# Patent

## Title
Pharmaceutical compositions and therapeutic applications for the use of a novel vitamin B12 derivative, N-acetyl-L-cysteinylcobalamin

## 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, N-acetyl-L-cysteinylcobalamin (NACCbl) to protect animal cells against oxidative stress damage. This invention also relates to the use of thiolatocobalamins, such as NACCbl, in lieu of current, commercially available forms of vitamin B12 for the treatment and prevention of conditions associated with oxidative stress damage and for dietary supplementation.

## Background
CROSS-REFERENCE
This application claims priority to U.S. Provisional Application Ser. No. 60/846,435 filed on Sep. 22, 2006.


FIELD OF THE INVENTION
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, N-acetyl-L-cysteinylcobalamin (NACCbl) 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 NACCbl, in lieu of current, commercially available forms of vitamin B12, for the treatment and prevention of conditions associated with oxidative stress damage.
BACKGROUND OF THE INVENTION
Three forms of vitamin B12 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 B12-dependent enzyme reactions and are frequently referred to as the B12 co-enzymes. Two known B12-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). B12; John Wiley & Sons, Inc.: New York, USA, 1982; Banerjee, R. (ed.) Chemistry and Biochemistry of B12; JohnWiley & Sons, Inc.: New York, USA, 1999). In short, methionine synthase and methylmalonyl-CoA mutase require the vitamin B12 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.
Thiol derivatives of B12, thiolatocobalamins, were first identified in the 1960'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 I). 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.
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 B12—AdoCbl and MeCbl. An alternative role for GSCbl was also recently proposed, in which the formation of GSCbl prevents B12 from being scavenged by xenobiotics.
The exact biochemical pathway(s) that lead to the incorporation of cobalamins into the B12-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.
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.
Formula I, below, depicts the structures of vitamin B12, including the two coenzyme forms of vitamin B12 and related B12 derivatives found in humans, all commonly referred to as the cobalamins.




The cobalamins belong to a family of compounds known as the corrinoids, which differ from one another in the specific nucleotide occupying the α 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.




