its hydroxylamine state occurs in vivo, with the equilibrium favoring the hydroxylamine state.  
           [0012]    Thus, a major disadvantage of unbound small molecular weight nitroxides is their rapid reduction in vivo to a less active hydroxylamine derivative. Further, timely delivery of therapeutic nitroxide agents to cells in the affected region where oxygen free radicals are rampant is required in order to produce a significant therapeutic benefit. Small molecule nitroxides suffer from the disadvantages mentioned above, principally a relatively short half-life in vivo, difficulty in formulating such compounds to be selectively compartmentalized within the skin, and the tendency of such compounds to be rapidly cleared from the dermal layers by bioreduction or clearance through the movement of fluids in the vascular interstitial tissue space. See, “In vivo EPR Imaging of a Distribution and Metabolism of Nitroxide Radicals in Human Skin,” He et al., J. of Magnetic Resonance 148, 155-164 (2001).  
           [0013]    Due to the above problems, a need exists for improved nitroxide compositions having high redox activity, tissue selectivity and compartmentalization, favorable imaging characteristics, and low toxicity. A need exists for nitroxide compositions and methods which detoxify free radicals and related toxic species and which are sufficiently active and persistent in the body to avoid being rapidly consumed through bioreduction, clearance, or when increases in free radical concentrations are encountered. Specifically, there is a need for biologically compatible nitroxide compositions having high redox activity which can be sequestered in the skin so that a higher therapeutic or diagnostically effective concentration of nitroxide can be attained and maintained.  
         SUMMARY OF THE INVENTION  
         [0014]    This invention discloses the use of novel compositions comprising stable esters of carboxylic acid linked nitroxides, or low molecular weight nitroxides, including derivatized molecules and precursors and derivatives thereof. The oxidation/reduction activity and compartmentalization of stable nitroxides can be regulated by linking a negatively charged anion, such as a mono- or dicarboxylic acid group to a stable nitroxide molecule. The linkage of the negatively charged ion yields a “gate” function such that the permeability of the species of crossed cell membranes is altered and the in vivo half life is increased substantially. In a preferred embodiment, the present invention exploits the use of carboxylic acid ester(s) of nitroxides for both sustained biological activity and targeted compartmentalization. The compounds can be used advantageously in topical applications as a pro-drug for targeting the intracellular compartment of esterase-containing cells. In the pro-drug form, the nitroxide compounds are administered, and after penetrating hydrophobic barriers (e.g. stratum corneum, brain blood barrier), the compounds are retained in target tissue cells due to reduced cell membrane permeability after hydrolysis of the ester groups by cellular esterases. These compositions may be used to alleviate oxidative stress and to avoid the biological damage associated with free radical toxicity including the effect of reactive oxygen species produced by ultraviolet and ionizing radiation, exposure to toxins or chemicals or other free radical generators. These compounds reduce inflammation, apoptosis, and other cellular or tissue damage, both acute and chronic. Due to their enhanced antioxidant and radioprotectant activity, the compositions disclosed herein have increased therapeutic value compared to other topical antioxidants, which, in combination with their diagnostic value, allows the novel compositions and methods of this invention to be used advantageously in a wide variety of applications. These compounds can be used alone or in combination with other nitroxide containing species in therapy and diagnosis.  
           [0015]    The nitroxide compositions of the present invention are formulated to be selectively membrane permeable and to be administered as a precursor or derivative of the form of the nitroxide that persists inside the cell. Upon administration, the precursor or derivative is converted in vivo by intracellular enzymes to a therapeutically or diagnostically active form, preferably a form that exhibits reduced membrane permeability compared to the precursor, thereby maximizing the in vivo half-life and utility. Thus, the capability to maintain the concentration of an active nitroxide in vivo pursuant to this invention offers advantages in virtually any application where administration of a nitroxide is beneficial but the utility is limited due to rapid bio-reduction.  
           [0016]    The drawbacks of conventional nitroxide formulations are overcome by selecting a nitroxide having high redox activity and converting the nitroxide into an esterified derivative. In particular, this invention discloses stable small molecule, or ion molecular weight nitroxides having unusually high redox activity and carboxylic acid and ester derivatives of these nitroxides formulated to be physiologically compatible and stable and non-toxic for pharmaceutical use. When formulated pursuant to the invention, the duration of diagnostic imaging potential, as well as the therapeutic action, of nitroxides in the body is extended compared to unmodified nitroxides. In another embodiment, a nitroxide ester, such as an ester of 2,2,6,6,-tetramethyl-1-oxyl-piperdinene-4-succinate (“TOPS”) is applied topically while a macromolecular polynitroxide, such as polynitroxylated albumin, is also injected subcutaneously, intraperitoneally, or intravascularly. The polynitroxide albumin serves as an acceptor nitroxide, and distributes predominantly in the vascular space and acts as a storehouse of activity. See Hsia U.S. Pat. No. 5,840,701, herein incorporated by reference in its entirety.  
           [0017]    The novel compositions described herein are also useful in diagnostic applications because the unpaired electron of a stable nitroxide is detectable by electron paramagnetic resonance spectroscopy and nuclear magnetic resonance spectroscopy. Imaging instrumentation capable of detecting these compounds yield high quality images of biological structures. Pursuant to this invention, composition containing nitroxide esters, polynitroxide macromolecules, and/or their pro-drug analogs or derivatives yield active nitroxide levels in the body that may be maintained for a prolonged period of time allowing both improved image contrast and longer signal persistence than with conventional nitroxide formulations. Furthermore, the selective modification of the chemical composition of the nitroxide molecule to allow predetermined compartmentalization and metabolism increases the diagnostic value of the nitroxide species.  
