Abstract:
Therapeutic applications, such as prevention, treatment and supplementation, for the use of novel and other thiolatocobalamins to protect human cells against the effects of oxidative stress. In particular, this invention relates to the use of a novel synthetic thiolatocobalamin, glutathionylcobalamin to protect animal cells against oxidative stress damage. This invention also relates to the use of thiolatocobalamins, such as glutathionylcobalamin, in lieu of current, commercially available forms of vitamin B 12  for the treatment and prevention of conditions associated with oxidative stress damage and for dietary supplementation.

Description:
CROSS-REFERENCE  
       [0001]     This application claims priority to U.S. Provisional Application Ser. No. 60/846,435 filed on Sep. 22, 2006. 
     
    
     FIELD OF THE INVENTION  
       [0002]     This invention relates to compositions having therapeutic applications, such as prevention, treatment and supplementation, for the use in protecting animal, and in particular, human cells against the effects of oxidative stress. This invention relates to a novel synthetic thiolatocobalamin, glutathionylcobalamin, which can be used to protect human cells against oxidative stress damage. This invention also relates to the use of thiolatocobalamins as a pharmaceutical composition and as a dietary supplement, such as glutathionylcobalamin, in lieu of current, commercially available forms of vitamin B 12 , for the treatment and prevention of conditions associated with oxidative stress damage.  
       BACKGROUND OF THE INVENTION  
       [0003]     Three forms of vitamin B 12  have long been recognized to occur in biology, aquacobalamin/hydroxycobalamin, methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) (Golding, B. T.  Chem. Brit.  1990, 950). (See Formula I). Methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) play crucial roles in the B 12 -dependent enzyme reactions and are frequently referred to as the B 12  co-enzymes. Two known B 12 -dependent enzymes exist in humans: methionine synthase, which is methylcobalamin (MeCbl)-dependent, and methylmalonyl-coenzyme A mutase, which is adenosylcobalamin (AdoCbl)-dependent. (Dolphin, D. (ed). B 12 ; John Wiley &amp; Sons, Inc.: New York, USA, 1982; Banerjee, R. (ed.)  Chemistry and Biochemistry of B   12 ; JohnWiley &amp; Sons, Inc.: New York, USA, 1999). In short, methionine synthase and methylmalonyl-CoA mutase require the vitamin B 12  derivatives methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl), respectively, for certain enzymatic reactions in the body. For example, in the MeCbl-dependent methionine synthase reaction, a methyl group is transferred from methyl-tetrahydrofolate (a metabolite of folate) to homocysteine (Hcy) via MeCbl to give methionine and tetrahydrofolate. This reaction results in the conversion of homocysteine, an amino acid found in humans, which has destructive, oxidative properties, back to methionine. This reaction has received much attention in the medical literature in recent years, because its impairment can lead to elevated levels of homocysteine, which is associated with an increased risk of cardiovascular, cerebrovascular and peripheral vascular disease, and other pathological conditions which are discussed below.  
         [0004]     Thiol derivatives of B 12 , thiolatocobalamins, were first identified in the 1960&#39;s, but have not attracted much attention until recently. They are characterized by having a cobalt-sulphur bond in the upper (beta) axial position. (See Formula 1). Glutathionylcobalamin (GSCbl (or GluSCbl), a thiolatocobalamin) has been recently isolated from mammalian cells. A method for preparing glutathionylcobalamin is the subject of U.S. Pat. No. 7,030,105, the contents of which are incorporated herein by reference.  
         [0005]     Glutathionylcobalamin (GSCbl) is an important cobalamin metabolite in mammals and is more active than other cobalamins in promoting methionine synthase activity in rabbit spleen extracts. It has been proposed that, in vivo, GSCbl (or a closely related thiolatocobalamin adduct) is a precursor of the two coenzyme forms of vitamin B 12 —AdoCbl and MeCbl. An alternative role for GSCbl was also recently proposed, in which the formation of GSCbl prevents B 12  from being scavenged by xenobiotics.  
         [0006]     The exact biochemical pathway(s) that lead to the incorporation of cobalamins into the B 12 -dependent enzymes have not yet been elucidated. It is known that thiolatocobalamins can be reduced by free thiols, yielding cob(l)alamin species, which can in turn be methylated by S-adenosylmethionine to form methylcobalamin. Whether this is an important biochemical pathway in humans needs further study.  
         [0007]     A variety of thiolatocobalamins have been synthesized. Recently, simple synthetic methods have been reported for the preparation of three additional thiolatocobalamins—D,L-homocysteinyl-cobalamin (HcyCbl), the sodium salt of N-acetyl-L-cysteinylcobalamin (Na[NACCbl]), and 2-N-acetylamino-2-carbomethoxy-L-ethanethiolatocobalamin (NACMECbl). (Suarez-Moreira, E., Hannibal, L., Smith, C., Chavez, R., Jacobsen, D. W., and Brasch, N. E., Dalton Trans., in press). However GSCbl is the only thiolatocobalamin that has been isolated in mammals to date. Furthermore, prior to this synthesis, Na[NACCbl] and NACMECbl were not even reported to exist.  
         [0008]     Formula I, below, depicts the structures of vitamin B 12 , including the two coenzyme forms of vitamin B 12  and related B 12  derivatives found in humans, all commonly referred to as the cobalamins.  
                         
 
         [0009]     The cobalamins belong to a family of compounds known as the corrinoids, which differ from one another in the specific nucleotide occupying the a axial site of the cobalt-corrin complex. The α (or lower) axial site is occupied by an intramolecularly-bound 5,6-dimethylbenzimidazole, and the β (or upper) axial site can be occupied by a variety of ligands. The various thiol ligand structures for the thiolatocobalamins mentioned herein are shown below.  
                         
 
         [0010]     It is known that vitamin B 12  and its derivatives play key roles in human, animal and microbial metabolism. In humans, vitamin B 12  helps maintain healthy nerve cells and red blood cells. It is also needed to produce DNA, the genetic material in all cells. (National Institutes of Health, Office of Dietary Supplements, Dietary Fact Sheet: Vitamin B 12 ). Cobalamins (Cbls) are bound to protein in food, and hydrochloric acid in the stomach releases vitamin B 12  from proteins during digestion. Once released, Cbls combine with a protein known as salivary haptocorrin (HC, also known as R-binder). Upon pancreatic proteolytic degradation of HC, Cbl is transferred in the duodenum to intrinsic factor (IF), which can then be absorbed by the GI tract. Cbl is transferred to transcobalamin (TC, TCII) within enterocytes. A substantial portion of TC-Cbl entering the portal vein after absorption is cleared by hepatocytes. Any free Cbl entering the circulation binds to either TC or HC.  
         [0011]     Vitamin B 12  deficiencies can occur in humans in a number of circumstances. Deficiencies can occur from malabsorption problems (damage to the GI tract lining, achlorhydria, inflammatory bowel conditions, infections, lack of intrinsic factor or other genetic anomalies), lack of a diet rich in vitamin B 12 , or the inability to utilize absorbed vitamin B 12  and enzymatic or amino acid deficiencies. Certain drugs can also interfere with the absorption of vitamin B 12 .  
         [0012]     Vitamin B 12  deficiency can manifest in several different ways, including but not limited to anemias (including megaloblastic anemia also known as pernicious anemia), weakness, fatigue, weight loss, neurological changes, such as neuropathies (numbness and tingling), depression, confusion, and cognitive decline (such as loss of memory and dementia).  
         [0013]     Vitamin B 12 , along with folate and vitamin B 6 , are involved in homocysteine metabolism. Homocysteine is a non-protein amino acid reversibly formed and secreted during human metabolism. Homocysteine is, however, a neurotoxin, and an abnormal increase in plasma homocysteine levels has been implicated in many pathological conditions, such as cardiovascular disease, neural tube defects, osteoporosis, stroke and other cerebrovascular disease, peripheral vascular disease, and certain forms of glaucoma and is now recognized in Alzheimer&#39;s disease. (Tchantchou, F., “Homocysteine metabolism and various consequences of folate deficiency”.  J.Alzheimees Dis . August 2006; Vol. 9, No. 4: 421-27). Homocysteine is eliminated from the body and is regulated by the transmethylation and transsulfuration pathways.  
         [0014]     Homocysteine, among other reactive species, plays a key role in inducing oxidative stress. Oxidative stress can be defined as a harmful condition that occurs when there is an excess of free radicals, a decrease in antioxidants, or both. (E.g., Halliwell B. Introduction: Free Radicals and Human Disease—Trick or Treat? In: Thomas, C. E., Kalyanaraman, B. (ed.) Oxygen Radicals and the Disease Process. 1 st  ed. Amsterdam. Harwood Academic Publishers. 1997. pp. 1-14). Free radicals cause damage to cells by attacking their lipids, proteins and DNA components. A free radical is any species that contains one or more unpaired electrons, which makes it more reactive so that it can react with other species to form new free radicals. (Goodall, H. Oxidative stress: an overview.) It is this cycle that can lead to damage to cells in the body from prolonged exposure to free radicals.  
         [0015]     The term reactive species is used to describe free radicals and other molecules that are themselves easily converted to free radicals or are powerful oxidizing agents. (Id.) Hydrogen peroxide is another example of a reactive species found intracellularly and extracellularly in humans.  
         [0016]     It is known that a deficiency of vitamin B 12 , folate, or vitamin B 6  may increase blood levels of homocysteine. Studies have shown that the reverse is also true. It was recently reported that vitamin B 12  and folic acid supplements decreased homocysteine levels in subjects with vascular disease and in young adult women, with the most significant drop in homocysteine levels being seen when folic acid was taken alone (Bronstrup, A. et al. “Effects of folic acid and combinations of folic acid and vitamin B 12  on plasma homocysteine concentrations in healthy, young women.”  Am J Clin Nutr  1998; 68: 1104-10; Clarke, R. “Lowering blood homocysteine with folic acid based supplements.  Brit Med J  1998; 316: 894-98). It has also been reported that a significant decrease in homocysteine levels occurred in older men and women who took a multivitamin/multimineral supplement for 8 weeks (McKay, D. et al. “Multivitamin/mineral Supplementation Improves Plasma B-Vitamin Status and Homocysteine Concentration in Healthy Older Adults Consuming a Folate-Fortified Diet.”  J.Nutrition  200; 130: 309-96).  
         [0017]     A question has been raised as to whether homocysteine levels correlate with actual disease, disease risk or are simply a marker reflecting an underlying process such as oxidative stress which is responsible for both high homocysteine levels and the development of disease. (Seshadri, S. “Elevated Plasma Homocysteine Levels: Risk Factor or Risk Marker for the Development of Dementia and Alzheimer&#39;s Disease”.  J. Alzheimer&#39;s Dis . August 2006; Vol. 9, No. 4: 393-398.) Furthermore, McCaddon et al. note that these mechanisms are not necessarily mutually exclusive—for example, elevated homocysteine levels may perhaps be both a cause and consequence of oxidative stress (McCaddon et al. “Functional Vitamin B 12  deficiency and Alzheimer&#39;s Disease.  Neurology  2002; 58 (9): 1395-99).  
         [0018]     It is well-accepted that many vitamin B 12 -related conditions, regardless of cause, can be easily (and reversibly) treated by administering vitamin B 12  or its hydroxycobalamin derivative, either orally or by injection into muscle tissue. As suggested by the above studies, vitamin B 12  may also play a role in conditions associated with oxidative stress by decreasing levels of homocysteine or other reactive species.  
         [0019]     Thiolatocobalamins may also present useful therapeutic alternatives to vitamin B 12  or hydroxycobalamin administration or supplementation. McCaddon and coworkers suggested that GSCbl and related thiolatocobalamins might be more effective than currently available pharmaceutical B 12  forms (CNCbl and hydroxycobalamin) in treating of conditions associated with oxidative stress such as Alzheimer&#39;s disease (AD) and other neurological diseases (McCaddon, A., Regland, B., Hudson, P.; Davies, G.  Neurol  2002; 58: 1395-1399). Numerous studies show that oxidative stress is an important neurodegenerative element in AD and several other neurological diseases. Glutathionylcobalamin is a naturally occurring intracellular form of cobalamin and is more readily absorbed and retained longer than cyanocobalamin. It has been proposed that, in vivo, GSCbl is an intermediate in the conversion of biologically inactive cyanocobalamin to the active coenzyme forms adenosylcobalamin and methylcobalamin. The reducing agent glutathione (GSH) is required for the formation of GSCbl, and is likely to be present in lower levels in AD patients as compared with healthy individuals due to oxidative stress. Thus, GSCbl has the potential to offer a valuable, direct source of cobalamin in therapeutic applications requiring administration of a vitamin B 12  derivative. Furthermore, reduced glutathione levels are associated with a wide range of pathophysiological conditions, including liver failure, malignancies, HIV infection, pulmonary disease, and Parkinson&#39;s disease. The following list is for example purposes only and, although extensive, is not exhaustive: Acetaminophen poisoning, Attention Deficit Disorder, Autistic Spectrum Disorders, Addison&#39;s disease, aging, Acquired Immunodeficiency Syndrome, Amyotrophic lateral sclerosis, ankylosing spondylitis, arteriosclerosis, arthritis (rheumatoid), asthma, autoimmune disease, Behcet&#39;s disease, burns, cachexia, cancer, candida, cardiomyopathy, chronic fatigue syndrome, chronic obstructive pulmonary disease, chronic renal failure, colitis, coronary artery disease, cystic fibrosis, diabetes mellitus, Crohn&#39;s disease, Down&#39;s syndrome, eczema, emphysema, Epstein Barr viral syndrome, fibromyalgia, glaucoma, Goodpasture syndrome, Grave&#39;s disease, hypercholesterolaemia, herpes, viral/bacterial/fungal infections, inflammatory bowel disease, systemic lupus erythematosis, senile and diabetic macular degeneration, malnutrition, Meniere&#39;s disease, Multiple Sclerosis, Myasthenia Gravis, neurodegenerative diseases, nutritional disorders, pre-eclampsia, progeria, psoriasis, rheumatic fever, sarcoidosis, scieroderma, shingles, stroke, vasculitis and vitiligo.  
         [0020]     McCaddon and Davies recently reported on observations concerning the co-administration of N-acetyl-L-cysteine (NAC, a glutathione precursor and potent antioxidant) with B vitamin supplements in cognitively impaired patients, all of whom had high serum homocysteine levels and two of whom had low reported glutathione levels. Improvements in agitation, alertness, and cognitive function were observed in these patients. (McCaddon, A. and Davies, G. “Co-administration of N-acetylcysteine, vitamin B 12 , and folate in cognitively impaired hyperhomocysteinaemic patients.”  Int. J. Geriatr Psychiatry  2005; 20: 998-1000).  
         [0021]     McCaddon also reported more recent observations concerning additional hyperhomocysteinanemic patients with cognitive impairment. The case reports demonstrate an apparent clinical efficacy of the addition of 600 mg N-acetyl-L-cysteine (NAC) to B 12  and/or folate regimens. (McCaddon, A. “Homocysteine and cognitive impairment; a case series in a General Practice setting.”  Nutrtion Journal  2006; 5:6).  
         [0022]     In view of the potential benefits reported with the use of naturally occurring glutathionylcobalamin the use of a synthetic version of the compound glutathionylcobalamin and salts of glutathionylcobalamin are of interest as a potential treatment or supplement.  
         [0023]     An understanding of the stability of thiolatocobalamins is essential if these compounds are to be used for treatment or supplemental applications. It is also important when exploring the biological relevance of these compounds. A range of thiolatocobalamins have been synthesized, some novel, and studies have been initiated on the stability and reactivity of these compounds as well. Interestingly, the stability of a specific thiolatocobalamin is very dependent on the thiol itself, and can vary over several orders of magnitude.  
         [0024]     In efforts to reduce the damaging effects of oxidative stress and to establish the role of thiolatocobalamin treatment or supplementation in conditions associated with oxidative stress, there is a need to identify useful, stable and reactive thiolatocobalamin species. There is also a need not only for simple convenient methods of preparing thiolatocobalamins for use in human and animal studies, but also to develop test protocols that better define the role of oxidative stress (including the effects of reactive species such as homocysteine and hydrogen peroxide) in cell damage. Finally, there is a need to demonstrate the effects of naturally occurring and novel thiolatocobalamins on both healthy cells and those subjected to oxidative stress, including among other things increased homocysteine or H 2 O 2  levels, in order to identify useful therapeutic applications for thiolatocobalamins.  
       SUMMARY OF THE INVENTION  
       [0025]     It has been discovered that glutathionylcobalamin protects cells from damage and death when exposed to oxidative stress conditions. Indeed, glutathionylcobalamin may be useful for a wide range of other diseases associated with oxidative stress. Glutathionylcobalamin may also be useful for dietary supplementation.  
         [0026]     The focus of a number of studies has been on the effects of vitamin B 12  and its derivatives on homocysteine levels. It is well-recognized that the hallmark of cardiovascular disease, cerebrovascular disease and peripheral vascular disease, among others, is endothelial cell damage. By using cell model data, we have discovered that that glutathionylcobalamin, regardless of whether they are administered alone or in combination with folate, effectively protects endothelial cells against homocysteine-induced oxidative damage. Furthermore, glutathionylcobalamin protects cells against hydrogen peroxide-induced oxidative damage. Importantly, this novel vitamin B 12  derivative shows superior protection compared with the currently available pharmaceutical forms of vitamin B 12  and folate and naturally occurring cobalamins.  
         [0027]     Thus, in accordance with the invention a pharmaceutical composition or dietary supplement respectively comprising glutathionylcobalamin is provided for the treatment of conditions of oxidative stress in animals, including mammals and birds, and specifically including humans; livestock, such as beef and diary cattle, horses, pigs, goats, rabbits and poultry; and domestic animals, such as cats and dogs. The pharmaceutical composition or dietary supplement preferably also comprises a folate composition and a vitamin B 6  composition. The pharmaceutical composition may include additional ingredients as is appropriate for the form of administration, including a pharmaceutically acceptable carrier or solvent.  
         [0028]     Further the glutathionylcobalamin of the pharmaceutical composition or dietary supplement may advantageously comprise a crystalline salt of glutathionylcobalamin, and in particular may comprise a biologically accepticalable salt, such as a sodium or potassium salt of said glutathionylcobalamin.  
         [0029]     The invention further relates to a method to treat a disease or condition associated with oxidative stress comprising administering an effective amount of glutathionylcobalamin as a biologicallyagent, and preferably of a synthetic glutathionylcobalamin (including a derivative or salt thereof). This agent may be administered in combination with effective amounts of compounds known to reduce serum homocysteine levels, such as one or more of a folate compound and vitamin B6. The disease or condition associated with oxidative stress may be one or more of cardiovascular disease, cerebrovascular disease, peripheral vascular disease, glaucoma, Alzheimer&#39;s disease, dementia, and combinations thereof. The invention also relates to a method for the inhibition or reduction of free radical formation comprising administering glutathionylcobalamin (or a derivative or salt thereof, and in particular where the free radical formation is due to high hydrogen peroxide levels, or notably where the free radical is hydrogen peroxide. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0030]      FIG. 1  is a graphic representation showing the effect of homocysteine and H 2 O 2  on endothelial cell viability;  
         [0031]      FIG. 2  is a graphic representation showing that NACCbl and GSCbl protect endothelial cells from the effect of homocysteine;  
         [0032]      FIG. 3  is a graphic representation showing that NACCbl and GSCbl protect endothelial cells from the effect of H 2 O 2 ;  
         [0033]      FIG. 4  is a further graphic representation showing that NACCbl and GSCbl protect endothelial cells from the effect of homocysteine;  
         [0034]      FIG. 5  is a further graphic representation showing that NACCbl and GSCbl protect endothelial cells from the effect of H 2 O 2 ;  
         [0035]      FIG. 6  is a graphic representation showing that NACCbl and GSCbl protect endothelial cells from apoptosis induced by homocysteine;  
         [0036]      FIG. 7  is a series of plots showing the effect of oxidative stress on Hsp32 and Hsp70 gene expression in SK-HEP-1 Cells;  
         [0037]      FIG. 8  illustrates a gene expression study for oxidative stress of SK-HEP-1 Cells;  
         [0038]      FIG. 9  illustrates Hsp32 gene expression as induced by homocysteine and NACCbl;  
         [0039]      FIG. 10  illustrates Hsp32 gene expression as inhibited by quercitin;  
         [0040]      FIG. 11  illustrates that NACCbl protects endothelial cells from the effect of homocysteine via a mechanism involving Hsp70 and Hsp32;  
         [0041]      FIG. 12  also illustrates that NACCbl protects endothelial cells from the effect of homocysteine;  
         [0042]      FIG. 13  illustrates the effect of high concentrations of NACCbl and GSCbl on SK-HEP-1 cells;  
         [0043]      FIG. 14  illustrates the effect of the free thiols NA and GSH versus NACCbl or GSCbl in the absence or presence of folate on protecting SK-HEP-1 cells from Hcy;  
         [0044]      FIG. 14   a  illustrates the effects of cobalamins in the presence of folate on protecting T SK-HEP-1 cells from Hcy;  
         [0045]      FIG. 15  illustrates the effect of variable Hcy, protection with NACCbl versus Hcy and N-acetyl-cysteine versus Hcy;  
         [0046]      FIG. 16  illustrates the normalized raw statistical data normalized to Log 2;  
         [0047]      FIG. 17  illustrates the normalized raw statistical data normalized to Log 10;  
         [0048]      FIG. 18  illustrates percentage survival rate of U937 cells at a variable concentration of Hcy with the administration of various compositions; and  
         [0049]      FIG. 19  illustrates percentage survival rate of Jurkat cells at a variable concentration of Hcy with the administration of various compositions. 
     