It is known that vitamin B12 and its derivatives play key roles in human, animal and microbial metabolism. In humans, vitamin B12 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 B12). Cobalamins (Cbls) are bound to protein in food, and hydrochloric acid in the stomach releases vitamin B12 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.
Vitamin B12 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 B12, or the inability to utilize absorbed vitamin B12 and enzymatic or amino acid deficiencies. Certain drugs can also interfere with the absorption of vitamin B12.
Vitamin B12 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).
Vitamin B12, along with folate and vitamin B6, 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's disease. (Tchantchou, F., “Homocysteine metabolism and various consequences of folate deficiency”. J. Alzheimer's Dis. August 2006; Vol. 9, No. 4: 421-27). Homocysteine is eliminated from the body and is regulated by the transmethylation and transsulfuration pathways.
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. 1st 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.
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.
It is known that a deficiency of vitamin B12, folate, or vitamin B6 may increase blood levels of homocysteine. Studies have shown that the reverse is also true. It was recently reported that vitamin B12 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 B12 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).
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's Disease”. J. Alzheimer'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 B12 deficiency and Alzheimer's Disease. Neurology 2002; 58 (9): 1395-99).
It is well-accepted that many vitamin B12-related conditions, regardless of cause, can be easily (and reversibly) treated by administering vitamin B12 or its hydroxycobalamin derivative, either orally or by injection into muscle tissue. As suggested by the above studies, vitamin B12 may also play a role in conditions associated with oxidative stress by decreasing levels of homocysteine or other reactive species.
Thiolatocobalamins present useful therapeutic alternatives to vitamin B12 or hydroxycobalamin administration or supplementation. McCaddon and coworkers suggested that GSCbl and related thiolatocobalamins might be more effective than currently available pharmaceutical B12 forms (CNCbl and hydroxycobalamin) in treating of conditions associated with oxidative stress such as Alzheimer'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 B12 derivative. Furthermore, reduced glutathione levels are associated with a wide range of pathophysiological conditions, including liver failure, malignancies, HIV infection, pulmonary disease, and Parkinson'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's disease, aging, Acquired Immunodeficiency Syndrome, Amyotrophic lateral sclerosis, ankylosing spondylitis, arteriosclerosis, arthritis (rheumatoid), asthma, autoimmune disease, Behcet'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's disease, Down's syndrome, eczema, emphysema, Epstein Barr viral syndrome, fibromyalgia, glaucoma, Goodpasture syndrome, Grave's disease, hypercholesterolaemia, herpes, viral/bacterial/fungal infections, inflammatory bowel disease, systemic lupus erythematosis, senile and diabetic macular degeneration, malnutrition, Meniere's disease, Multiple Sclerosis, Myasthenia Gravis, neurodegenerative diseases, nutritional disorders, pre-eclampsia, progeria, psoriasis, rheumatic fever, sarcoidosis, scleroderma, shingles, stroke, vasculitis and vitiligo.
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 B12, and folate in cognitively impaired hyperhomocysteinaemic patients.” Int. J. Geriatr Psychiatry 2005; 20: 998-1000).
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 B12 and/or folate regimens. (McCaddon, A. “Homocysteine and cognitive impairment; a case series in a General Practice setting.” Nutrition Journal 2006; 5:6).
In view of the potential benefits reported with the use of glutathionylcobalamin, other thiolatocobalamins are also of interest. In particular, the novel compound Na[NACCbl] and other salts of NACCbl are of interest as a potential treatment or supplement, especially considering the above-noted observations associated with co-administrating hydroxycobalamin and N-acetyl-L-cysteine to Alzheimer's patients. There is an impetus to further test these novel, synthetic compounds for biological activity.
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.
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 H2O2 levels, in order to identify useful therapeutic applications for thiolatocobalamins.
SUMMARY OF THE INVENTION
It has been discovered that NACCbl protects cells from damage and death when exposed to oxidative stress conditions. Indeed, NACCbl may provide superior results in patients having, or prone to, oxidative stress conditions associated with neurological disease. It may also be useful for a wide range of other diseases associated with oxidative stress. NACCbl may also be a useful form of vitamin B12 for dietary supplementation. NACCbl has been synthesized as a sodium salt (Na[NACCbl]); however, other salts of NACCbl, such for example a potassium salt, may also prove useful in the treatment of oxidative stress-related disorders or as dietary supplements and are well within the intent and spirit of the invention.
The focus of a number of studies has been on the effects of vitamin B12 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 N-acetyl-L-cysteinylcobalamin, regardless of whether administered alone or in combination with folate, effectively protects endothelial cells against homocysteine-induced oxidative damage. Furthermore, N-acetyl-L-cysteinylcobalamin protects cells against hydrogen peroxide-induced oxidative damage. Importantly, this novel vitamin B12 derivative shows superior protection compared with the currently available pharmaceutical forms of vitamin B12 and folate and naturally occurring cobalamins.
Thus, in accordance with the invention a pharmaceutical composition or dietary supplement respectively comprising N-acetyl-L-cysteinylcobalamin 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 may also comprise a folate composition and a vitamin B6 composition. The pharmaceutical composition may include additional ingredients as is appropriate for the form of administration, including a pharamaceutically acceptable carrier or solvent.
Further the N-acetyl-L-cysteinylcobalamin of the pharmaceutical composition or dietary supplement may advantageously comprise a crystalline salt of N-acetyl-L-cysteinylcobalamin, and in particular may comprise a biologically acceptable salt, such as a sodium or potassium salt of said N-acetyl-L-cysteinylcobalamin.
The invention further relates to a method to treat a disease or condition associated with oxidative stress comprising administering an effective amount of N-acetyl-L-cysteinylcobalamin (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's disease, dementia, and combinations thereof. The invention also relates to a method for the inhibition or reduction of free radical formation comprising administering N-acetyl-L-cysteinylcobalamin (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
FIG. 1 is a graphic representation showing the effect of homocysteine and H2O2 on endothelial cell viability