           [0018]    Nitroxides having the structural compositions defined herein contain chemical leaving groups that are metabolized in biological systems to yield a modified nitroxide compound having different cell permeability than the unmodified compound. Thus, the invention may also be characterized by the difference between two nitroxide species. The first species contains a chemical group that is capable of being modified by intracellular biological processes such that a second nitroxide species is produced in vivo. Typically, the first nitroxide species will be characterized by increased cell membrane permeability relative to the second nitroxide species and the operative chemical modification takes advantage of this difference and the ability for the chemical modification to be susceptible to intracellular processes. Compounds of the invention include succinate, aspartate, and glutimate-linked forms of the nitroxide as well as other medium-sized fatty acids capable of forming modified esters. The compounds may also be synthesized as mono or diesters and homo or hetero-esters. For example, experimental results presented herein show that topical applications of an ethyl ester or di-ethyl ester of 2,2,6,6,-tetramethyl-1-oxyl-piperdinenel-4-succinate yields a targeted, diagnostic or therapeutic dose of nitroxide. The di-ethyl form of the ester is a particularly useful form of the nitroxide for intracellular localization because the ester groups functions as leaving groups when exposed to enzymes within cells. Pharmacokinetic results are provided to show human skin penetration, compartmentalization, and retention of DE-TOPS, increased plasma half-life ion toxicity, spectroscopic activity, and a protective effect against reactive oxygen species. Topical application of DE-TOPS is also shown Animal data shows skin thickening, reduced apoptosis, and reduced skin wrinkling in acute and chronic radiation exposure models. 
       
    
    
     DESCRIPTION OF THE FIGURES  
       [0019]    FIGS.  1 A- 1 C show the structures of selected examples of specially formulated nitroxide compounds of the invention.  
         [0020]    [0020]FIG. 1A is 2,2,6,6-tetramethyl-1-oxyl-piperidinenel-4-succinate (TOPS). FIGS. 1B and 1C are mono- (E-TOPS) and di-ethyl esters (DE-TOPS) of carboxylic acid derivatives.  
         [0021]    [0021]FIG. 2 shows a liquid chromatograph spectrum identifying TOPS and its ester(s) of carboxylic acid derivatives, E-TOPS and DE-TOPS.  
         [0022]    [0022]FIG. 3 shows the hydrolysis of DE-TOPS to TOPS in vitro sodium hydroxide (NaOH) in an aqueous environment showing the promotion of intracellular retention. As is apparent from the EPR signals, pre- and post-hydrolysis, near complete conversion from DE-TOPS to TOPS occurs.  
         [0023]    [0023]FIG. 4 compares the plasma half-life of a mono-ethyl esterified carboxylic acid derivative of  14 N-E-TOPS to  15 N-TEMPOL.  
         [0024]    [0024]FIG. 5 shows the extended in vivo plasma half-life of  14 N E-TOPS compared to  15 N TEMPOL when both are co-administrated intraperitoneally.  
         [0025]    [0025]FIG. 6 shows the extended in vivo plasma half-life of E-TOPS compared to TEMPOL when both are co-administrated intravenously.  
         [0026]    [0026]FIG. 7 shows the extended in vivo plasma half-life of E-TOPS compared to TEMPOL when both are co-administrated intramuscularly.  
         [0027]    [0027]FIG. 8 shows the extended in vivo plasma half-life of E-TOPS compared to TEMPOL when both are co-administrated orally.  
         [0028]    FIGS.  9 A-C shows skin penetration and compartmentalization of DE-TOPS in a Franz Diffusion cell when DE-TOPS and TEMPOL are co-applied on hairless mouse skin:  9 A—before application;  9 B—in receptor cell; and  9 C in skin.  
         [0029]    [0029]FIG. 10 shows human skin penetration and compartmentalization of DE-TOPS.  
         [0030]    [0030]FIG. 11 shows the relationship between the survival rate and dosage for E-TOPS compared to TEMPOL in an LD 50  study in mice.  
         [0031]    [0031]FIG. 12 shows the metabolism of DE-TOPS through excretion in urine and reduction in plasma after topical application at 6 hours.  
         [0032]    [0032]FIG. 13 shows the superoxide dismultase activity of E-TOPS determined by inhibition of the reduction of Cytochrome c.  
         [0033]    [0033]FIG. 14 show that E-TOPS dose dependent inhibition of hemoglobin-induced toxicity of cortical neurons.  
         [0034]    [0034]FIG. 15 shows TOPS, E-TOPS, and DE-TOPS dose dependent inhibition of peroxynitrite-induced toxicity on cortical neurons. Peroxynitrite generated from 3-morpholinosydnonimine (SIN-1). Rat cortical neurons were exposed to SIN-1 (1 mM) for 24 hours with or without the nitroxides TOPS, E-TOPS, or DE-TOPS at 50 or 500 μM. Following exposure to SIN-1, cell viability was determined based on the cell-dependent formation of purple formazan from MTT with a one-hour incubation. Data are means±standard deviation for 12 determinations.  
         [0035]    [0035]FIG. 16 shows that the nitration of hydroxy phenol acetic acid (HPA) by peroxynitrite was inhibited by TOPS, E-TOPS, and DE-TOPS in a dose-dependent manner.  