    
     DESCRIPTION OF THE INVENTION  
       [0050]     This invention relates to a novel synthetic thiolatocobalamin, glutathionylcobalamin which can be used to protect cells against oxidative stress damage. This composition relate to a pharmaceutical composition or a dietary supplement which advantageously further comprises a folate or folate compound which is used here to includes folate and any natural isomer of reduced folate, such as (6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahydrofolic acid, 5-formyl-(6S)-tetrahydrofolic acid, 10-formyl-(6R)-tetrahydrofolic acid, 5,10-methylene-(6R)-tetrahydrofolic acid, 5,10-methenyl-(6R)-tetrahydrofolic acid, 5-formimino-(6S)-tetrahydrofolic acid, and their polyglutamyl derivatives as described in U.S. Pat. No. 5,997,915. A “pharmaceutical composition” is used herein to mean a composition in a biologically acceptable carrier as is appropriate for the means of administration and at a concentration to provide an acceptable dosage for the intended therapeutic or prophalatic result. A “dietary supplement” is used herein to mean a form that can be acceptably administered as a supplement to the customary dietary intake of the subject animal such as, for example, multivitamin preparations (with or without minerals and other nutrients); breakfast foods such as prepared cereals, toaster pastries and breakfast bars; infant formulas; dietary supplements and complete diet and weight-loss formulas and bars; animal feed (for example pet foods) and animal feed supplements (such as for poultry feed). The amount of the natural isomer of a reduced folate in a composition for human consumption can range between about 5% and about 200% of the daily requirement for folic acid per serving or dose. The animals to which the compositions can be applied for therapeutic effect are advantageously birds or mammals, such as livestock, domestic animal or most advantageously humans.  
         [0051]     The invention further relates to a method of treatment of diseases of conditions related to oxidative stress comprising administering a therapeutically effective amount of a composition comprising glutathionylcobalamin (meaning specifically glutathionylcobalamin, its derivatives and salts thereof, preferably with one or more or of a folate compound (as previously discussed) and vitamin B 6 . The term “therapeutically effective amount” as used herein refers to an amount of an “glutathionylcobalamin” sufficient to affect the symptoms due to oxidative stress, or free radical presence or inhibit free radical formation to a statistically significant degree. The term “effective amount” therefore includes, for example, an amount sufficient to prevent or treat a condition of oxidative stress, such as dementia or stroke. The dosage ranges for the administration of glutathionylcobalamin are those that produce the desired effect. Generally, the dosage will vary with the age, weight, condition, and sex of the patient. A person of ordinary skill in the art, given the teachings of the present specification, may readily determine suitable dosage ranges. The dosage can be adjusted by the individual physician in the event of any contraindications. In any event, the effectiveness of treatment can be determined by monitoring the extent of oxidative conditions or diseases by methods well known to those in the field. Moreover, the glutathionylcobalamin can be applied in pharmaceutically acceptable carriers known in the art. The glutathionylcobalamin can be used to treat conditions or diseases associated with oxidative stress in animals and in humans in vivo. The application can be oral, by injection, or topical, providing that in an oral administration the glutathionylcobalamin is preferably protected from digestion.  
         [0052]     The glutathionylcobalamin may be administered to a patient by any suitable means, including oral, parenteral, subcutaneous, intrapulmonary, topically, and intranasal administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or intravitreal administration. The glutathionylcobalamin may also be administered transdermally, for example in the form of a slow-release subcutaneous implant, or orally in the form of capsules, powders, or granules. Although direct oral administration may cause some loss of activity, the glutathionylcobalamin could be packaged in such a way to protect the active ingredient(s) from digestion by use of enteric coatings, capsules or other methods known in the art.  
         [0053]     Pharmaceutically acceptable carrier preparations for parenteral administration include sterile, aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer&#39;s dextrose, dextrose and sodium chloride, lactated Ringer&#39;s, or fixed oils. The active therapeutic ingredient may be mixed with excipients that are pharmaceutically acceptable and are compatible with the active ingredient. Suitable excipients include water, saline, dextrose, and glycerol, or combinations thereof. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer&#39;s dextrose, and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases, and the like.  
         [0054]     The glutathionylcobalamin may be formulated into therapeutic compositions as pharmaceutically acceptable salts. These salts include the acid addition salts formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, or tartaric acid, and the like. Salts also include those formed from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like.  
         [0055]     Controlled delivery may be achieved by admixing the active ingredient with appropriate macromolecules, for example, polyesters, polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, prolamine sulfate, or lactide/glycolide copolymers. The rate of release of the glutathionylcobalamin may be controlled by altering the concentration of the macromolecule.  
         [0056]     Another method for controlling the duration of action comprises incorporating the glutathionylcobalamin or a salt or derivative thereof into particles of a polymeric substance such as a polyester, peptide, hydrogel, polylactide/glycolide copolymer, or ethylenevinylacetate copolymers. Alternatively, the glutathionylcobalamin may be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, by the use of hydroxymethylcellulose or gelatin-microcapsules or poly(methylmethacrylate) microcapsules, respectively, or in a colloid drug delivery system. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.  
         [0057]     The present invention provides a method of preventing, treating, or ameliorating a disease that results from development of oxidative stress in the body, such as cardiovascular disease, neural tube defects, osteoporosis, stroke and other cerebrovascular disease, peripheral vascular disease, glaucoma, Alzheimer&#39;s disease and dementia, comprising administering to a subject at risk for a disease or displaying symptoms for such disease, an effective amount of the glutathionylcobalamin I. The present invention also provides a method of preventing, treating, or ameliorating a disease that results from an increase in free radical activity, such as inflammation, oxidative stress, rheumatoid arthritis, aging, arthrosclerosis, multiple sclerosis, asthma, inflammatory bowel disease, chronic inflammatory demyelinating polyradioculoneuritis, and cancer. The term “ameliorate” refers to a decrease or lessening of the symptoms or signs of the disorder being treated. The symptoms or signs that may be ameliorated, for example, include those associated with dementia or AD.  
         [0058]     For purposes of the inventions described herein, the structure and purity of the synthetic compound glutathionylcobalamin was characterized using UV/Vis Spectrophotometry,  1 H NMR spectroscopy, X-ray crystallography, XAS (spectrum not shown) and ES-MS (data not shown) for purposes of providing a thorough characterization of the new compound and evaluating its purity, stability and reactivity. The synthesis and characterization of glutathionylcobalamin has been previously reported in U.S. Pat. No. 7,030,105, the entire contents of which are incorporated herein by reference. %). The synthesis is carried out in aqueous solution by the addition of a small excess of thiol to a highly concentrated solution of aquacobalamin, followed by the addition of acetone to precipitate the product after completion of the reaction. The aqueous solvent is water alone. Where the reaction is carried out in a mixture of water and water miscible solvent, the proportion of water to water miscible solvent may depend on the kinetics and/or thermodynamics of the reaction. The reaction mixture may also optionally contain additional agents such as buffers, for example, MES. The resultant cobalamin derivatives may be slightly light-sensitive, therefore, preferably, the reaction is carried out under red light only conditions.  
         [0059]     The reaction may be performed at a temperature from 0° to about 60°. The reaction may The reaction is performed in an aqueous solvent, being water alone or a mixture of water and a water miscible solvent (such as MeOH, EtOH, PrOH &amp; BuOH). Preferably be carried out at ambient room temperature, such as from about 15° C. to about 30° C., for example about 20-25° C. The reaction is allowed to proceed for a time sufficient to achieve substantial completion. Reference to substantial completion of the reaction is intended to refer to the substantial consumption (e.g. greater than 95%) of the HOCbl•HX. Precipitation of the resultant products may be performed under cooling, for example ice cooling, e.g. to about −10-10° C. However, yield of the cobalamin products can be increased by the addition of a precipitate inducing solvent. The precipitate inducing solvent used to precipitate the formed product which is preferably a water miscible solvent less polar then water and includes alcohols (such as MeOH, EtOH, PrOH &amp; BuOH) and acetone, is added in an amount sufficient to induce precipitation of the formed glutothionyllcalamin. A preferred precipitate inducing solvent is acetone. In a preferred embodiment of the invention, the reaction is carried out with from 1.1 to about 2.5 equivalents of hydroxycobalamin, (“GluSH”), more preferably from about 1.2 to about 2 equivalents. Further in an alternative embodiment, the amount of hydroxycobalamin approaches saturation in the aqueous solvent, or in yet again another alternative embodiment, the reaction is run with a slight excess of GluSH and a concentration of at least about 0.025M hydroxycobalamin in the aqueous solvent is used. The synthesis is carried out under red light only conditions. Hydroxycobalamin hydrochloride (HOCbl•HCl, 98% (stated purity by manufacturer) was purchased from Fluka. The percentage of water in HOCbl•HCl (•nH 2 O) (12±2%) was determined by converting HOCbl•HCl to (CN) 2 Cbl- and the concentration of (CN) 2 Cbl- determined by UV-vis spectroscopy (Barker, H. A.; Smyth, R. D.; Weissbach, H; Toohey, J. I.; Ladd, J. N.; Volcani, B. E.  J. Biol. Chem.,  1960, 235, 480). Glutathione (GluSH, 98%; i.e., in its reduced form) was purchased from Aldrich.  1 H NMR spectra were recorded on an Inova 500 MHz spectrometer equipped with a 5 mm thermostatted (25.0±0.2° C.) probe. All solutions were prepared in D 2 O and TSP (3-(trimethylsilyl)propionic-2,2,3,3-d 4  acid, sodium salt) was used as an internal standard. Visible spectra were recorded on a Cary 1E spectrophotometer equipped with a thermostatted cell changer (25.0±0.1° C.) and operated with WinUV Bio software (version 2.00).  
         [0060]     According to this method of synthesis, a final product with greater than 90% purity, preferably greater than about 95% purity, more preferably 97, 98 or 99% purity as determined by the any of methods described herein such as, for example,  1 H NMR spectroscopy or the dicyanocobalamin test described by Barker et al.,  J. Biol. Chem.  1960, 135, 181-190 incorporated herein by reference as if fully set forth herein. The precipitated Na[NACCbl] is collected by filtration, preferably under suction, and optionally washing the precipitate with a suitable solvent or mixture of solvents such as acetone and/or ether. In another embodiment of the invention, the precipitate can be collected by decanting off the solvents or removing them by suction. Preferably, the precipitate is further dried to remove any remaining solvent. This may be carried out by under vacuum, optionally with heating (at a temperature which does not decompose the Na[NACCbl], for example from about 25-40° C.). The hydrochloride salt of hydroxycobalamin, HOCbl•HCl (81.13 mg, 5.16×10 −5  moles (12% H 2 O)) was dissolved in distilled water (0.800 ml) in a vial with gentle heating i.e. using a heat gun on a low setting. At these concentrations the solution is an intense red color and quite thick in appearance. After the addition of 0.382 ml of GluSH (0.261 M, 9.97×10 −5  moles), the vial was capped, vigorously shaken and left in the dark for 3 hr. A purple precipitate formed upon the addition of 1.00 ml acetone. After cooling in an ice bath for 30 min, the purple precipitate was filtered under suction (water aspirator), washed with acetone and ether and dried at 50° C. for 2 days under vacuum (0.13 mbar). Yield 63.6 mg (78%). Duplicate syntheses gave yields of 76 and 86%.  
         [0000]     Structure  
         [0061]     UV/Vis spectrophotometry can be used to characterize thiolatocobalamins. All spectra were recorded using a Varian Cary 5000 spectrophotometer. Data in Table 1 below showed that all thiolatocobalamins have a similar electronic spectrum with characteristic bands at 333, 372, 428 and 534 nm that are in agreement with previous reports for other thiolatocobalamins.  
                                                         TABLE 1                                   Cbl   γ max (nm)                                            GSCbl   333   372   428   534           NACCbl   333   372   428   534           HcyCbl   333   372   428   534           NACMECbl   333   372   428   534                      
 