FIG. 2 is a graphic representation showing that NACCbl and GSCbl protect endothelial cells from the effect of homocysteine;
FIG. 3 is a graphic representation showing that NACCbl and GSCbl protect endothelial cells from the effect of H2O2;
FIG. 4 is a further graphic representation showing that NACCbl and GSCbl protect endothelial cells from the effect of homocysteine;
FIG. 5 is a further graphic representation showing that NACCbl and GSCbl protect endothelial cells from the effect of H2O2;
FIG. 6 is a graphic representation showing that NACCbl and GSCbl protect endothelial cells from apoptosis induced by homocysteine;
FIG. 7 is a series of plots showing the effect of oxidative stress on Hsp32 and Hsp70 gene expression in SK-HEP-1 Cells;
FIG. 8 illustrates a gene expression study for oxidative stress of SK-HEP-1 Cells;
FIG. 9 illustrates Hsp32 gene expression as induced by homocysteine and NACCbl;
FIG. 10 illustrates Hsp32 gene expression as inhibited by quercitin;
FIG. 11 illustrates that NACCbl protects endothelial cells from the effect of homocysteine via a mechanism involving Hsp70 and Hsp32;
FIG. 12 also illustrates that NACCbl protects endothelial cells from the effect of homocysteine;
FIG. 13 illustrates the effect of high concentrations of NACCbl and GSCbl on SK-HEP-1 cells;
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;
FIG. 14a illustrates the effects of cobalamins in the presence of folate on protecting T SK-HEP-1 cells from Hcy;
FIG. 15 illustrates the effect of variable Hcy, protection with NACCbl versus Hcy and N-acetyl-cysteine versus Hcy;
FIG. 16 illustrates the normalized raw statistical data normalized to Log 2;
FIG. 17 illustrates the normalized raw statistical data normalized to Log 10;
FIG. 18 illustrates percentage survival rate of U937 cells at a variable concentration of Hcy with the administration of various compositions; and
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
This invention relates to a novel synthetic thiolatocobalamin, N-acetyl-L-cysteinylcobalamin (NACCbl), which can be used to protect cells against oxidative stress damage. NACCbl can be a pharmaceutical composition or a dietary supplement which advantageously further comprises a folate compound which is used here to include 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 birds or mammals, such as livestock, domestic animal or most advantageously humans.
The invention further relates to a method of treatment of diseases of conditions related to oxidative stress comprising administering an effective amount of a composition comprising NACCbl (meaning specifically NACCbl, its derivatives and salts thereof), preferably with one or more of a folate compound (as previously discussed) and vitamin B6. The term “effective amount” as used herein refers to an amount of an NACCbl sufficient to affect the symptoms due to oxidative stress or free radical presence 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 NACCbl 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 NACCbl can be applied in pharmaceutically acceptable carriers known in the art. The NACCbl 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 NACCbl is preferably protected from digestion.
The NACCbl 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 NACCbl 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 NACCbl 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.
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's dextrose, dextrose and sodium chloride, lactated Ringer'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'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.
The NACCbl 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.
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 NACCbl may be controlled by altering the concentration of the macromolecule.
Another method for controlling the duration of action comprises incorporating the NACCbl 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 NACCbl 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.
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's disease and dementia, comprising administering to a subject at risk for a disease or displaying symptoms for such disease, an effective amount of NACCbl. 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.
For purposes of the inventions described herein, the structure and purity of the novel compound NACCbl (sodium salt) was characterized using UV/Vis Spectrophotometry, 1H 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 thiolatocobalamin derivative, Na[NACCbl], is synthesized herein and isolated in high purity (>95%) and in good yield (>70%). 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 synthesis of the sodium salt of N-acetyl-L-cysteinylcobalamin (Na[NACCbl]) involves reacting a salt of hydroxycobalamin with a slight excess (i.e. from about 1.2 to about 2 or about 1.5, and preferably from about 1.2 to about 1.5 of equivalents) of N-acetyl-L-cysteine in an aqueous solvent; precipitating the formed Na[NACCbl] from the aqueous solvent, preferably by the addition of a precipitate inducing solvent; and collecting the precipitated Na[NACCbl] and all of the above steps are carried out under aerobic conditions.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise” and variations such as “comprises” and “comprising” will be understood to imply the inclusion of a stated integer or step or group of integers but not the exclusion of any other integer or step or group of integers. As used herein, a salt of hydroxycobalamin refers to a compound H2Ocbl.X (or HOCbl.HX) wherein X is a counter anion such as a halide (particularly Cl−) or an O-acyl group such as acetate.
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 & BuOH). Preferably 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.
The reaction may be performed at a temperature from 0° to about 60°. The reaction may 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, eg to about −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 Na[NACCbl], which is preferably a water miscible solvent less polar then water and includes alcohols (such as MeOH, EtOH, PrOH & BuOH) and acetone, is added in an amount sufficient to induce precipitation of the formed Na[NACCbl]. A preferred precipitate inducing solvent is acetone.
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, 1H 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.).
Synthesis of N-acetyl-L-cysteinylcobalamin (Na[NACCbIl])
The synthesis was carried out under aerobic conditions, notwithstanding the potential light sensitivity of thiolatocobalamins. A solution of N-acetyl-L-cysteine (263μml, 284 mM, 74.7 mmol, 1.1 mol equiv.) in MES buffer (0.1 M, pH  ̃6) was added drop wise to a solution of HOCbl.HCl (107 mg, 67.9 mmol) in MES buffer (0.80 ml, 0.1 M, pH  ̃6) with stirring, and the reaction was allowed to react for 30 min at 0° C. The product precipitated upon dripping into a chilled acetone solution (−20° C.), and was filtered, washed with chilled acetone (20 ml, −20° C.) and diethyl ether (10 ml, −20° C.). The product was dried at 50° C. under vacuum (2×10−2 mbar) overnight. Yield: 90 mg (87%). The percentage of non-corrinoid products (salts) in the product can be determined by converting the thiolatocobalamin to dicyanocobalamin after drying the product at 50° C. under vacuum (λ367 nm=30.4 mM−1 cm−1), and was found to be ≦5%.
Structure
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.