         [0036]    [0036]FIG. 17 shows that E-TOPS reduces apoptosis induced by tumor necrosis factor alpha (TNF-α) on Y-79 cell line at different incubation times.  
         [0037]    [0037]FIG. 18 is a histopathological sample of mouse skin after 10 days of chronic UVB radiation showing the inhibition of UVB-induced apoptosis with topical application of DE-TOPS.  
         [0038]    [0038]FIG. 19 shows that topical application of DE-TOPS prevents skin thickening induced by chronic UVB radiation compared with a placebo-treated mouse. No significant difference appears between normal and DE-TOPS treated ice following 11 days of ultraviolet exposure.  
         [0039]    [0039]FIG. 20 shows that the topical application of DE-TOPS accelerates wound healing compared with placebo treated mouse.  
         [0040]    [0040]FIG. 21 shows that topical application of DE-TOPS prevents UVB induced skin damage on mice.  
         [0041]    [0041]FIG. 22 shows a comparison of DE-TOPS, a placebo, and Retin A 15 days after treatment with DE-TOPS and ultraviolet radiation. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0042]    The term “nitroxide” is used herein to describe molecules having stable nitroxide free radicals including precursors (such as the N-H form), and derivatives thereof including their corresponding hydroxylamine derivative (N-OH) where the oxygen atoms are replaced with a hydroxyl group and exist in a hydrogen halide form. Stability of the unpaired electron is provided at the nitrogen nucleus by two adjacent carbon atoms that are substituted with strong electron donor groups. With the partial negative charge on the oxygen of the N—O bond, the two adjacent carbon atoms together localize the unpaired electron on the nitrogen nucleus. Nitroxides generally may have either a heterocyclic or linear structure.  
         [0043]    The physiological compartmentalization of the nitroxide pursuant to this invention can be achieved through several discrete chemical structures or molecular modifications. The molecules are designed pursuant to the criteria disclosed herein to provide the selected permeability and reactivity characteristics. Modified nitroxide formulations may be topically applied and targeted to specific cells because the membrane solubility of the nitroxide is altered by converting the nitroxide to the modified form as described herein. Once the modified nitroxide has entered the cell, the ordinary intracellular hydrolysis mechanisms of endogenous enzymes create derivatives of the nitroxide, which have reduced membrane permeability. Thus, these compounds readily enter the cell, but resist leaving the cell, and as a consequence, exhibit increased permeability for transmembrane entry into a target area and a decreased tendency to be removed from tissue by ordinary physiological clearance processes. This feature yields selectable preferential compartmentalization in vivo and sustained therapeutic or diagnostic effect.  
         [0044]    As described herein, accumulation and sequestration or compartmentalization of nitroxide species may be enhanced by converting the nitroxide to carboxylic acid ester. This result can be optimized using negatively charged anions such as nono- or dicarboxylic acids. Topical applications prefer a diester, which may be asymmetric, and where one esterified group is more labile than the other. For example, the t-butyl ester nitroxide will be more readily hydrolyzed than ethyl ester because it is more labile than ethyl ester. Hydrolysis of an asymmetric nitroxide comprising these two esters will thus first yield a less membrane permeable mono-carboxyl TOPS. The selection of the particular ester, e.g., methyl, ethyl, butyl, and mono or di-carboxylic acid derivative, e.g., succinate, aspartate, glutamate determines the compartmentalization and intracellular accumulation characteristics and thus may be tailored to be higher or lower for specific diagnostic or therapeutic indications. Likewise, preparation of nitroxide-diesters of naturally occurring dicarboxylic acids will permit increased accumulation and sequestration intracellularly and have an added advantage in that naturally occurring amino acids are well characterized and bioreactivity is known for biodistribution, metabolism and excretion (ADME) studies.  
         [0045]    The nitroxides that can be employed in this invention are structurally diverse because the requisite property of the nitroxide is its ability to be chemically modified with carboxylic acids and via esterification or to form the desired, modified nitroxide. Thus, nitroxides in their monocarboxylic, dicarboxylic, or polycarboxylic state may be employed. Selected embodiments of the present invention have the following structures, although the invention contemplates derivatives, isomers, substitutions, polymers, and other routine chemical modifications that preserve the functionality herein.  
                         
 
         [0046]    The carboxyl acid may be substituted with aspartate, glutimate, or another medium sized fatty acid and wherein R is a hydrogen molecule or ester, a homo- or hetero- or mono- or diester, including the methyl-, ethyl-, propyl, isopropyl, butyl, and t-butyl forms, or other functional equivalent.  
         [0047]    Since the compounds described herein provide a targeted therapeutic dose of antioxidant nitroxide, they are highly effective in preventing and alleviating the effects of oxidative stress. E-TOPS was found to have an increased in vivo half life compared to TEMPOL via intravenous administration, intramuscular, oral, and intraperitoneal administration at 200 mg/kg. See FIGS. 4, 5,  6 ,  7 , and  8 . The E-TOPS has also been demonstrated to have a very low acute toxicity profile compared to TEMPOL in an LD 50  model in mice. See FIG. 11. In transdermal applications, the DE-TOPS formulation reduces ultraviolet light induced apoptosis, and ultraviolet light induced skin thickening in mice. These formulations also compare favorably to Retin A. See FIGS.  18 - 22 . As described in more detail below, a major advantage of the nitroxide compounds of the present invention is the ability to administer a physiologically compatible solution containing these nitroxides in a variety of routes. Also, depending on the target cells and the diagnostic or therapeutic goal, the activity of these compounds may be enhanced by concurrent subcutaneous administration of a polynitroxide.  