         [0062]     The  1 H NMR spectrum of the cobalamins was also recorded (500 MHz Varian spectrometer, D 2 O, 25° C.). NACMECbl IS 2-N-acetylamino-2-carbomethoxy-L-ethanethiolatocobalamin. Thiolatocobalamins have five characteristic signals in the aromatic region (B 7 , B 2 , B 4 , R 1  and C 10  protons, see  FIG. 1  for assignment). Table 2 below summarizes the results. It can be seen that all thiolatocobalamins have similar chemical shifts, and they are in agreement with reported values.  
                                                       TABLE 2                             1 H NMR Data                Chemical Shift (ppm)                Cobalamin   B 7     B 2     B 4     R 1     C 10                         GSCbl   7.19   6.95   6.39   6.28   6.09           NACCbl   7.19   6.95   6.40   6.28   6.09           HcyCbl   7.20   6.95   6.38   6.28   6.10           NACMECbl   7.19   6.95   6.40   6.28   6.09                      
 
 Purity 
 
         [0063]     The purity of the products was also assessed by  1 H NMR spectroscopy and by the dicyanocobalamin test. All B 12  derivatives are converted to dicyanocobalamin ((CN) 2 Cbl) upon the addition of cyanide; hence, the percentage of non B 12  impurities can be determined. Table 3 below shows the results obtained.  
                                 TABLE 3                           Purity Assay                          1 H NMR           Cobalamin   (CN) 2 Cbl Test   Spectroscopy                       GSCbl   99%   98%           NACCbl   95%   98%           HcyCbl   97%   98%           NACMECbl   96%   98%                      
 
 X-Ray Diffraction Studies 
 
         [0064]     Crystals of Na[NACCbl] 18H 2 O were grown in water. Diffraction experiments were carried out on beamline BL11-1 at the Stanford Synchrotron Radiation Laboratory (SSRL). Data were collected on an ADSC Q-315 CCD detector using X-rays produced by a 26 pole wiggler insertion device, with a wavelength of 0.81798 Å (15160 eV) from a side scattering bent asymmetric cut Si (111) crystal monochromator. Table 4, below, shows bond length data for NACCbl. Bond length data for γ glutamylcysteinylcobalamin (γ-GluCysCbl) is also given for comparison purposes.  
                                                 TABLE 4                                   NACCbl   γ-GluCysCbl   Δ(NACCbl-γGluCysCbl)                                    Co—S   2.25   2.27   −0.02       Co—NB 3     2.06   2.05   +0.01       Co—N 21     1.88   1.89   −0.01       Co—N 22     1.92   1.90   +0.02       Co—N 23     1.93   1.91   +0.02       Co—N 24     1.88   1.89   −0.01                  
 
 Stability Studies 
 
         [0065]     The decomposition of GSCbl, NACCbl, and HcyCbl in PBS at 37° C. was monitored by UV/Vis spectrophotometry. Table 5 below shows t 1/2  and observed rate constant, k obs , calculated for each derivative.  
                                                           TABLE 5                                   t 1/2  (hr)   K obs  (min −1 )                                        GSCbl   No decomposition within 20 hours           NACCbl   No decomposition within 20 hours                HcyCbl   6.6   0.0018                      
 
         [0066]     After characterization, NACCbl was subjected to several experiments to determine if it offered protection to endothelial and other cells subject to oxidative stress under variable concentrations of homocysteine or H 2 O 2 . Experiments were also conducted to determine what, if any, detrimental effects NACCbl and GSCbl have on endothelial cells at increasing concentrations. While not wishing to be bound by any specific theory, experiments were conducted to determine potential mechanisms by which NACCbl&#39;s protective effects occur. Finally, experiments were conducted to determine if NACCbl offered any advantage in protection over other thiolatocobalamins, cobalamins or folate. These experiments are set forth in the examples below.  
       EXAMPLES  
     Reagents  
       [0067]     The conduct of the experiments required a number of reagents, which are set forth below. However, the experiments are not limited to the specific reagents listed, and other reagents, useful in the described methods, are well within the spirit and intention of the invention.  
       Reagents/Materials Used in Experiments  
       [0068]     The reagents, assays, kits and other materials used in the experiments are set forth in the lists below. All chemicals were obtained from Sigma-Aldrich Company Limited, Poole, Dorset, UK, unless otherwise indicated.  
       Reagents  
       [0000]    
       
         
           
              MegaCell® M.E.M. Media. Sigma. Product Code: M4067  
              DMEM without L-glutamine and phenol red. BioWhittaker, Cambrex Bioscience, Nottingham, UK.  
              FetalClone 1 Serum; Triple 0.1 μM filtered. HyClone, Logan, Utah, USA. Cat No. SH30080.03  
              5-Methyltetrahydrofolic acid disodium salt: [5-Methyl-5,6,7,8-tetrahydropteroyl-L-glutamic acid disodium salt]; C 20 H 23 N 7 Na 2 O 6 ; F.W. 503.42; EC No. 2.1.1.13  
              DL-Homocysteine: [2-Amino-4-mercaptobutyric acid] HSCH 2 CH 2 CH(NH 2 )COOH; F.W. 135.18; EC No. 207-222-9  
              Dimethyl sulfoxide: [DMSO, Methyl sulfoxide]; (CH 3 ) 2 SO; F.W. 78.13; EC No. 200-664-3  
              Pyridoxine: [Pyridoxol; Vitamin B 6 ]; C 8 H 11 NO 3 ; F.W. 169.18; EC No. 200-603-0  
              Vitamin B 12 : [CN-Cbl; Cyanocobalamin]; C 63 H 88 CoN 14 O 14 P; F.W. 1355.37; EC No. 200-680-0  
              Methylcobalamin: C 63 H 91 CoN 13 O 14 P; F.W. 1344.38; EC No. 236-535-3  
              Vitamin B 12  a: [Aquocobalamin chloride]; C 62 H 90 CIC o N 13 O 15 P; F.W. 1382.82; EC No. 261-200-3  
              Hydroxocobalamin: [Hydroxocobalamin acetate salt, Vitamin B 12 a]; C 64 H 91 CoN 13 O 16 P; F.W. 1388.39; EC No. 236-533-2  
              Bilirubin Mixed Isomers [Bilirubin IX-alpha] C 33 H 36 N 4 O 6 ; M.W. 584.68; EC No. 211-239-7  
           
         
       
     
         [0081]     Glutathionylcobalamin: GSCbl M.W. 1635.0 synthesized by the inventors as described in U.S. Pat. No. 7,030,105  
         [0082]     N-acetyl-L-cysteinylcobalamin: NACCbl M.W. 1491.0 synthesized by the inventors 
        Quercetin dehydrate: [2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one dihydrate]; C 15 H 10 O 7 .2H 2 O; F.W. 338.27; EC No. 204-187-1     Sn Protoporphyrin(IX) dihydrochloride 
            8,13-Bis(vinyl)-3,7,12,17-tetramethyl-21H, 23H-porphine-2,18dipropionic acid tin(IV) dichloride; [Sn(IX) PP]; C 34 H 32 N 4 O 4 SnCl 2 ; M.W. 750.26; Frontier Scientific Inc., Carnforth, UK.    
            Hydrogen peroxide solution: 30% (w/w): H 2 O 2 : F.W. 34.01; EC No. 231-765-0     Propidium iodide: [3,8-Diamino-5[3-(diethylmethylammonio)propyl]-6-phenyl-phenanthridinium diiodide]; C 27 H 34 I 2 N 4 ; M.W. 668.41; EC No. 247-081-0     Necrosis Inhibitor: IM 54; [2-(1H-Indol-3-yl)-3-pentylamino-maleimide]; C 19 H 23 N 3 O 2 ; M.W. 325.4; Cat No. 480060     Etoposide: [VP-16]; C 29 H 32 O 13 ; M.W. 588.6; EC No. 251-509-1; Calbiochem, Nottingham, UK.     Hemin: C 34 H 32 C 1 FeN 4 O 4 ; M.W. 651.96; EC No. 240-140-1     Trypan Blue: [Direct Blue 14]; C 34 H 24 N 6 Na 4 O 14 S 4 ; F.W. 960.81; EC No. 200-786     Z-VAD-FMK: C 22 H 30 O 7 N 3 F; M.W. 467.5     Trypsin-EDTA solution 0.25%: 0.25%, 2.5 g porcine trypsin, 0.2 g EDTA; M.W. 23.8 kDa; EC No. 3.4.21.4     N-Acetyl-L-Cysteine: [LNAC; NAC]; HSCH 2 CH(NHCOCH 3 )CO 2 H; F.W. 163.19; EC No. 210-498-3      L -Glutathione reduced: (γ-Glu-Cys-Gyl; CSH); [γ- L -Glutamyl- L -cysteinyl-glycine]; H 2 NCH(CO 2 H)CH 2 CH 2 CONHCH(CH 2 SH)CONHCH 2 CO 2 H; F.W. 307.32; EC No. 200-725-4 
 
 Assays and Kits 
    EnzoLyte™ Rh110 Caspase-3 Assay Kit; AnaSpec, San Jose, Calif., USA; Cat No.     CellTiter® Aqueous One Solution Cell Proliferation Assay; Promega Corporation, Madison, Wis., USA. 
 