Cbl
γ max (nm)























GSCbl
333
372
428
534



NACCbl
333
372
428
534



HcyCbl
333
372
428
534



NACMECbl
333
372
428
534



[definition?]










The 1H NMR spectrum of the cobalamins was also recorded (500 MHz Varian spectrometer, D2O, 25° C.). NACMECbl is 2-N-acetylamino-2-carbomethoxy-L-ethanethiolatocobalamin. Thiolatocobalamins have five characteristic signals in the aromatic region (B7, B2, B4, R1 and C10 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







1H NMR Data









Chemical Shift (ppm)














Cobalamin
B7
B2
B4
R1
C10







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

The purity of the products was also assessed by 1H NMR spectroscopy and by the dicyanocobalamin test. All B12 derivatives are converted to dicyanocobalamin ((CN)2Cbl) upon the addition of cyanide; hence, the percentage of non B12 impurities can be determined. Table 3 below shows the results obtained.







TABLE 3







Purity Assay













1H NMR



Cobalamin
(CN)2Cbl Test
Spectroscopy







GSCbl
99%
98%



NACCbl
95%
98%



HcyCbl
97%
98%



NACMECbl
96%
98%










X-Ray Diffraction Studies

Crystals of Na[NACCbl] 18H2O 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—NB3
2.06
2.05
+0.01


Co—N21
1.88
1.89
−0.01


Co—N22
1.92
1.90
+0.02


Co—N23
1.93
1.91
+0.02


Co—N24
1.88
1.89
−0.01









Stability Studies

The decomposition of GSCbl, NACCbl, and HcyCbl in PBS at 37° C. was monitored by UV/Vis spectrophotometry. Table 5 below shows t1/2 and observed rate constant, kobs, calculated for each derivative.










TABLE 5







t1/2 (hr)
Kobs (min−1)



















GSCbl
No decomposition within 20 hours



NACCbl
No decomposition within 20 hours











HcyCbl
6.6
0.0018










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 H2O2. 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'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
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
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:


    
    
        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]; C20H23N7Na2O6; F.W. 503.42; EC No. 2.1.1.13
        DL-Homocysteine: [2-Amino-4-mercaptobutyric acid] HSCH2CH2CH(NH2)COOH; F.W. 135.18; EC No. 207-222-9
        Dimethyl sulfoxide: [DMSO, Methyl sulfoxide]; (CH3)2SO; F.W. 78.13; EC No. 200-664-3
        Pyridoxine: [Pyridoxol; Vitamin B6]; C8H11NO3; F.W. 169.18; EC No. 200-603-0
        Vitamin B12: [CN-Cbl; Cyanocobalamin]; C63H88CoN14O14P; F.W. 1355.37; EC No. 200-680-0
        Methylcobalamin: C63H91CoN13O14P; F.W. 1344.38; EC No. 236-535-3
        Vitamin B12 a: [Aquocobalamin chloride]; C62H90ClCoN13O15P; F.W. 1382.82; EC No. 261-200-3
        Hydroxocobalamin: [Hydroxocobalamin acetate salt, Vitamin B12a]; C64H91CoN13O16P; F.W. 1388.39; EC No. 236-533-2\
        Bilirubin Mixed Isomers [Bilirubin IX-alpha] C33H36N4O6; M.W. 584.68; EC No. 211-239-7
    
    


Glutathionylcobalamin: GSCbl M.W. 1635.0 synthesized by the inventors as described in U.S. Pat. No. 7,030,105
N-acetyl-L-cysteinylcobalamin: NACCbl M.W. 1491.0 synthesized by the inventors as described herein

    
    
        Quercetin dehydrate: [2-(3,4-Dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one dihydrate]; C15H10O7.2H2O; 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]; C34H32N4O4SnCl2; M.W. 750.26; Frontier Scientific Inc., Carnforth, UK.
        