         [0048]    Compositions of the invention may be used in dermatologically/cosmetically acceptable vehicles for transdermal such as ointments, lotions, or gels, and other solvents or carriers acting as a dilutant, dispersant or carrier. The vehicle may comprise materials commonly employed in skin care products such as water, liquid or solid emollients, silicone oils, emulsifiers, solvents, humectants, thickeners, powders, propellants and the like.  
         [0049]    As noted above, the unpaired nitroxyl electron gives nitroxides other useful properties in addition to the antioxidant activity. In particular, nitroxides in their free radical form are paramagnetic probes whose EPR signal can reflect metabolic information in biological systems, e.g., oxygen tension or tissue redox states. Because naturally occurring unpaired electrons are essentially absent in vivo, EPR imaging has the further advantage that there is essentially no background noise. Nitroxides also decrease the relaxation times of hydrogen nuclei, and are useful as contrast agents in proton or nuclear magnetic resonance imaging (MRI). Nitroxides can also act as contrast agents to add metabolic information to the morphological data already available from MRI. For example, by substituting various functional groups on the nitroxide, it is possible to manipulate properties including relaxivity, solubility, biodistribution, in vivo stability and tolerance. Contrast enhancement obtained from nitroxide can improve the performance of MRI by differentiating isointense, but histologically dissimilar, tissues and by facilitating localization and characterization of lesions, such as blood brain barrier damage, abscesses and tumors.  
         [0050]    In view of the stable chemical nature of the nitroxides, the compositions disclosed herein can be administered to a patient by various routes. For the purposes of this invention, “pharmaceutical” or “pharmaceutically acceptable” compositions are formulated by known techniques to be non-toxic and, when desired, used with carriers or additives that are approved for administration to humans. As described in the Examples below, the routes of administration include intravenous, occular or intraoccular, oral, intramuscular, topical or transdermal, intraperitoneal or sub-cutaneous application or injection. Such compositions may include buffers, salts, or other solvents known to these skilled in the art to preserve the activity of the vaccine in solution.  
       EXAMPLE 1  
       [0051]    Synthesis and Preparation of Mono or Dicarboxylic Acid And Esterified Nitroxide Species  
         [0052]    The following chemical synthesis protocols yield stable nitroxide free radicals whose physiological compartmentalization, as a function of membrane permeability and clearance in vivo, is regulated by a negatively charged anion such as mono- or di-carboxylic acids. Topical applications are particularly advantageous with ester derivatives that provide differential permeability across hydrophobic barriers with a first nitroxide species having increased membrane permeability relative to a second species having increased intracellular retention and antioxidant therapeutic utility after hydrolysis by intracellular esterases.  
         [0053]    (a) Synthesis of Monoethyl and Diethyl2,2,6,6,-Teteramethyl-1-oxyl-4-piperidinyl succinate (E-TOPS) and (DE-TOPS).  
         [0054]    A dry, two-necked flask fitted with a reflux condenser and magnetic stirrer is charged with 45 ml of absolute tert-butanol and 6.72 g of potassium tert-butoxide under nitrogen atmosphere. The mixture is boiled and heated under reflux until all solids are dissolved. The flask is then cooled and 6.8 grams of 4-oxo-[TPO], 12 ml of diethyl succinate and 15 ml of tertiary butanol is added. The reaction mixture is then heated for 10 minutes. After cooling with ice, and neutralizing with dilute HCL, the bulk of the alcohol is distilled off under reduced pressure. The residue is poured into 350 ml of ice water and acidified with dilute hydrochloric acid to pH 3, and extracted with methylene chloride. The combined extracts are washed several times with a 1% ammonia solution. The solutions are cooled with ice, acidified and extracted again with methyl chloride. The resulting extract is then dried with sodium sulfate. After evaporation of the methylene chloride, the remaining oily red liquid is triturated with hexane. The crystal of the monoester is pressed on a porous porcelain plate and recrystallized from a mixture of ether and hexane. The product is a yellow prism with a melting point of 103° C., and the expected yield is in the 60-70% range.  
         [0055]    (b) Synthesis of t-butyl, ethyl 2,2,6,6,-tetramethyl-1-oxyl-4-piperidinenel succinate (BE-TOPS).  
         [0056]    DCC (dicyclohexylcarbodiimide, 1 equivalent) is added to a solution of E-TOPS and t-butanol (1 equivalent) in dry pyridine (50 ml). The reaction mixture is stirred for 4 hours and the precipitates are removed by filtration. The filtrate is then evaporated to dryness and the residue is purified by column chromatography.  
         [0057]    [0057]FIG. 2 shows the separation of TOPS, E-TOPS, and DE-TOPS by liquid chromatography.  
       EXAMPLE 2  
       [0058]    Hydrolysis of DE-TOPS to TOPS by Sodium Hydroxide (NaOH)  
         [0059]    Referring to FIG. 3, the hydrolysis of DE-TOPS to the non-esterified TOPS form will yield selective cell membrane permeability and increased intracellular retention when the nitroxide compounds are exposed to esterases or any intracellular enzyme or other biochemical reaction that cleaves the ester group. The application of DE-TOPS as a hydrophobic pro-drug will penetrate the stratum corneum (dead cells) into the metabolically active base-membrane layers. Enzymatic hydrolysis of DE-TOPS will allow the product TOPS to be retained in the aqueous phase and hopefully and primarily in the intracellular volume. To demonstrate this reaction, chemical hydrolysis of DE-TOPS is shown to yield a compound which preferentially distributes in water vs. octanol.  