 RT-PCR Reagents 
    QuickPrep micro mRNA Purification Kit; Amersham Pharmacia Biotech; Cat. No. 27-9255-01; Buckinghamshire, UK.     Ready-To-Go You-Prime First-Strand Beads; Amersham Pharmacia Biotech; Cat. No. 27-9261-01; Buckinghamshire, UK.     puReTaq™ Ready-To-Go™ PCR Beads; Amersham Biosciences; Cat. No. 27-9558-01; Piscataway, N.J. USA.     BenchTop 100 bp DNA Ladder; Promega Corporation, Madison, Wis., USA; Cat. No. G8291.     Trackit™ 100 bp DNA Ladder: 0.1 μg/μl; Invitrogen; Cat. No. 10488-058; Paisley, UK.     Agarose 1 Biotechnology Grade; Amresco, Solon, Oh., USA; Product No. 0710-500 G     Ethidium Bromide Fluorescence λ ex 530 nm; λ em 600 nm; [3,8-Diamino-5-ethyl-6-phenylphenanthridinium bromide]; C 21 H 20 BrN 3 ; F.W. 394.32; EC No. 1239-45-8; Appligene Oncor, Graffenstaden, Germany.     DAPI: [4′,6-Diamidino-2-phenylndole,dilactate]; C 22 H 27 N 5 O 6 ; F.W. 457.48     Pd(N) 6  Sodium salt; Amersham Pharmacia, N.J. USA; Cat No. 27-2166-01     Primers for RT-PCR: All primer sequences obtained from Alta Biosciences University of Birmingham, UK. 
 
 siRNA Reagents 
    Lipofectamine™ 2000 
            3:1 (w/w) liposome formulation of the polycationic lipid 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-M, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE) in membrane filtered water. Invitrogen, Paisley, UK; Cat No. 11668-019    
            DNase-, RNase- protease-free Water DEPC treated. Dihydrogen oxide H 2 O M r  18.02 filtered 0.2 μM membrane. EC No. 231-791-2; BioChemika, Fluka, Poole, UK.     RNase Removing Solution Biotechnology Grade; Amresco, Solon, Ohio. USA; Cat No. 6440     OptiMem1 Media; Invitrogen. Paisley, UK; Cat No. 31985-062     siControl RISC-Free siRNA; Dharmacon Inc. Boulder, Colo. USA; Cat No. D-001220-01-20     siControl Non-Targeting siRNA #1; Dharmacon Inc. Boulder, Colo. USA; Cat No. D-001210-01-20     siControl Tox Transfection Control; Dharmacon Inc. Boulder, Colo. USA; Cat No. D-001500-01-20     Individual siRNA Primer Sequences; Dharmacon Inc, Boulder, Colo. USA. 
 
 Cell Lines 
        A. SK-HEP-1 Cells; ECACC (European Collection of Cell Cultures); No. 91091816 
        Cell Line Name     SK-HEP-1 Human liver adenocarcinoma     Cell Line Description 
            Derived from an ascites sample from a 52 year old male suffering from adenocarcinoma of the liver. The cells have now been shown to be endothelial in origin (In Vitro 1992; 28A: 136).    
            Species:Human. Tissue:Liver. Morphology:Endothelial     Sub Culture Routine 
            Split sub-confluent cultures (70-80%) 1:2 to 1:4 i.e. seeding at 2-4×10,000 cells/cm 2  using 0.25% trypsin or trypsin/EDTA; 5% CO 2 ; 37° C.    
            Karyotype: Hyperdiploid to hypotriploid    
        B. U937 (monocytes)—Purchased from ECACC and cultured under known standard conditions.     C. Jurkat (T-cells)—T-lymphocyte leukemic cell line, purchased from ECACC and cultured under known, standard conditions. 
 
 Gene Accession 
        Heme Oxygenase-1 (decyclizing) Human EC 1.14.99.3 
            Reaction: heme+3AH 2 +3O 2 =biliverdin+Fe 2 +CO+3A+3H 2 O     Swiss-Prot: P09601 Gene Name: HMOX1 Location: Microsomal Sequence Information Length 288 M. M.W. 32819 Da. Chromosome 22q12    
            Heat Shock 70 kDa Human 
            Swiss-Prot: P17066 Gene Name: HSPA6 Location: Cytosolic     Sequence Information Length 643AA. M.W. 71028 Da. Chromosome 1q23    
            Transcription regulator protein BACH1 Human 
            Swiss-Prot: 014867 Gene Name: BACH-1 Location: Nucleus     Sequence Information Length 736 M. M.W. 81958 Da. Chromosome 21q22.11    
            78 kDA glucose-regulated protein Human 
            Swiss-Prot: P11021 Gene Name: HSPA5: GRP78 Location: Endoplasmic reticulum.     Sequence Information Length 654 M. M.W. 72333 Da. Chromosome 9q33-q34.1    
            Heat-shock protein beta-1 Human 
            Swiss-Prot: P04792 Gene Name: HSP27 Location: Cytoplasm, translocates to nucleus during heat-shock.     Sequence Information Length 205AA M.W. 22783 Da. Chromosome 7q 11.23    
            Heat shock protein HSP 90-alpha Human 
            Swiss-Prot: P07900 Gene Name: HSP90A Location: Cytoplasm     Sequence Information Length 731M. M.W. 84529 Da. Chromosome 14q32.33    
            Heat shock protein HSP 90-beta Human 
            Swiss-Prot: P08238 Gene Name: HSP90B Location: Cytoplasm     Sequence Information Length 723M. M.W. 83133 Da. Chromosome 6q12    
            Beta-actin, cytoplasmic 1 Human 
            Swiss-Prot: P60709 Gene Name: ACTB Location: Cytoplasm     Sequence Information Length 375M. M.W. 41737 Da. Chromosome 7q22.1    
            94 kDa Glucose-regulated protein Human 
            Swiss-Prot: P14625 Gene Name: GRP94 Location: Endoplasmic reticulum    
           
       
 
         [0154]     Sequence Information Length 803AA. M.W. 92469 Da. Chromosome 12q23.3 
                                                                   RT-PCR Primer Sequences.                    Sense Primer   Antisense Primer                        β-   TGC-TAT-CCC-TGT-   AGT-ACT-TGC-GCT-CAG-GAG-GA           actin   ACG-CCT-CT               Hsp 27   ATG-GCG-TGG-TGG-   CAA-AAG-AAC-ACA-CAG-GTC-GC           AGA-TCA-CC               HO-1   CAG-GCA-GAG-AAT-   GCT-TCA-CAT-AGC-GCT-GCA           GCT-GAG-TTC               Hsp 70   TTC-CGT-TTC-CAG-   CGT-TCA-GCC-CCG-CGA-TGA-CA           CCC-CCA-ATC               Hsp   AGA-AGG-TTG-AGA-   AAG-AGT-GAG-GGA-ATG-GG       90β   AGG-TGA-CAA               grp 78   GAT-AAT-CAA-CCA-   GTA-TCC-TCT-TCA-CCA-GTT-GG           ACT-GTT-AC               gp 96   TGC-CAA-GGA-AGG-   GTT-GCC-AGA-CCA-TCC-GTA-CT           AGT-GAA-GT               BACH-1   GGA-CAC-TCC-TTG-   TGA-CCT-GGT-TCT-GGG-CTC-TCA           CCA-AAT-GCA                  
 
         [0155]     Primer Sequences Used for RT-PCR. 
        DNA sequences are indicated from 5′ to 3′ terminus according to convention. Hsp and grp 78 primers from Wang et al. (1999).     HO-1, gp 96, β-actin and BACH-1 primers designed from gene sequences obtained from the NCBI website, (http://www.ncbi.nlm.nih.gov/).     Primers were designed using Primer 3 software (http:/www-genome.wi.mit.edu/cgi-bin/primer/primer3www.cqi). 
 