         [0060]    A 20 mg sample of DE-TOPS was added to a mixture of 1 ml water and 1 ml octanol and allowed to partition. After 15 minutes, a 40 μl sample of water or oil was taken for EPR spectral analysis. Next, 20 mg of DE-TOPS was mixed in 1 ml of 10 mM NaOH and allowed to incubate overnight at room temperature. The solution was neutralized with hydrochloric acid and added to 1 ml of water and 2 ml of octanol. The mixture was allowed to partition, and after 15 minutes, a 40 μl sample of water or oil was taken for EPR spectral analysis. EPR spectral were taken using a Varian E9 spectrophotometer. Sweep width was 100 G, frequency was 9.535 GHz, microwave power was 2 mV and the modulation frequency was 100 Hz.  
         [0061]    Before hydrolysis, DE-TOPS was preferentially distributed in octanol. The partition coefficient (LogP) for DE-TOPS was found to be 1.7. After hydrolysis, the presumed product TOPS partitions in the aqueous phase. The partition coefficient (LogP) for TOPS was found to be −0.9. This shows that following hydrolysis, the product thus formed is significantly hydrophilic and would be more readily compartmentalized intracellularly.  
       EXAMPLE 3  
       [0062]    In Vivo Plasma Half-Life of DE-TOPS and TEMPOL  
         [0063]    As noted above, a principal drawback in existing nitroxide-based compositions for in vivo therapeutic or diagnostic use is the limited half-life of these molecules and their rapid in vivo bioreduction and clearance. The result of the comparatively short half life of TEMPOL is a need to administer larger and larger doses to yield a profound therapeutic or diagnostic effect. As shown in FIG. 11 below, the increased dosages of nitroxides can yield acute and chronic toxicity. However, where the plasma half-life of a compound is increased, the overall dosage in both acute and chronic indications can be reduced.  
         [0064]    [0064]FIG. 4 is an EPR spectra showing TEMPOL, and E-TOPS simultaneously recorded at distinguished magnetic field positions. Plasma half-life is measured by collecting the spectrum each minute for 60 to 90 minutes. The peak height of  15 N TEMPOL or  14 N E-TOPS EPR signal is calculated from each spectrum. The peak height of TEMPOL or E-TOPS is plotted against time as shown in FIGS.  5 - 8 . Referring to FIG. 5, in an intraperitoneal administration, the effective therapeutic activity of  14 N E-TOPS (upper line) is substantially enhanced for the entire half-life of the compounds. Although  15 N TEMPOL (lower line) has a measurable half-life exceeding 40 minutes, E-TOPS has substantially higher activity for at least 80 minutes. The doses are 125 mg/kg of E-TOPS and 80 mg/kg TEMPOL at a 1:1 molar ratio.  
         [0065]    [0065]FIG. 6 shows a measurement of the in vivo plasma half-life of an intravenous administration of E-TOPS and TEMPOL at 125 mg/kg. In the intravenous infusion example, TEMPOL (lower line) is rapidly reduced in vivo such that, by the five minute mark after infusion, very little active TEMPOL remains in the intravascular space. By contrast, the activity of E-TOPS (upper line) is measurably increased, compared to TEMPOL, for at least 50 minutes. Referring to FIG. 6, the extension of the in vivo plasma half-life of E-TOPS compared to TEMPOL is shown when both compounds are co-administered intravenously at a dose of 125 mg/kg of E-TOPS and 80 mg/kg of TEMPOL for a 1:1 molar ratio.  
         [0066]    As is shown in FIG. 7, beyond the first few minutes the in vivo plasma half-life of E-TOPS is dramatically extended over TEMPOL for at least 60 minutes following intramuscular co-administration.  
         [0067]    Referring to FIG. 8, the in vivo plasma half-life of E-TOPS compared to TEMPOL is shown to be extended when both compounds are administered orally. As in FIGS.  5 - 7 , the doses are 125 mg/kg of E-TOPS and 80 mg/kg of TEMPOL at 1:1 molar ratio. As is indicated by FIG. 8, the in vivo plasma half-life of E-TOPS is dramatically extended compared to TEMPOL following co-administration of the compounds and for at least 40 minutes thereafter.  
       EXAMPLE 4  
       [0068]    Penetration and Compartmentalization in Human Skin  
         [0069]    Referring to FIG. 9, DE-TOPS (100 mM) in a petroleum base was applied on fresh human skin. The receptor buffer is PBS with 0.01% sodium azide. The buffer was constantly stirring. The cell was maintained at 37C with a circulating water bath. FIG. 9 shows the degree of skin penetration. Mouse skin (or donor human skin) to cover the top of the receptor and cell DE-TOPS is applied on top of the skin. Under the skin, PBS buffer is applied to keep the skin alive. 24 hours later the buffer will be collected for EPR assay. The surface of the skin is cleaned and the skin sample tested for EPR Signal. Although TEMPOL and DE-TOPS have same signal intensity prior to application, twenty-four hours after application a stronger TEMPOL signal exists in the buffer compound to DE-TOPS. However, in the skin the E-TOPS signal is stronger than TEMPOL. Thus, DE-TOPS is localized in skin compared with TEMPOL. DE-TOPS will penetrate into the cells of the epidermis and dermis where it will be enzymatically hydrolyzed and become compartmentalized. Compared to the freely soluble Tempone, this would result in a more uniform distribution of DETOPS in these skin layers.  
         [0070]    Preliminary S-band EPR spectroscopy and imaging experiments using DETOPS were performed on the skin of a human volunteer. The human volunteer&#39;s forearm skin, about 6 mm diameter circular spot, typically at the ulnar surface of the wrist, was washed thoroughly with alcohol and 3 μL of 100 mM DE-TOPS solution (about 2×10 17  spins) was applied to the marked skin area. Five minutes later, when the deposited solution dried, a specially designed positioning holder with a 7 mm diameter well and bottom disk that locked into a well in the resonator cap was attached to the skin to fix this region of skin to the surface resonator See, “In vivo EPR Imaging of a Distribution and Metabolism of Nitroxide Radicals in Human Skin,” He et al., J. of Magnetic Resonance 148, 155-164 (2001). EPR and EPRI measurements were then started. Measurements on the volunteer were performed for 15 to 20 minutes periods after which there were 30-minute rest periods in which the volunteer removed the arm from the resonator and the magnet. This holder fixed the skin positioning and assured a constant filling factor of the loaded resonator. The positioning holder was left attached to the arm for the entire series of measurements lasting up to 8 hours.  
         [0071]    Referring to FIG. 10, a color-coded image of CNO penetration and compartmentalization in human skin is shown. The 1-D spatial images were obtained from the skin of the fore-arms of the same human volunteer at 1 hr and 8 hr post-topical application of 3 μL or DE-TOPS (100 mM in DMSO). The measurements were performed using S-band (2.2 GHz) EPR imaging system with a specially designed surface resonator as reported (He et al., supra). The dotted line marks the surface of the skin. The estimated skin depths are marked as epidermis, dermis and subcutaneous layers. Compartmentalization results in a more diffuse distribution of DE-TOPS throughout the skin layers. The 1-D EPR spatial image of DE-TOPS compared to tempone show an enhanced visual distribution throughout the dermis and epidermis by eight hours.  
       EXAMPLE 5  
       [0072]    Acute Toxicity LD 50  of TEMPOL and E-TOPS.  
         [0073]    As noted above, the practical, clinical application of unbound, small molecule nitroxides has been limited by the reduced activity in vivo and comparatively short in vivo half-life. The result of the reduced in vivo half-life is the need to administer a larger dose to achieve the same therapeutic or diagnostic effect. The toxicity of TEMPOL may be measured with an LD 50  model to determine the survival rate of mice at varying dosages. FIG. 11 shows the acute toxicity curve for TEMPOL as a function of survival rate with increasing dosages. The E-TOPS formulation shows essentially no decrease in survival rate at dosages up to 3.5 mmol/kg whereas TEMPOL shows zero survival rate at the lower dosage of 2.0 mmol/kg. Significant differences in survival rate appear between the dosage of 2.0-2.5 mmol/kg. FIG. 12 shows the stronger EPR signal (proportional to concentration) from DE-TOPS in urine compound for plasma after 6 hours, showing excretion and clearance through normal metabolism.  
       EXAMPLE 6  
       [0074]    Inhibition of Cytochrome Reduction  
         [0075]    E-TOPS is shown to be a hydrolytic intermediate of DE-TOPS and to function as a SOD-mimetic based on the inhibition of cytochrome reduction by superoxide. The superoxide scavenging (SOD-mimetic) activity of E-TOPS was measured as the capacity to inhibit the reduction of cytochrome by superoxide radical. The reactions were analyzed calorimetrically at 550 nm and 25° C. with an HP spectrophotometer. The reactions were assayed with various concentrations of E-TOPS, 8×10 −5  M cytochrome, 1×10 −4  M xanthine, 6 μg/ml catalase, and 28 mU/ml xanthine oxidase. All of the chemicals and enzymes were diluted into Hank&#39;s Balanced Salt Solution (HBSS, Sigma). A reaction cocktail was immediately mixed by inversion in a cuvette and the absorption increase at 550 nm was recorded over 5 minutes. A blank sample (without nitroxide) showed the maximum rate of absorption. The IC 50  was calculated from the dose response curves as the concentration of bound or free nitroxide giving 50% inhibition of the maximum absorption rate.  
         [0076]    Referring to FIG. 13, reduction of cytochrome by superoxide occurs with increasing concentrations of E-TOPS. The correlation coefficient by linear regression is r 2 =0.998 demonstrating that E-TOPS has SOD mimetic activity.  
       EXAMPLE 7  
       [0077]    Hemoglobin Toxicity in Cultured Rat Cortical Neurons  
         [0078]    E-TOPS is neuroprotective in a model of hemorrhagic transformation in stroke. Primary neuronal cultures were made from forebrains of fetal rat pups (embryonic day 15). The cells were dispersed by repeated mechanical trituration in neuronal culture medium (MEM Eagle (Sigma, M4526), supplemented with glutamine (2 mM), penicilin-streptomycine (50 Units/ml -0.05 mg/ml), heat-inactivated horse serum (10%), fetal bovine serum (10%), glucose (0.5% or 28 mM). Following centrifugation (900 g; 5 min), the cells were placed onto poly-L-lysine-coated 96 well plates at a density of 5×10 6  cells/well. Hemoglobin in saline was added at 10 uM final concentration and ETOPS were added at 10 uM, 1 uM, and 0.1 uM final concentration. 24 hours after incubation neuronal viability was quantitatively determined using the calorimetric MTT assay. MTT was added to each well such that the final concentration of the dye was 0.15 mg/ml. Plates were then returned to the incubator for 1 hour at which time unincorporated MTT was removed, and the plates allowed to air dry. The purple formazan product present in viable cells was then dissolved by adding acidified isopropanol (with 0.1 N HCl in) and the absorbance intensity (540 nm) was measured using a 96 well plate reader. % Control=(Test A 540 /Mean Control)×100%. FIG. 14 shows increased neuronal viability with the E-TOPS samples.  