 Experimental Methods 
       
 
         [0159]     The methods used for cell preparation and the various tests are set forth in detail below. The reagents, including assays, kits, and cell lines are described above.  
       Cell Lines  
       [0160]     The SK-HEP-1 (ECACC No. 91091816) is a human liver adenocarcinoma cell line. It is derived from an ascites sample from a 52 year old male human suffering from adenocarcinoma of the liver. The cells have now been shown to be endothelial in origin. SK-HEP-1 cells are very sensitive to homocysteine. Jurkat (T-cells) and U937 (monocytes) cell lines were also used. Jurkat and U937 cells are more resistant to homocysteine than SK-HEP-1 cells, but do demonstrate adverse effects when exposed to homocysteine.  
       Preparation of Cell Culture Media  
       [0161]     Megacell MEM media was supplemented with 3% serum (Fetalclone® 1) and 200 mM L-glutamine. Aliquots (2 ml) were regularly transferred to a 24-well plate and examined under a light microscope for infections and integrity of the culture media.  
       Cell Culture  
       [0162]     Cells were subcultured 1.2 in culture flasks or seeded into various plates as required for experimental use. For passaging of SK-HEP-1, the medium was removed and the cells were washed with serum free medium. After addition of 1 ml or 2 ml of trypsin/EDTA 0.4% solution per 25 cm 2  or 75 cm 2  flask, respectively, the trypsin was removed after 90 seconds. The digestion was stopped after a further 3 minutes by the addition of 5 ml of fresh complete medium. For experimental use, cells between passage three and fifteen were grown in a monolayer until approx. 90% confluent in 6-, 12-, 24-, 48-, or 96-well plates. For siRNA experiments, cells between passage three and ten were grown until approximately 60-70% confluence was reached in 6-, 12-, 24-, or 48-well plates. The cells were allowed to adhere to the plastic surface of the culture vessels for a period of 24 hours prior to experimentation. Cells were grown at 37° C. in a 5% CO 2  humidified Heracell incubator.  
       Determination of Cell Counts and Viability  
       [0163]     Routine evaluation of the quality and growth rate of cultured SK-HEP-1 cells was accomplished by use of an inverted phase-contrast microscope at 100× magnification. Endothelial cells display “cobblestone” morphology at confluence. After prolonged maintenance at full confluence, these cells may acquire a ‘sprouting’ phenotype and infiltrate under other cells. Characteristics of endothelial cells include a flat irregular shape, multiple small vesicles, and pleiomorphic oval nuclei and are approximately 10-20 μm in diameter.  
       Trypan Blue Exclusion Test of Viability  
       [0164]     Regular cell counts were performed and cells were stained with Trypan Blue to determine viability and cell counts. Cells suspended in media were diluted 1:1 with Trypan Blue and incubated for 20 minutes. A cell count was performed using 20 μl of this suspension with a haemocytometer according to the manufacturer&#39;s instructions.  
       Freezing, Storage and Thawing of SK-HEP-1 Cells  
       [0165]     For long-term storage, confluent SK-HEP-1 cells were detached with trypsin/EDTA 0.4% solution. Cells were transferred to a centrifuge tube and centrifuged at 218 g for 3 minutes. The culture media was removed and the cell pellet was resuspended in 1.0 ml of freeze media (complete MegaCell media supplemented with 10% [v/v] sterile DMSO). The cell suspension was transferred to cryovials and frozen immediately at −20° C. for 24 hours, then at −80° C. for 7 days and then transferred to −96° C. in vapour phase liquid nitrogen. This procedure was performed in order to ensure gradual freezing of the cells to avoid ice-crystal formation within the cell structure. For thawing of cells, SK-HEP-1 cells were warmed quickly in a 37° C. water bath and the cell suspension was immediately transferred to a 25 cm 2  cell culture flask containing 9 ml fresh complete MegaCell MEM media. Cells were grown at 37° C. in a 5% CO 2  humidified Heracell incubator.  
       Sterilization of Equipment  
       [0166]     Filter units containing 0.2 μm filters were autoclaved for filter sterilization of all reagents used under experimental conditions in culture media.  
       Cell Treatments  
       [0167]     Cells were plated on a 96-well plate at approximately ±5000 cells per well and cultured for 24 hours. Then, subconfluent cells were exposed to test treatments for times indicated. Media was removed from test wells and replaced with 100 μl phenol-red free media containing various concentrations of test compounds. Plates were incubated for either 2 hours or 24 hours.  
       MTS Assay  
       [0168]     MTS® assay is a standard measure of cell activity. The CellTiter 96® Aqueous One Solution Cell Proliferation Assay was used according to the manufacturer&#39;s instructions. Reduction of the MS tetrazolium compound to formazan was detected by color development at 490 nm using a Bio-Tek Synergy H. T. Multi-Detection Microplate Reader, running KC-4 v 3.4 software. After treatment, all media was removed and 100 μl of fresh media was added to each well. 20 μl of the CellTiter 96® solution was added to each test well, and the plate was further incubated for 3 hours.  
       Gene Expression Experiments  
       [0169]     Cells were plated into 6-, 12-, or 24-well cell culture clusters, at 2×10 3 , 2×10 5 , or 1×10 5  cells per well and incubated until 90% confluent. Treatments were applied as above.  
         [0170]     Following incubation, cells were subjected to mRNA extraction followed by cDNA synthesis for each sample under test. Polymerase chain reaction cDNA templates were prepared for simplex PCR protocol. PCR products were then visualized using a UV Transilluminator and images captured using a Kodak ID gel imaging system. Densitometry and statistical analysis was then performed on each gene expression band image using PSP® v 10.0 running under Windows XP®.  
       Preparation of mRNA from Cell Cultures  
       [0171]     mRNA extraction was performed using the Quickprep micro mRNA Purification Kit. Following incubation of cells post-treatment, the media was removed from the cells, adherent cells were re-suspended in 0.4 ml extraction buffer and 0.8 ml elution buffer at 65° C. was added.  
         [0172]     Cell suspension was mixed and transferred to a 1 ml microcentrifuge tube. For each sample, 1 ml of oligo(dt)-cellulose was added to a separate microcentrifuge tube. The cell suspension and oligo(dt)-cellulose were centrifuged for 2 minutes at 15,130 g. Supernatant from the oligo(dt)-cellulose was removed and discarded.  
         [0173]     Subsequently, 1 ml of cleared homogenate from cell suspensions was added to the pelleted oligo(dt)-cellulose. The sample was re-suspended by inversion for 3 minutes and further mixed in a WhirliMixer for 30 seconds. This mixing step allows binding to occur between the poly-A-tail of the mRNA and the T bases on the oligo-(dt)-cellulose. The oligo(dt)-cellulose was pelleted by centrifugation at 15,130 g for 10 seconds. The supernatant was then discarded.  
         [0174]     Each sample was further re-suspended in 1 ml of HIGH salt buffer, the oligo(dt)-cellulose containing cell sample was pelleted by centrifugation at 15,130 g for 10 seconds.  
         [0175]     This HIGH salt washing step was carried out a further four times to remove cell debris.  
         [0176]     Each sample was then re-suspended in 1 ml LOW salt buffer, and oligo(dt)-cellulose pelleted by centrifugation at 15,130 g for 10 seconds. The supernatant was discarded. This LOW salt washing step was carried out three times in total.  
         [0177]     Each sample was then re-suspended in 0.5 ml LOW salt buffer and the slurry transferred to a clean microcentrifuge tube containing a spin column. Samples were centrifuged at 15,130 g for 5 seconds. The eluant was discarded and 0.5 ml LOW salt buffer was carefully added to the spin column. Samples were centrifuged at 15,130 g for 5 seconds. This final step was repeated three times in total.  
         [0178]     The spin columns were transferred to clean microcentrifuge tubes. Pre-warmed elution buffer (0.2 ml) at 65° C. was added to the spin column. Samples were further centrifuged at 15,130 g for 5 seconds. This step elutes the mRNA from the oligo(dt)-cellulose into the microcentrifuge tube. The microcentrifuge tubes were then incubated at 65° C. for ten minutes and then placed on ice to preserve the integrity of the mRNA and to prevent base pairing of the mRNA.  
       cDNA Synthesis  
       [0179]     A reaction mixture was prepared in individual microcentrifuge tubes containing 2 cDNA synthesis beads, 32 μl of the mRNA solution and 1 μl of pd(N) 6 . This mixture was incubated at 37° C. in a water bath for 60 minutes. After incubation, 27 μl of RNase-free DEPC treated water was added to each tube to make a total volume 60 μl. cDNA samples were prepared in duplicate. Sample 1 was used immediately in RT-PCR protocol. Sample 2 was prepared for qPCR protocol by the addition of 150 μl of 95% ice-cold ethanol and stored at −20° C.  
       Polymerase Chain Reaction  
       [0180]     Microcentrifuge tubes from puReTaq™Ready-To-Go™ PCR Beads containing one PCR bead were labeled for each sample required. Added to the PCR bead were 17 μl of RNase-free DEPC treated water, 1 μl sense primer, 1 μl anti-sense primer and 5 μl of the cDNA solution. RT-PCR conditions were as follows: Hsp70 and β-actin, pre-treatment step, 94° C. for 1 minute, followed by denaturing at 92° C. for 1 minute, annealing at 60° C. for 1 minute and extension at 72° C. for 1 minute. Total cycles, 30, post-treatment was then carried out at 72° C. for 10 minutes. For Hsp32, pre-treatment step, 95° C. for 2 minutes, followed by denaturing at 94° C. for 30 seconds, annealing at 58° C. for 1 minute and extension at 72° C. for 1 minute. Total cycles 45, post treatment was then carried out at 72° C. for 10 minutes. Following RT-PCR, samples were stored at −20° C.  
       Gel Electrophoresis of RT-PCR Products  
       [0181]     Agarose (0.56 g) and 0.56 ml TAE buffer (50×) was added to 27.44 ml ddH 2 O. The solution was brought to boiling point for 40 seconds in a microwave. Then, 10 μl ethidium bromide (concentration 1 mg/ml) was added and the solution swirled to mix. Ethidium bromide intercalates with RNA and therefore allows visualization of the bands under UV light. The gel solution was immediately poured into a casting chamber of the electrophoresis kit containing 8-well combs. The gel was allowed to set at room temperature for 30 minutes. The combs were then removed and 100 ml of agarose running buffer was poured into the casting chamber.  
         [0182]     Amplification products were separated on a 1.8% agarose gel (m/v) in TAE buffer. The size of the PCR products was determined by comparison to DNA fragments of a well-defined size; therefore, 5 μl of the DNA Ladder was carefully pipetted into the first well of the gel. Successive 10 μl of each PCR sample was then pipetted into subsequent wells on the agarose gel. The gel was connected to the Power Pack and run at 100V for 30 minutes.  
         [0183]     Gels were visualised on a UV Transilluminator. Photographs were stored using a Kodak Digital camera system fitted with a UV filter set connected to a PC. Images were then transferred to the Kodak ID gel imaging system. Densitometry was performed on each gene expression band using PSP™ v10 running under Windows XP®.  
       Caspase-3 Activity (Apoptosis Assay)  
       [0184]     Caspase 3 is activated when cells undergo apoptosis. Caspase-3 assay is a standard for apoptosis assay. Homocysteine is a known inducer of apoptosis. Cells were cultured in 96-well plates for 24 hours and then treated with reagents under test conditions. Cytotoxic agents, H 2 O 2  and etoposide were added to negative control wells. The assay was performed according to the manufacturer&#39;s protocol, as follows: Cells were re-suspended in 100 μl of clear media and 50 μl of Caspase-3 substrate solution was immediately added to each test well. Plates were incubated at 37° C. for 60 minutes and formation of free 7-amino-4-trifluoro-methylcoumarin (AFC) was acquired by fluorescence measurement at 496/520 nm by Microplate reader.  
       Propidium Iodide Assay (Necrosis Assay)  
       [0185]     Propidium iodide is a standard assay for necrosis. H 2 O 2  is a known inducer of necrosis. Cells were cultured in 96-well plates for 24 hours and then treated with reagents under test conditions. Cytotoxic agents, H 2 O 2  and etoposide were added to negative control wells. Cells were re-suspended in 50 μl of clear media and 50 μl of 5 μg/ml propidium iodide solution was added under red-light conditions as the propidium iodide is light sensitive. Plates were incubated at 37° C. for 20 minutes and then absorbance was measured at λex 535 nm/λem 617 nm by a Microplate reader.  
       RNA Interference RNAi  
       [0186]     siRNAs for human HO-1 were synthesized in 2′-deprotected, duplexed, desalted and purified form by Dharmacon Research Inc., published sequences from Zhang et al., (Zhang, Shan et al. 2004). Human Hsp70 primers were from proprietary sequences, and all control non-targeting primer sequences were also synthesized by Dharmacon Research Inc.  
         [0187]     First, 200 μl of the 2′-deprotection buffer was added to each 2′-ACE protected, single-stranded complementary RNA strand which was then combined, vortexed and centrifuged. The combined RNA was then incubated at 60° C. for 45 minutes in a dry-heat block. The complexes were then briefly centrifuged for 1-2 seconds and cooled at room temperature for 30 minutes to allow the RNA duplexes to anneal.  
         [0188]     Following annealing of the duplexes, 40 μl of the 10M ammonium acetate and 1.5 ml of 100% ethanol was added to 400 μl of siRNA duplex solution and vortexed. The solution was placed at −20° C. for &gt;16 hours or at −70° C. for 2 hours. Following freezing, the solution was centrifuged at 14000 g for 30 minutes at 4° C., then the supernatant was carefully pipetted away from the pellet. The pellet was then rinsed with 200 μl of cold 95% ethanol. The sample was finally dried under vacuum and then re-suspended in 1×siRNA Universal buffer and stored in small aliquots at −20° C. until used.  
         [0189]     Transfection was optimized using a standard siControl Tox protocol. All transfection experiments included non-targeted siRNA.  
         [0190]     Stock solutions of 2 μM siRNA were removed from a −20° C. freezer 30 minutes before transfection experiments. For triplicate transfections in 96-well plate format, the following master mix of reagents was prepared in RNase-free tubes for distribution of 100 μl per well:  
         [0191]     Tube 1-17.5 μl of 2 μM siRNA was added to 17.5 μl OptiMEM media. Total volume 35 μl.  
         [0192]     Tube 2-4.8 μl Lipofectamine was added to 30.2 μl OptiMEM media. Total volume 35 μl.  
         [0193]     The contents of each tube were mixed and incubated at room temperature for 20 minutes. These tubes were then combined, mixed by pipetting and further incubated for 30 minutes at room temperature. Following incubation, 280 μl OptiMEM media was added to the combined solution.  
       Forward Transfection Protocol: 2 Day Method  
       [0194]     Cells were trypsinized and plated into 12- or 96-well plates at cell density of 2×10 5 , then incubated for 24 hours until adherent. Complexed siRNA and transfection agent at 100 nm was added directly to each experimental well. Plates were incubated at 37° C. for 32.5 hours for mRNA gene analysis experiments or 72 hours for protein analysis by Western Blot.  
       Reverse Transfection Protocol: 1 Day Method  
       [0195]     Complexed siRNA and transfection agent was added to each well of either 12- or 96-well plates. Cells were then trypsinized and added directly into each test well at cell density of 2×10 5 . Plates were incubated at 37° C. for 32.5 hours for mRNA gene analysis experiments or 72 hours for protein analysis by Western Blot.  
         [0000]     Statistics  
         [0196]     GraphPad Prism™ 4.0 running under Microsoft Windows XP®. All calculations n=6. For single variable comparisons, Student&#39;s t-test was used. For multiple variable comparisons, data were analysed by one-way ANOVA with Dunnett test performed post-hoc, where data was compared to control data; p&lt;0.05 (95% confidence interval) or p&lt;0.01 (99% confidence interval) was considered significant.  
         [0000]     Results  
         [0197]     The effects of homocysteine and H 2 O 2  on endothelial cell viability were assessed first. SK-HEP-1 cells were exposed to increasing concentrations of homocysteine or H 2 O 2  (range 0-50 μM) for two hours. Cell viability was determined by MTS® assay. Values are shown in  FIG. 1  for the means ±SE of six independent samples.  FIG. 1  illustrates the effect of homocysteine and H 2 O 2  on endothelial cell viability. SK-HEP-1 cells were exposed to increasing concentrations of homocysteine or H 2 O 2  (0-50 μM) for 2 hours. Cell viability was determined by MTS® assay. Values shown are the means ±SE of 6 independent samples. The results showed that concentrations of 3 μm or higher of H 2 O 2  were sufficient to kill cultured endothelial cells (P&lt;0.001). Greater than 90% (&lt;10% survival) death was achieved by 12.5 μM H 2 O 2 . Concentrations of 5 μM and higher of homocysteine killed cells (p&lt;0.001), and greater than 90% death was achieved by 25 μM homocysteine ( FIG. 1 ).  
                                                                           TABLE 7                       n = 6                                Concentration                               μM   Homocysteine   Homocysteine   Homocysteine   Homocysteine   Homocysteine   Homocysteine               50   0.284276   −0.43099   0.944523   1.439709   3.255389   0.284276       25   1.494729   −1.31132   −0.32095   −0.48601   0.504359   0.119214       12.5   4.906009   −0.21091   0.394317   5.181112   3.860618   17.12059       6.25   62.01742   28.39982   38.2485   48.6474   67.62952   55.0298       3.125   95.96514   91.61852   82.81522   78.30353   80.94451   91.78358       1.562   100.5319   102.4026   85.18111   81.32966   81.60476   98.82622       0.781   99.76157   98.11095   102.5676   102.4576   98.71617   98.38605                    Concentration                               μM   H 2 O 2     H 2 O 2     H 2 O 2     H 2 O 2     H 2 O 2     H 2 O 2                 50   −0.02255   0.428443   −0.24805   0.067649   −0.69904   0.473542       25   3.179495   3.630486   3.630486   3.224594   3.269693   3.585387       12.5   12.42483   11.25225   10.07967   11.83854   11.74834   12.37973       6.25   32.58418   33.89206   32.31359   35.19994   32.53908   30.46452       3.125   46.88063   52.02194   50.62387   52.38274   82.7345   84.53848       1.562   96.44466   96.08387   96.44466   96.67016   98.97022   85.98165       0.781   99.42121   102.4429   98.06824   98.74472   102.3076   99.01532                  
 
         [0198]     The effect of increasing concentrations of NACCbl and GSCbl to protect endothelial cells from the effects of homocysteine was assessed. SK-HEP-1 cells were exposed to increasing concentrations of NACCbl or GSCbl for two (2) hours prior to exposure to 30 μM homocysteine for 24 hours. Cell viability was determined by MTS® assay. Values shown in  FIG. 2  are the means ±SE of six (6) independent samples. Results showed that pre-incubation with ≧2.0 μM of NACCbl protected cells from homocysteine-induced cell death.  FIG. 2  illustrates a that NACCbl and GSCbl protect endothelial cells from the effect of homocysteine. SK-HEP-1 cells were exposed to increasing concentrations of NACCbl (▪) or GSCbl (▴) for 2 hours prior to exposure to 30 μm homocysteine for 24 hours. Cell viability was determined by MTS® assay. Values shown are the means ±SE of 6 independent samples. The level of protection increased with increasing NACCbl and survival was 80% at ≧12.5 μM NACCbl. GSCbl was also effective in protecting cells, but required a higher concentration (&gt;80% survival protection required 50 μM GSCbl). At concentrations below 12.5 μM, the protection provided by NACCbl was significantly greater than that provided by GSCbl (p&lt;0.001).  
                                       TABLE 8*                           Concentration                               NACCbl   NACCbl   NACCbl   NACCbl   NACCbl   NACCbl   NACCbl               200   99.44568   100.0554   105.0998   99.55655   96.11974   99.8337       100   86.86253   88.13747   99.61198   100.0554   96.72949   91.18626       50   87.47228   89.80045   86.6408   85.25499   90.63194   93.12639       25   69.56763   79.93348   65.2439   87.08426   79.60089   87.41685       12.5   79.93348   79.93348   70.78714   78.10421   76.38582   93.45898       6.25   63.41463   75.55432   68.62527   72.56098   74.72284   68.56985       3.125   44.95566   55.93126   59.70067   60.25499   53.49224   58.75832       1.78   26.44124   20.898   32.03991   31.54102   33.75832   30.09978       0   131.153   111.918   108.7583   87.41685   95.34369   93.84702               Concentration       GSCbl   GSCbl   GSCbl   GSCbl   GSCbl   GSCbl   GSCbl               200   81.87362   76.38582   99.6674   96.72949   92.23947   93.18182       100   51.99557   87.86031   89.91132   93.51441   108.5366   132.2062       50   81.3193   103.6585   84.31264   94.62307   102.2727   95.50999       25   60.36586   65.13304   75.77605   62.74945   69.73393   104.102       12.5   56.76275   71.50776   60.47672   56.153   51.49668   75.49889       6.25   60.42128   62.02883   64.41241   52.32816   58.64745   57.76053       3.125   30.76497   14.74501   25.44346   23.61419   20.898   22.56098       1.78   14.13525   9.811529   6.485587   0.997783   11.58537   9.09091       0   132.2062   51.99557   87.86031   89.91132   93.51441   108.5366                 *Varying Concentrations of NACCbl and GSCbl, followed by exposure to 30 μM homocysteine for 24 hours.             
 