       EXAMPLE 8  
       [0079]    TOPS or TOPS-ester Neuroprotection of Cortical Neurons Exposed to the Peroxynitrite Generator SIN-1.  
         [0080]    SIN-1 toxicity is studied in cultured rat cortical neurons based on the generation of peroxynitrite. The demonstration that TOPS or TOPS-ester are neuroprotective in this model, translates into nitroxide-dependent blockade of EGFR activation caused by SIN-1.  
         [0081]    Primary neuronal cultures were made from forebrains of fetal rat pups (embryonic day 15). The cells were dispersed by repeated mechanical trituration in neuronal culture medium (MEM Eagle (Sigma, M4526), supplemented with glutamine (2 mM), penicilin-streptomycine (50 Units/ml-0.05 mg/ml), heat-inactivated horse serum (10%), fetal bovine serum (10%), glucose (0.5% or 28 mM). Following centrifugation (900 g; 5 min), the cells were placed onto poly-L-lysine-coated 96 well plates at a density of 5×10 6  cells/well. Cytotoxicity was induced in the cells according to a published procedure (Carroll et al 2000). SIN-1 (3-morpholinosydnonimine, Sigma), a PN generator, was dissolved in 50 mM phosphate (pH 5.0) just prior to use, and added to each well to give the final concentration of 1 mM. TOPS, E-TOPS or DE-TOPS were added at the indicated concentration to each culture 15 minutes prior to SIN-1. Neuronal viability was quantitatively determined using the colorimetric MTT assay. MTT was added to each well such that the final concentration of the dye was 0.15 mg/ml. Plates were then returned to the incubator for 1 hour at which time unincorporated MTT was removed, and the plates allowed to air dry. The purple formazan product present in viable cells was then dissolved by adding acidified isopropanol (with 0.1 N HCl in) and the absorbance intensity (540 nm) was measured using a 96 well plate reader. % Control=(Test A 540 /Mean Control)×100%.  
         [0082]    Referring to FIG. 15, TOPS or TOPS-esters prevented the toxicity of SIN-1 in a dose dependent manner. Neuroprotective concentrations of these compounds will prevent peroxynitrite-dependent EGFR activation by preventing the covalent dimerization of receptors and their subsequent autophosphorylation.  
       EXAMPLE 9  
       [0083]    Antioxidant Activity by Inhibition of Nitration  
         [0084]    To demonstrate antioxidant activity of DE-TOPS, E-TOPS and TOPS in vitro, these compounds are compared with TEMPOL as the gold standard. TEMPOL was shown to prevent the nitration of 4-hydroxyphenylacetic acid (HPA) by peroxynitrite in vitro (Carroll et al., 2000). In this preliminary experiment, % inhibition by TEMPOL, DE-TOPS, E-TOPS or TOPS of peroxynitrite-dependent nitration of HPA was measured.  
         [0085]    Peroxynitrite was made by a procedure described previously (Reed et al., 1974). Solutions of 1 mM 4-hydroxyphenylacetic acid (HPA, Sigma) were made in 100 mM sodium phosphate at pH 6.5. Certain amount of TOPS, E-TOPS and DE-TOPS were added to 1 ml of HPA solution mentioned above to give a final concentration of nitroxide at 0.98, 3.91, 15.5, 62.5 and 250M. Peroxynitrite was added at a final concentration of 1 mM to start the nitration. Reactions were also carried out using inactive peroxynitrite as blank and zero nitroxide as positive control. The nitration was followed spectrophotometrically at 405 nm. The concentration of 4-hydroxy-3-nitrophenylacetate was determined spectrophotometrically (430=4400 M −1 cm −1 ) after the pH of reaction mixtures were increased to 10-11 with NaOH.  
         [0086]    Referring to FIG. 16, 40% to 60% of HPA nitration by 1 mM peroxynitrite are inhibited by the nitroxides of the invention at 3 μM. The mechanism of inhibition by nitroxides is catalytic as was reported by Carroll et al., 2000. As peroxynitrite is suggested to be an important player in radiation-induced cellular damage, TOPS-esters has utility as a therapy for skin exposed to reactive oxygen species such as peroxynitrite.  