         [0199]     The effect of increasing concentrations of NACCbl and GSCbl to protect endothelial cells from the effect of H 2 O 2  was assessed. SK-HEP-1 cells were exposed to increasing concentrations of NACCbl or GSCbl for two (2) hours prior to exposure to 25 μM H 2 O 2  for 24 hours. Cell viability was determined by MTS® assay. Values shown in  FIG. 3  are the means ±SE of 6 independent samples.  FIG. 3  illustrates that NACCbl and GSCbl protect endothelial cells from the effect of H 2 O 2 . SK-HEP-1 cells were exposed to increasing concentrations of NACCbl (▪) or GSCbl (▴) for 2 hours prior to exposure to 25 μM H 2 O 2  for 24 hours. Cell viability was determined by MTS® assay. Values shown are the means ±SE of 6 independent samples. The results show that preincubation with ≧2.0 μM NACCbl protected cells from H 2 O 2 -induced cell death. There was no difference between the protection afforded by NACCbl and that by GSCbl. Both required 100 μM to achieve &gt;80% survival.  
                                                                           TABLE 9*                       n = 6                                Concentration                               NACCbl   NACCbl   NACCbl   NACCbl   NACCbl   NACCbl   NACCbl               200   100.7246   99.7365   101.8445   107.3123   92.68775   97.56258       100   61.5942   56.58762   70.22398   74.37418   64.22925   61.79184       50   56.65349   49.93412   60.2108   63.24111   57.11463   49.93412       25   46.90382   46.77207   45.25692   42.68774   45.71805   48.15547       12.5   45.25692   41.69961   44.99342   45.5863   42.22661   47.36496       6.25   42.8195   42.09486   39.39394   30.89592   27.99737   38.4058       3.125   26.8116   19.49934   35.83663   30.50066   35.24374   33.00395       1.562   4.677207   1.844532   5.270093   1.646904   4.347825   5.599474       0   128.4585   124.1107   134.3215   115.8103   128.4585   108.3663                    Concentration                               GSCbl   GSCbl   GSCbl   GSCbl   GSCbl   GSCbl   GSCbl               200   94.07115   100.0659   102.8327   93.87352   105.5995   94.72991       100   64.62451   64.62451   77.86562   71.60738   72.59553   76.5481       50   42.68774   45.71805   48.15547   46.90382   46.77207   51.84454       25   41.23847   45.5863   42.22661   45.25692   41.69961   44.99342       12.5   44.26878   54.34783   50.52701   47.03558   44.13702   42.09486       6.25   48.68248   56.25824   45.98156   43.34651   42.09486   41.96311       3.125   20.88274   30.89592   21.34387   26.8116   19.49934   29.24901       1.562   6.060607   1.119894   −1.58103   2.635046   −0.46113   −7.83926       0   117.3254   129.9737   133.5968   122.4638   120.4875   115.1515                 *Variable concentrations of NACCbl and GSCbl, followed by exposure to 25 μM H 2 O 2  for 24 hours.             
 
         [0200]     The effect of a constant concentration of NACCbl and GSCbl to protect cells against variable concentrations of homocysteine was assessed. Cells were pre-treated with NACCbl or GSCbl (30 μM) for two hours and then exposed to variable concentrations of homocysteine for a further two hours. Cell viability was determined by MTS® assay. Data shown in  FIG. 4  are representative of means ±SE of 6 independent samples.  FIG. 4  illustrates that NACCbl (▪) and GSCbl (▴) protect endothelial cells from the effect of homocysteine. Cells were pre-treated with NACCbl or GSCbl (30 μM) for two hours and then exposed to variable concentrations of homocysteine for a further two hours. Cell viability was determined by MTS® assay. Data shown are representative of means ±SE of 6 independent samples. The results show that pre-incubation of cells with 30 μM NACCbl or GSCbl protected cells from homocysteine-induced cell death. There was a decrease in protection as homocysteine concentration increased, but there was still ˜60% survival at 50 μM homocysteine.  
                                                                                                   TABLE 10*                           Variable                               Concentration   NACCbl   NACCbl   NACCbl   NACCbl   NACCbl   NACCbl       Hcy   30 μM   30 μM   30 μM   30 μM   30 μM   30 μM               200   −8.4058   −18.6473   11.11111   12.75363   4.444445   −1.25604       100   43.28503   33.42996   40.96619   44.92754   37.77778   41.15942       50   61.5459   59.22706   55.94203   57.68116   56.61837   50.5314       25   71.0145   77.68117   86.47344   83.96136   73.81644   66.2802       12.5   66.57005   67.24638   77.48792   72.36715   77.00484   66.37682       6.25   89.46861   80.86957   90.62803   78.4541   85.70049   86.18359       3.125   94.87923   85.89373   87.24638   81.35267   90.24156   84.05798       1.78   88.40581   92.85025   90.72464   88.21256   79.03381   81.64253       0.781   105.314   107.7295   97.2947   93.33334   86.85991   109.4686                    Variable                               Concentration   GSCbl   GSCbl   GSCbl   GSCbl   GSCbl   GSCbl       Hcy   30 μM   30 μM   30 μM   30 μM   30 μM   30 μM               200   5.794027   13.84914   −4.27487   6.535942   −5.86469   −16.0396       100   8.019773   20.95035   15.43896   28.05157   23.2821   33.03303       50   66.52534   53.91274   66.41935   56.35047   59.6361   51.47501       25   64.29959   52.85284   53.38278   46.70552   51.26302   54.76063       12.5   76.38226   66.73732   72.56669   77.12418   75.42837   76.80621       6.25   76.38226   76.80621   77.33616   79.03197   67.9032   80.19784       3.125   88.35894   76.9122   97.8979   89.52482   83.58947   94.71825       1.78   104.3632   94.2943   99.6997   101.5015   100.8656   99.27575       0.781   111.4644   100.9716   92.91645   97.47395   93.3404   100.9716                    Variable                               Concentration       Hcy   Control   Control   Control   Control   Control   Control               200   −3.784322   0.825153   −0.625979   2.361645   4.666382   3.300612       100   3.385972   −3.784322   −5.150093   6.032153   0.44103   −0.924741       50   7.09916   4.666382   −3.912363   7.09916   1.508038   8.934416       25   0.825153   3.215251   3.727415   7.568644   5.391947   2.745767       12.5   10.55627   14.90966   17.47048   15.16574   16.27543   17.47048       6.25   43.50548   62.37018   50.80381   43.2494   41.75558   49.69412       3.125   54.517   58.35823   58.40091   50.88917   63.01038   56.77906       1.78   63.05307   55.37061   67.5345   59.63864   67.1077   59.42524       0.781   100.441   102.2336   107.782   95.44743   98.5631   95.53279                 *Constant concentrations of NACCbl and GSCbl (30 μM), followed by variable concentrations of homocysteine for a further two hours.             
 
         [0201]     Protection of endothelial cells by NACCbl (at a constant concentration of 30 μM) was also observed when cells were exposed to variable concentrations of H 2 O 2 , however, the protection decreased below 60% survival above 25 μM H 2 O 2  ( FIG. 5 ).  FIG. 5  illustrates that NACCbl (▪) and GSCbl (▴) protect endothelial cells from the effect of H 2 O 2 . Cells were pre-treated with NACCbl or GSCbl (30 μM) for two hours and then exposed to variable concentrations of H 2 O 2  for a further two hours. Cell viability was determined by MTS® assay. Data shown are representative of means ±SE of 6 independent samples. GSCbl (at a constant concentration of 30 μM) did not provide significant protection above 7.5 μM H 2 O 2  (also  FIG. 5 ).  
                                                                                                         TABLE 11*                       n = 6                                H 2 O 2                                 Concentration   NACCbl   NACCbl   NACCbl   NACCbl   NACCbl   NACCbl       μM   30 μM   30 μM   30 μM   30 μM   30 μM   30 μM               200   5.2108   −0.61582   7.153008   −1.65798   1.989578   0.663193       100   29.98579   23.96968   19.56419   18.901   17.52724   20.89057       50   50.21317   48.27095   44.955   45.71293   48.17622   44.57603       25   67.12459   66.74561   61.86642   56.18191   55.3766   55.94505       12.5   77.16722   76.97774   76.59877   67.12459   77.6883   74.13548       6.25   88.20464   87.58881   82.14117   81.57272   78.06727   77.11985       3.125   88.20464   93.17859   92.89436   99.76315   92.42065   91.80482       1.56   98.53151   91.37849   98.72099   100.1421   89.76788   96.11559       0.781   93.46281   102.7949   99.24207   101.3738   103.2686   95.45239       0   102.9844   100.8053   103.2212   107.9583   101.8948   98.43676                    H 2 O 2                                 Concentration   GSCbl   GSCbl   GSCbl   GSCbl   GSCbl   GSCbl       μM   30 μM   30 μM   30 μM   30 μM   30 μM   30 μM               200   1.326385   7.153008   3.60019   1.942207   5.921364   0.757935       100   10.80057   12.36381   11.5585   9.995263   6.679299   8.526765       50   15.01658   20.8432   18.94837   13.12174   15.6324   17.62198       25   23.54334   26.52771   22.92752   24.44339   22.02748   20.41686       12.5   29.7963   32.02274   36.09664   32.40171   29.98579   33.91758       6.25   36.52297   39.7442   34.48603   34.72288   34.86499   36.09664       3.125   40.31265   91.33112   87.3046   82.33065   79.77262   77.59356       1.56   93.55756   90.81004   92.32591   88.25201   90.81004   91.8522       0.781   96.11559   98.38939   97.3946   98.0578   96.02085   95.31028       0   99.24207   102.2264   105.5898   103.6002   104.0265   104.5476                    H 2 O 2                                 Concentration       μM   Control   Control   Control   Control   Control   Control               200   4.263382   −1.61061   −0.52108   −2.17906   1.56324   −1.51587       100   1.326385   0.757935   4.547607   3.458076   5.447654   6.679299       50   3.837044   7.153008   −1.56324   3.60019   3.837044   2.321175       25   19.42208   10.27949   13.73757   19.75367   15.58503   8.810989       12.5   34.86499   40.31265   44.71814   30.22264   32.49645   28.61203       6.25   72.38276   73.04596   67.17196   72.28801   78.63572   69.58788       3.125   87.92043   87.54145   83.98862   90.81004   84.41497   87.25723       1.56   93.46281   98.29465   99.33681   101.0422   104.6897   103.2212       0.781   93.46281   102.3212   96.11559   100.8053   103.2686   101.5632       0   110.2795   108.8584   109.9479   113.6902   109.9479   114.35354                 *Constant concentrations of NACCbl at 30 μM, followed by exposure to variable concentrations of H 2 O 2 .             
 