       EXAMPLE 10  
       [0087]    Cell Apoptosis Measured by Exposure to the TNF α 
         [0088]    The UV light induced apoptosis may be measured by a model in which apoptosis is induced by exposure to tumor necrosis factor alpha (TNF-α). As shown in FIG. 17, measurement of apoptosis severity over time for cells exposed to TNF-α plus E-TOPS is measured against a control. Cultured human Y-79 cells were maintained at pH 7.4 in culture flasks in a mixture of amphotericin/penicillin/streptomycin treated (1% v/v) RPMI 1640 media with L-glutamine and 10% fetal bovine serum in an incubator under 10%-CO 2 /90%-air brood conditions at 37 degrees Celsius and 20% humidity. E-TOPS was prepared as a 10 mg/ml formulation. From this stock solution, 10 μl was added to 1 ml of the cell suspension solution so that the final concentration of ETOPS was 100 μg/ml of ETOPS. Human TNF-α (10 g/1 ml) was used to prepare serial media dilutions to obtain TNF-α concentrations of 5.0 ng/ml. Cell densities and viability were determined by trypan blue exclusion assay on a Zeiss inverted microscope to ensure cell concentrations prior to cytometric assaying. After gentle mixing of the TNF-α, vial sets at the given concentrations for 6, 24, and 48 hours, the cytokine-treated cells were resuspended in PBS. The cells were resuspended in annexin V-FITC conjugate solution and incubated at room temperature in the dark for fifteen minutes. Binding buffer was then added to each of the samples to bring the cell densities up to approximately 1.0×10 6  cell/ml. The binding buffer was prepared by mixing the following together: 10 ml of 1M HEPES/NaOH, ph 7.4, 30 ml of 5M NaCl, 5 ml of 1M KCl, 1 ml of 1M MgCl 2 , 1.8 ml of 1M CaCl 2  and 52.2 ml of DDW.  
         [0089]    Flow cytometric methods were employed to take advantage of annexin V&#39;s reversible and calcium-dependent binding to negatively charged phosphatidylserine (PS) residues in a 1:50 annexin to PS ratio. Assay at 488 nm on a Becton-Dickinson FACStar flow cytometer was then conducted on the cells from the culture vials to determine the relative proportions of cells that were nonviable. A two dimensional x-y contour plot was used to show populations of early apoptotic events separate from late apoptotic events. Compensation was set before the cytometric trials by using an annexin-only stained population, a propidium-only stained population, and an unstained population to delimit the ceilings of detection in the respective FL-1/Fl-2 quadrants. Total samplings averaged 5000 cell events counted out of sample populations averaging well over 1.0×10 6  cells/ml with statistics and regression analysis given for each set of sample quadrants using CellQuest software. Fluorescein stained populations alone demarcated apoptotic detection while dual counterstaining with propidium iodide indicated necrotic populations.  
         [0090]    Referring again to FIG. 17, FL1/FL2 plots demonstrate light scattering showing that E-TOPS rescued (more cell survival) Y-79 cells from increased apoptosis over time at 5.0 ng/ml TNF-α doses when compared to control Forward and lacteral cytometric light scattering characteristics also showed that E-TOPS exhibited protective effects in Rb cell types. Negligible amounts of cells died due to necrosis as defined by PI cytometric gating.  
       EXAMPLE 11  
       [0091]    Uvb Induced Apoptosis  
         [0092]    Measurement of the caspase 3 enzyme in SKH-1 mouse skin is a quantitative analysis of ultraviolet light induced apoptosis. See Hurwitz et al.,  Experimental Dermatology  2000 June; 9(3):185-91. Topical application was daily 10 minutes prior to UVB radiation. UVB dose was 200 mJ/cm2 during 15 minutes. UVB lamp has spectrum distribution of 290-31 0 nm at 80% below 310 nm at 20%. The DE-TOPS formulations are 100 mM in a petroleum base. Referring to FIG. 18, the topical administration of DE-TOPS inhibits UV induced apoptosis 10 days after chronic radiation with ultraviolet B light.  
       EXAMPLE 12  
       [0093]    Protection Against Ultraviolet Induced Skin Thickening and Enhanced Wound Healing  
         [0094]    Referring to FIG. 19, 11 days exposure to UVB causes significant skin thickening compared to normal mouse skin without UVB exposure. Histopathological skin sections reveal that topical application of DE-TOPS dramatically reduces skin thickening induced by UVB. Topical application was daily 10 minutes prior to UVB radiation. UVB dose was 200 mJ/cm2 during 15 minutes. UVB lamp has spectrum distribution of 290-310 nm at 80% below 310 nm at 20%. The DE-TOPS formulations are 100 mM in a petroleum base. Referring to FIG. 21, with a similar protocol, wound healing is enhanced.  
       EXAMPLE 13  
       [0095]    Effectiveness of DE-TOPS for Alleviating UVB-Induced Skin Damage.  
         [0096]    Hairless Rhino mice were subjected to a fifteen-day UV irradiation experiment in which test materials were applied to the skin. The nitroxide concentrations in this study were 100 mM in a petroleum base applied topically and daily 10 minutes before UVB radiation. The mice were exposed to UVB irradiation of 200 mJ/cm2 for 15 minutes with a spectrum distribution of 80% 290-310 nm and 20% below 310 nm. Skin morphology (extent of wrinkling) was examined daily and recorded by photography prior to any daily treatment. Treatment of the dorsal skin with DE-TOPS resulted in reduction of wrinkling and an improved cutaneous histological appearance (see compare FIG. 21).  
         [0097]    To determine the advantage of DE-tOPS compared to commercially available topical therapies, BE-TOPS, Retin-A, and a placebo were administered to the skin of mice exposed to alternate days of UVB radiation. Treatment with Retin-A resulted in a reduction in skin wrinkling compared with control animals (See FIG. 22). Although the photo-sensitivity of Retin-A caused discoloration and flaking of the dorsal skin, cutaneous histological sections of the dorsal skin also revealed Retin-A treatment resulted in a reduction in the density of open and deep cysts.  
         [0098]    BE-TOPS also has the advantage over Retin-A that DE-TOPS treated skin is not UVB sensitive.