         [0202]     The effects of NACCbl, GSCbl and folate to protect endothelial cells from apoptosis induced by homocysteine were also assessed. Homocysteine-induced apoptosis in cells was measured by Caspase-3 activity ( FIG. 6 ).  FIG. 6  illustrates that NACCbl, GSCbl and folate protect endothelial cells from apoptosis induced by homocysteine. Cells were pre-treated with NACCbl, GSCbl or folate for two hours and then exposed to 0 or 30 μM homocysteine for a further two hours. Cell viability was determined by MTS® assay. Data shown are representative of means ±SE of 6 independent samples. Cells were pretreated with NACCbl, GSCbl or folate (all at 30 μM) for two hours and then exposed to 0 or 30 μM homocysteine for a further two hours. Cell viability was determined by MTS® assay. Data shown in  FIG. 6  are representative of means ±SE of 6 independent samples. The results showed that pre-incubation with folate provided partial protection against apoptosis induced by homocysteine, whereas both NACCbl and GSCbl provided total protection.  
         [0203]     Efforts were made to elicit the mechanism by which NACCbl affords protection to endothelial cells. Experiments were conducted using Hsp32 and Hsp 70 gene expression as the basis for study. Homocysteine is an oxidative stress inducer and as such should induce the expression of heat shock protein (Hsp) Hsp32.  FIG. 7  shows the results of oxidative stress on Hsp32 and Hsp 70 gene expression with no treatment, Hcy 30 μM, folate 30 μM, folate 30 μM plus Hcy 30 μM, and heat shock at 42° C. (all treatments for 2 hours). The densitometric data are presented in  FIG. 6  as means ±SEM of n=3 separate experiments (p&lt;0.05 or p&lt;0.01, treatment vs. control). The results showed that the Hsp70 gene was expressed in control cells (no treatment), whereas the Hsp32 gene was not ( FIG. 7 ).  FIG. 7  illustrates that the effect of oxidative stress on Hsp32 and Hsp70 gene expression in SK-HEP-1 Cells. All panels: 1, no treatment; 2: Hcy 30 μM, 2 hr; 3: folate 30 μM, 2 hr; 4: folate 30 μM, 2 hr+Hcy 30 μM, 2 hr; 5: Heat shock 42° C., 2 hr. The densitometric data are presented as means ±SEM of n=3 separate experiments. *p&lt;0.05 or **p&lt;0.01, treatment vs. control. An increase in expression of both genes was induced by 30 μM homocysteine, by 30 μM folate and by folate plus homocysteine (both at 30 μM concentration). A 42° C. heat shock increased expression of Hsp70, but not Hsp32, confirming that Hsp32 is specifically induced by oxidative stress.  FIG. 8  illustrates a Gene Expression Study: Oxidative stress of SK-HEP-1 Cells. Panel A, B and C: Lane 1 shows the control, no treatment; lane 2: H 2 O 2  25 μM, 2 hr; lane 3: folate 30 μM, 2 hr+H 2 O 2  25 μM, 1 hr; lane 4: Sn(IX) PP 25 μM, 2 hr; lane 5: Sn(IX) PP 25 μM, 2 hr+folate 30 μM, 2 hr+H 2 O 2  25 μM, 1 hr; lane 6: Heat shock 42° C., 2 hr. Panel B shows fold induction difference in β-actin gene expression from Control=1.  
         [0204]      FIG. 9  illustrates that Hsp32 gene expression is induced by homocysteine and NACCbl, and can be inhibited by Sn(IX) protoporphyrin. SK-HEP-1 cells were treated with various compounds prior to PCR. (note HO1=Hsp32).  FIG. 9  also shows that the Hsp32 gene can be inhibited by Sn (IX) protoporphyrin (25 μM). Other molecules, including NACCbl and GSCbl, that protect against homocysteine-induced cell death also induce Hsp32 ( FIGS. 7, 9  and  10 ). Quercitin 15 μM inhibits Hsp70 gene expression, but does not inhibit Hsp32 gene expression ( FIGS. 9 and 10 ).  
         [0205]     Two alternative approaches were used to determine whether Hsp32 or Hsp70 have a role in the mechanism by which NACCbl protects against homocysteine induced cell death. Hsp32 was inhibited using either Sn (IX) protoporhyrin or using an siRNA which specifically knocks out Hsp32. Hsp70 was inhibited using quercitin or using an siRNA which specifically knocks out Hsp70. In addition, two methods were used to inhibit both Hsp32 and Hsp70: chemically Sn (IX) protoporphyrin plus quercitin was used; and then, directly, a combination of the siRNA for both genes was used simultaneously.  
         [0206]     In one set of experiments, cells were pre-treated with NACCbl (30 μM) for two hours in the presence or absence of Sn (IX) protoporphyrin or quercitin and then exposed to variable concentrations of homocysteine for a further two hours. Cell viability was determined by MTS® assay. Data shown in  FIG. 11  are representative of means ±SE of 6 independent samples. In another set of experiments, cells were pre-treated NACCbl (30 μM) for two (2) hours in the presence or absence of siRNA specific for Hsp32 or Hsp70 and then exposed to variable concentrations of homocysteine for a further two hours.  FIG. 11  illustrates that NACCbl protects endothelial cells from the effect of homocysteine via a mechanism involving Hsp70 and Hsp32. Cells were pre-treated with NACCbl (30 μM) for two hours in the presence or absence of Sn(IX) protoporphyrin or quercitin and then exposed to variable concentrations of homocysteine for a further two hours. Cell viability was determined by MTS® assay. Data shown are representative of means ±SE of 6 independent samples.  FIG. 12  illustrates NACCbl protects endothelial cells from the effect of homocysteine. Cells were pre-treated NACCbl (30 μM) for two hours in the presence or absence of siRNA specific for Hsp70 and then exposed to variable concentrations of homocysteine for a further two hours. Cell viability was determined by MTS® assay. Data shown are representative of means ±SE of 6 independent samples. Cell viability was determined by MTS® assay. Data shown are representative of means ±SE of 6 independent samples. The results showed that inhibition of Hsp32 by both methods (chemically and directly) reduced the protection by NACCbl by 10-20% (p&lt;0.001,  FIGS. 11 and 12 ). Inhibition of Hsp70 by both methods (chemically and directly) reduced the protection by NACCbl by 20-40% (p&lt;0.001,  FIGS. 11 and 12 ). Inhibition of both Hsp32 and Hsp70 together by both methods (chemically and directly) totally removed protection by NACCbl ( FIGS. 11 and 12 ).  
                                                                                                                                                                                                                       TABLE 12                       ( FIG. 11 )                                Variable   NACCbl + Hcy (N = 6)            Conc. of Hcy   1   2   3   4   5   6               200   1.378   1.167   1.33   1.456   1.491   1.537       100   1.678   1.592   1.694   1.773   1.732   1.639       50   1.678   1.832   1.881   1.73   1.74   1.855       25   2.1   2.19   1.879   1.937   1.779   1.837       12.5   2.21   2.11   2.045   2.065   2.101   2.324       6.25   2.41   2.4   2.322   2.201   2.115   2.187       3.125   2.453   2.338   2.36   2.317   2.418   2.535       1.78   2.647   2.551   2.789   2.89   2.549   2.611       0   2.885   2.698   2.821   2.771   2.691   2.7                    Variable   Variable Hcy (n = 6)            Conc. of Hcy   1   2   3   4   5   6               200   0.33   0.312   0.39   0.302   0.421   0.406       100   0.654   0.449   0.466   0.501   0.522   0.51       50   0.678   0.881   0.602   0.77   0.724   0.779       25   0.692   0.956   0.876   0.882   0.937   0.74       12.5   1.104   1.001   1.042   1.114   1.324   1.479       6.25   1.304   1.406   1.227   1.176   1.15   1.119       3.125   1.543   1.487   1.656   1.63   1.593   1.558       1.78   1.746   1.932   1.95   1.83   1.837   1.884       0   2.109   2.11   1.999   2.056   2.1   1.936                    Variable   NACCbl + Sn(IX)PP + Hcy (n = 6)            Conc. of Hcy   1   2   3   4   5   6               200   0.679   0.837   0.882   0.91   0.723   0.577       100   1.29   1.117   1.104   1.002   0.994   1.336       50   1.488   1.47   1.43   1.379   1.337   1.402       25   1.876   1.749   1.678   1.63   1.557   1.921       12.5   2.123   2.301   2.244   2.221   2.109   2.008       6.25   2.443   2.331   2.156   2.004   2.078   2.14       3.125   2.578   2.432   2.29   2.278   2.21   2.11       1.78   2.567   2.69   2.733   2.779   2.821   2.879       0   2.6788   2.897   2.701   2.857   2.891   2.945                    Variable   NACCbl + Quercetin + Hcy (n = 6)            Conc. of Hcy   1   2   3   4   5   6               200   0.573   0.423   0.444   0.301   0.567   0.583       100   1.111   1.678   0.732   0.883   0.891   0.693       50   1.021   1.247   1.207   1.197   1.177   1.022       25   1.478   1.501   1.376   1.321   1.297   1.25       12.5   2.123   2.116   2.046   2.1   2.187   1.794       6.25   2.467   2.409   2.337   2.365   2.401   2.588       3.125   2.765   2.631   2.661   2.589   2.603   2.509       1.78   2.678   2.889   2.921   2.703   2.831   2.899       0   2.991   2.851   2.956   2.756   2.764   2.678                    Variable   NACCbl + Sn(IX)PP + Quercetin + Hcy (n = 6)            Conc. of Hcy   1   2   3   4   5   6               200   0.278   0.389   0.41   0.336   0.333   0.417       100   0.367   0.401   0.476   0.507   0.551   0.367       50   0.746   0.478   0.539   0.337   0.439   0.551       25   1.123   1.034   0.786   1.809   1.922   0.664       12.5   1.678   1.89   1.709   1.902   1.834   1.88       6.25   2.123   2.39   2.167   2.117   2.17   1.963       3.125   2.456   2.389   2.301   2.489   2.447   2.567       1.78   2.789   2.657   2.489   2.699   2.798   2.662       0   2.888   2.79   2.901   2.979   2.991   2.804                  
 
         [0207]    
       
         
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
               
             
               
               
               
             
           
               
                 TABLE 13 
               
             
             
               
                   
               
               
                   
               
               
                 ( FIG. 12 ) 
               
             
          
           
               
                 Variable 
                   
                   
               
               
                 Conc. of 
               
               
                 Hcy 
                 Mean 
                 SEM 
               
               
                   
               
             
          
           
               
                 NACCbl + Hcy 
               
             
          
           
               
                 200 
                 43.9595 
                 2.101288 
               
               
                 100 
                 55.16888 
                 1.013695 
               
               
                 50 
                 59.06556 
                 1.271858 
               
               
                 25 
                 73.75504 
                 1.724869 
               
               
                 12.5 
                 82.83664 
                 1.460632 
               
               
                 6.25 
                 89.85452 
                 2.128654 
               
               
                 3.125 
                 91.34142 
                 1.305542 
               
               
                 1.78 
                 97.04544 
                 1.198051 
               
               
                 0 
                 100 
                 0.929276 
               
             
          
           
               
                 Variable Hcy 
               
             
          
           
               
                 200 
                 6.114708 
                 1.68879 
               
               
                 100 
                 14.81961 
                 1.644149 
               
               
                 50 
                 27.14154 
                 2.160151 
               
               
                 25 
                 33.14524 
                 2.430397 
               
               
                 12.5 
                 51.47086 
                 4.194286 
               
               
                 6.25 
                 54.41258 
                 2.442547 
               
               
                 3.125 
                 73.70027 
                 1.393837 
               
               
                 1.78 
                 89.53746 
                 1.702477 
               
               
                 0 
                 100 
                 1.608582 
               
             
          
           
               
                 NACCbl + RNAi + Hsp 32 + Hcy 
               
             
          
           
               
                 200 
                 22.71906 
                 2.604406 
               
               
                 100 
                 45.81881 
                 1.243889 
               
               
                 50 
                 55.51684 
                 2.269596 
               
               
                 25 
                 70.24131 
                 1.135635 
               
               
                 12.5 
                 79.12633 
                 1.428551 
               
               
                 6.25 
                 85.54007 
                 2.120552 
               
               
                 3.125 
                 88.81791 
                 2.042473 
               
               
                 1.78 
                 91.044 
                 1.296631 
               
               
                 0 
                 100 
                 2.045876 
               
             
          
           
               
                 NACCbl + RNAi + Hsp 70/32 + Hcy 
               
             
          
           
               
                 200 
                 4.156479 
                 0.830308 
               
               
                 100 
                 7.328696 
                 1.192364 
               
               
                 50 
                 20.61939 
                 1.645706 
               
               
                 25 
                 47.21334 
                 3.858868 
               
               
                 12.5 
                 58.88659 
                 1.501209 
               
               
                 6.25 
                 60.86139 
                 2.375267 
               
               
                 3.125 
                 74.12074 
                 1.424911 
               
               
                 1.78 
                 83.07944 
                 2.454529 
               
               
                 0 
                 100 
                 1.008469 
               
             
          
           
               
                 NACCbl + RNAi + Hsp 70 + Hcy 
               
             
          
           
               
                 200 
                 23.74355 
                 0.881615 
               
               
                 100 
                 24.32296 
                 1.313255 
               
               
                 50 
                 48.75299 
                 1.06419 
               
               
                 25 
                 57.9481 
                 2.211338 
               
               
                 12.5 
                 75.53218 
                 1.459312 
               
               
                 6.25 
                 80.8918 
                 0.7901 
               
               
                 3.125 
                 83.51177 
                 1.823074 
               
               
                 1.78 
                 86.53482 
                 1.290047 
               
               
                 0 
                 100 
                 1.468781 
               
               
                   
               
             
          
         
       
     
         [0208]     The concentrations needed for clinical treatments and supplementation may well need to be higher than those used for the cellular experiments described herein. Equally important to the use of thiolatocobalamins to protect endothelial and other cells from the effects of oxidative stress is their safety or lack of a detrimental effect on exposed cells. The effect of high concentrations of NACCbl and GSCbl on SK-HEP-1 cells was evaluated. Cells were exposed to both compounds in increasing concentrations over twenty-four (24) hours. Cell viability was determined by MTS® assay. Data shown in  FIG. 13  are representative of means ±SE of 3 independent samples.  FIG. 13  illustrates that the effect of high concentrations of NACCbl and GSCbl on SK-HEP-1 cells. Cells were exposed to the compounds for 24 hours and cell viability was determined by MTS® assay. Data shown are representative of means ±SE of 3 independent samples. The results showed that increasing the concentrations of NACCbl and GSCbl did not affect cell viability until reaching concentrations above 0.2 mM. Above this concentration, there was a decrease in survival but even at 10 mM over 60% survival was observed in the NACCbl treated cells ( FIG. 13 ). Above 0.2 mM concentration, GSCbl caused greater cell death than NACCbl ( FIG. 13 ).  
                                                                     TABLE 14                       Variable                               Conc.   NACCbl   NACCbl   NACCbl   GSCbl   GSCbl   GSCbl                                10000   1.908   1.82   1.732   1.322   1.342   1.366       5000   2.081   2.003   2.004   1.728   1.67   1.623       2500   1.989   1.891   2.085   1.775   1.81   1.896       1250   2.2   2.055   2.3   1.973   1.846   1.71       625   2.358   2.242   2.347   2.197   1.912   1.787       312.5   2.409   2.301   2.437   2.306   2.204   2.138       156.25   2.484   2.42   2.492   2.407   2.37   2.318       78.125   2.521   2.488   2.475   2.444   2.406   2.402       39.062   2.503   2.474   2.453   2.492   2.484   2.468       19.531   2.485   2.466   2.502   2.553   2.476   2.42       9.765   2.505   2.474   2.514   2.532   2.476   2.42       4.882   2.532   2.463   2.457   2.543   2.528   2.519       2.441   2.592   2.536   2.499   2.527   2.434   2.46       1.22   2.546   2.524   2.538   2.347   2.433   2.503       0.61   2.572   2.498   2.473   2.554   2.376   2.53       0   2.61   2.688   2.634   2.487   2.441   2.52                  
 
         [0209]     A direct comparison of the protection against homocysteine-induced damage demonstrated that NACCbl and GSCbl are superior to the free thiols. (FIGS.  14 ,  14 ( a ).  FIG. 14  illustrates the effect of the free thiols NAC (45 μm) and GSH (100 μM) versus NACCbl (12.5 μM) or GSCbl (15 μM) in the absence or presence of folate (25 μm) on protecting SK-HEP-1 cells from Hcy (30 μM).  FIG. 14   a  illustrates the effects of cobalamins in the presence of folate (25 μM) on protecting T SK-HEP-1 cells from Hcy (30 μM). GSCbl=15 μM; NACCbl=12.5 μM; CNCbl=15.0 μM; MeCbl=12.5 μM; HOCbl=15.5 μM. The protective effects of NAC (45 μM) and GSH (100 μM) in the presence of folate (25 μM) are also shown for comparison purposes. The data is set forth below.  
                                                                             Condition   [Hcy] (μM)   Mean   SDM               Control   0   3.06117   0.10953       Hcy   30   0.616   0.09193       NAC   30   1.29633   0.07974       NACCbl   30   1.69583   0.11965       NAC + Folate   30   1.43783   0.08293       NACCbl + Folate   30   2.66117   0.09405                    [NAC] = 45 μM       [Folate] = 25 μM       [NACCbl] = 12.5 μM            Condition   [Hcy] (μM)   Mean   SDM               Control   0   3.06117   0.10953       HCy   30   0.616   0.09193       GSCbl   30   2.52433   0.15311       NACCbl   30   2.66117   0.09405       CNCbl   30   1.38333   0.12226       MeCbl   30   1.32217   0.10209       HOCbl   30   0.94983   0.10809       NAC   30   1.43783   0.08293       GSH   30   1.40933   0.09033                    [GSH] = 100 μM       [NAC] = 45 μM       [Folate] = 25 μM       [GSCbl] = 15 μM; [NACCbl] = 12.5 μM; [CNCbl] = 15 μM;       [MeCbl] = 12.5 μM; [HOCbl] = 17.5 μM.          
 
         [0210]     A comparison of the protective effects of NACCbl, NAC and cyanocobalamin+NAC against variable Hcy concentration is shown in  FIG. 15 . The results demonstrate that NACCbl (30 μM) is superior to either NAC (75 μM) alone or in combination with CNCbl (cyanocobalamin+NAC) in preventing cell death. Table 15 and Table 15 (a) below show the results for cell death as characterized by absorbance at 490 nm for NACCbl, variable Hcy, NAC and cyanocobalamin+NAC. The data was normalized (log 2 and log 10) as shown in  FIGS. 16 and 17  and Tables 16 and 17, respectively, below. Normalization reflects that protection with NACCbl is superior to that of NAC or cyanocobalamin+NAC.  
                                                                                                                                                                               TABLE 15                       Absorbance at 490 nm (n = 6)                                Variable   Variable Hcy            Conc. of Hcy   Y1   Y2   Y3   Y4   Y5   Y6               200.000   0.330   0.312   0.390   0.302   0.421   0.406       100.000   0.654   0.449   0.466   0.501   0.522   0.510       50.000   0.678   0.881   0.602   0.770   0.724   0.779       25.000   0.692   0.956   0.876   0.882   0.937   0.740       12.500   1.104   1.001   1.042   1.114   1.324   1.479       6.250   1.304   1.406   1.227   1.176   1.150   1.119       3.125   1.543   1.487   1.656   1.630   1.593   1.558       1.780   1.746   1.932   1.950   1.830   1.837   1.884       0.000   2.109   2.110   1.999   2.056   2.100   1.936                    Variable   NACCbl + Hcy            Conc. of Hcy   Y1   Y2   Y3   Y4   Y5   Y6               200.000   1.378   1.167   1.330   1.456   1.491   1.537       100.000   1.678   1.592   1.694   1.773   1.732   1.639       50.000   1.678   1.832   1.881   1.730   1.740   1.855       25.000   2.100   2.190   1.879   1.937   1.779   1.837       12.500   2.210   2.110   2.045   2.065   2.101   2.324       6.250   2.410   2.400   2.322   2.201   2.115   2.187       3.125   2.453   2.338   2.360   2.317   2.418   2.535       1.780   2.647   2.551   2.789   2.890   2.549   2.611       0.000   2.885   2.698   2.821   2.771   2.691   2.700                    Variable   N-Acetyl-L-cysteine + Hcy            Conc. of Hcy   Y1   Y2   Y3   Y4   Y5   Y6               200.000   0.567   0.668   0.623   0.490   0.721   0.788       100.000   0.789   0.842   0.743   0.589   0.490   0.799       50.000   0.890   0.821   1.034   1.227   0.799   1.045       25.000   1.345   1.501   1.632   1.378   1.226   1.447       12.500   1.675   1.232   1.339   1.590   1.602   1.622       6.250   1.678   1.789   1.897   1.899   1.933   1.905       3.725   1.890   2.003   1.784   1.946   1.958   1.801       1.780   2.225   1.903   1.947   2.310   2.187   2.206       0.000   2.592   2.476   2.437   2.678   2.336   2.447                    Variable   Cyanocobalamin + NAC + Hcy            Conc. of Hcy   Y1   Y2   Y3   Y4   Y5   Y6               200.000   0.345   0.421   0.456   0.567   0.378   0.471       100.000   0.789   0.634   0.456   0.447   0.336   0.490       50.000   0.890   0.576   0.678   0.732   0.602   0.693       25.000   1.345   0.898   0.936   1.121   1.044   1.117       12.500   1.675   1.056   1.210   1.226   1.402   1.339       6.250   1.678   1.538   1.336   1.669   1.722   1.590       3.725   1.890   1.578   1.773   1.609   1.793   1.993       1.780   2.225   1.567   1.875   1.690   1.884   1.921       0.000   2.592   2.389   2.449   2.201   2.108   2.557                  
 
         [0211]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 15(a) 
               
             
             
               
                   
               
               
                   
               
               
                 Statistical Analysis (n = 6) 
               
               
                 Absorbance at 490 nm 
               
             
          
           
               
                   
                   
                   
                 N-Acetyl-L- 
                 Cyanoco- 
               
               
                   
                 NACCbl + 
                   
                 cysteine + 
                 balamin + 
               
               
                 Variable 
                 Hcy 
                 Variable Hcy 
                 Hcy 
                 NAC + Hcy 
               
             
          
           
               
                 Conc. Hcy 
                 Mean 
                 SEM 
                 Mean 
                 SEM 
                 Mean 
                 SEM 
                 Mean 
                 SEM 
               
               
                   
               
             
          
           
               
                 200.000 
                 1.393 
                 0.055 
                 0.360 
                 0.021 
                 0.643 
                 0.044 
                 0.440 
                 0.032 
               
               
                 100.000 
                 1.685 
                 0.026 
                 0.517 
                 0.030 
                 0.709 
                 0.057 
                 0.525 
                 0.066 
               
               
                 50.000 
                 1.786 
                 0.033 
                 0.739 
                 0.039 
                 0.969 
                 0.067 
                 0.695 
                 0.046 
               
               
                 25.000 
                 1.954 
                 0.065 
                 0.847 
                 0.044 
                 1.421 
                 0.057 
                 1.077 
                 0.065 
               
               
                 12.500 
                 2.142 
                 0.043 
                 1.177 
                 0.076 
                 1.510 
                 0.073 
                 1.318 
                 0.086 
               
               
                 6.250 
                 2.273 
                 0.050 
                 1.230 
                 0.044 
                 1.850 
                 0.040 
                 1.589 
                 0.057 
               
               
                 3.125 
                 2.404 
                 0.033 
                 1.578 
                 0.025 
                 1.897 
                 0.036 
                 1.773 
                 0.065 
               
               
                 1.780 
                 2.673 
                 0.056 
                 1.863 
                 0.031 
                 2.130 
                 0.067 
                 1.860 
                 0.092 
               
               
                 0.000 
                 2.761 
                 0.032 
                 2.052 
                 0.029 
                 2.494 
                 0.050 
                 2.383 
                 0.079 
               
               
                   
               
             
          
         
       
     
         [0212]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 16 
               
             
             
               
                   
               
               
                   
               
               
                 Normalized Data Log 2 (n = 6) 
               
             
          
           
               
                   
                   
                   
                 N-Acetyl-L- 
                 Cyanocobalamin + 
               
               
                 Variable 
                 NACCbl + Hcy 
                 Variable Hcy 
                 cysteine + Hcy 
                 NAC + Hcy 
               
             
          
           
               
                 Conc. Hcy 
                 Mean 
                 SEM 
                 Mean 
                 SEM 
                 Mean 
                 SEM 
                 Mean 
                 SEM 
               
               
                   
               
             
          
           
               
                 200.000 
                 40.781 
                 2.159 
                 −0.033 
                 0.832 
                 11.135 
                 1.727 
                 3.108 
                 1.261 
               
               
                 100.000 
                 52.298 
                 1.042 
                 6.164 
                 1.170 
                 13.738 
                 2.233 
                 6.493 
                 2.595 
               
               
                 50.000 
                 56.302 
                 1.307 
                 14.935 
                 1.538 
                 24.035 
                 2.637 
                 13.203 
                 1.803 
               
               
                 25.000 
                 62.926 
                 2.573 
                 19.208 
                 1.730 
                 41.900 
                 2.249 
                 28.283 
                 2.585 
               
               
                 12.500 
                 70.387 
                 1.704 
                 32.253 
                 2.986 
                 45.397 
                 2.896 
                 37.811 
                 3.412 
               
               
                 6.250 
                 75.524 
                 1.973 
                 34.347 
                 1.739 
                 58.837 
                 1.578 
                 48.512 
                 2.265 
               
               
                 3.125 
                 80.699 
                 1.323 
                 48.077 
                 0.992 
                 60.687 
                 1.432 
                 55.775 
                 2.575 
               
               
                 1.780 
                 91.341 
                 2.227 
                 59.351 
                 1.212 
                 69.880 
                 2.655 
                 59.239 
                 3.625 
               
               
                 0.000 
                 94.824 
                 1.284 
                 66.798 
                 1.145 
                 84.288 
                 1.965 
                 79.876 
                 3.120 
               
               
                   
               
             
          
         
       
     
         [0213]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 17 
               
             
             
               
                   
               
               
                   
               
               
                 Normalized Data Log 10 (n = 6) 
               
             
          
           
               
                   
                   
                   
                 N-Acetyl-L- 
                 Cyanocobalamin + 
               
               
                 Variable 
                 NACCbl + Hcy 
                 Variable Hcy 
                 cysteine + Hcy 
                 NAC + Hcy 
               
             
          
           
               
                 Conc. Hcy 
                 Mean 
                 SEM 
                 Mean 
                 SEM 
                 Mean 
                 SEM 
                 Mean 
                 SEM 
               
               
                   
               
             
          
           
               
                 200.000 
                 45.526 
                 2.176 
                 6.115 
                 1.169 
                 17.503 
                 1.948 
                 8.893 
                 1.497 
               
               
                 100.000 
                 57.135 
                 1.050 
                 14.820 
                 1.644 
                 20.439 
                 2.518 
                 12.910 
                 3.079 
               
               
                 50.000 
                 61.171 
                 1.317 
                 27.142 
                 2.160 
                 32.051 
                 2.974 
                 20.874 
                 2.140 
               
               
                 25.000 
                 67.848 
                 2.594 
                 33.145 
                 2.430 
                 52.198 
                 2.537 
                 38.770 
                 3.067 
               
               
                 12.500 
                 75.368 
                 1.717 
                 51.471 
                 4.194 
                 56.141 
                 3.266 
                 50.078 
                 4.049 
               
               
                 6.250 
                 80.546 
                 1.989 
                 54.413 
                 2.443 
                 71.298 
                 1.779 
                 62.777 
                 2.688 
               
               
                 3.125 
                 85.763 
                 1.333 
                 73.700 
                 1.394 
                 73.385 
                 1.615 
                 71.397 
                 3.056 
               
               
                 1.780 
                 96.489 
                 2.245 
                 89.537 
                 1.702 
                 83.752 
                 2.994 
                 75.508 
                 4.302 
               
               
                 0.000 
                 100.000 
                 1.294 
                 100.000 
                 1.609 
                 100.000 
                 2.216 
                 100.000 
                 3.703 
               
               
                   
               
             
          
         
       
     
         [0214]     Additional cell studies were conducted using Jurkat (T-cells) and U937 (monocyte) cell lines. These experiments confirmed that other cell types are killed by homocysteine, although these cell lines are not as sensitive as the SK-HEP-1 cell line discussed in the experiments above and are thus more resistant to homocysteine. Cells were exposed to NACCbl 30 μM, NAC 75 μM, CNCbl 15 μM, and folate 30 μM. Data set forth in FIGS.  18  (U937) and 19 (Jurkat cells) showed that NACCbl is more effective at protecting these cells from death than NAC, CN Cbl or folate, especially at higher homocysteine concentrations, and that the protective effect is not just limited to SK-HEP-1 cells. In these Figures, the monocytes are N=6 and concentrations are as follows: NACCbl 30 μM, NAC 75 μM, CNCbl 15 μM, Folate 30 μM  
       CONCLUSIONS  
       [0215]     NACCbl has been shown to be stable and biologically active and to protect cells from oxidative stress damage. This novel, synthetic thiolatocobalamin was more effective than any of the other cobalamins in this activity for both homocysteine and H 2 O 2 -induced oxidative stress.  
         [0216]     It will be understood by those who practice the invention and those skilled in the art that various modifications and improvements may be made to the invention without departing from the spirit of the disclosed concept. The scope of protection afforded is to be determined by the claims and by the breadth of interpretation allowed by law.