        
        Hydrogen peroxide solution: 30% (w/w): H2O2; F.W. 34.01; EC No. 231-765-0
        Propidium iodide: [3,8-Diamino-5[3-(diethylmethylammonio)propyl]-6-phenyl-phenanthridinium diiodide]; C27H34I2N4; M.W. 668.41; EC No. 247-081-0
        Necrosis Inhibitor: IM 54; [2-(1H-Indol-3-yl)-3-pentylamino-maleimide]; C19H23N3O2; M.W. 325.4; Cat No. 480060
        Etoposide: [VP-16]; C29H32O13; M.W. 588.6; EC No. 251-509-1; Calbiochem, Nottingham, UK.
        Hemin: C34H32C1FeN4O4; M.W. 651.96; EC No. 240-140-1
        Trypan Blue: [Direct Blue 14]; C34H24N6Na4O14S4; F.W. 960.81; EC No. 200-786-7
        Z-VAD-FMK: C22H30O7N3F; 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]; HSCH2CH(NHCOCH3)CO2H; F.W. 163.19; EC No. 210-498-3
        L-Glutathione reduced: (γ-Glu-Cys-Gyl; CSH); [γ-L-Glutamyl-L-cysteinyl-glycine]; H2NCH(CO2H)CH2CH2CONHCH(CH2SH)CONHCH2CO2H; F.W. 307.32; EC No. 200-725-4

Assays and Kits

        EnzoLyteTM Rh110 Caspase-3 Assay Kit; AnaSpec, San Jose, Calif., USA; Cat No. 71141
        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-GoTM You-Prime First-Strand Beads; Amersham Pharmacia Biotech; Cat. No. 27-9261-01; Buckinghamshire, UK.
        puReTaqTM Ready-To-GoTM 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.
        TrackitTM 100 bp DNA Ladder: 0.1 μg/μl; Invitrogen; Cat. No. 10488-058; Paisley, UK.
        Agarose 1 Biotechnology Grade; Amresco, Solon, Ohio, USA; Product No. 0710-500G
        Ethidium Bromide Fluorescence λ ex 530 nm; λ em 600 nm; [3,8-Diamino-5-ethyl-6-phenylphenanthridinium bromide]; C21H20BrN3; F.W. 394.32; EC No. 1239-45-8; Appligene Oncor, Graffenstaden, Germany.
        DAPI: [4′,6-Diamidino-2-phenylndole,dilactate]; C22H27N5O6; F.W. 457.48 Pd(N)6 Sodium salt; Amersham Pharmacia, NJ. USA; Cat No. 27-2166-01
        Primers for RT-PCR: All primer sequences obtained from Alta Biosciences University of Birmingham, UK.

siRNA Reagents

        LipofectamineTM 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 H2O Mr 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/cm2 using 0.25% trypsin or trypsin/EDTA; 5% CO2; 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+3AH2+3O2=biliverdin+Fe2++CO+3A+3H2O Swiss-Prot: P09601 Gene Name: HMOX1 Location: Microsomal Sequence Information Length 288 AA. 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 7q11.23
        
        
        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
            Sequence Information Length803AA. M.W. 92469 Da. Chromosome 12q23.3
        
        
    
    












RT-PCR Primer Sequences










Sense Primer
Antisense Primer














β-actin
TGC-TAT-CCC-TGT-ACG-CCT-CT
AGT-ACT-TGC-GCT-CAG-GAG-GA



 


Hsp 27
ATG-GCG-TGG-TGG-AGA-TCA-CC
CAA-AAG-AAC-ACA-CAG-GTC-GC


 


HO-1
CAG-GCA-GAG-AAT-GCT-GAG-TTC
GCT-TCA-CAT-AGC-GCT-GCA


 


Hsp 70
TTC-CGT-TTC-CAG-CCC-CCA-ATC
CGT-TCA-GCC-CCG-CGA-TGA-CA


 


Hsp 90β
AGA-AGG-TTG-AGA-AGG-TGA-CAA
AAG-AGT-GAG-GGA-ATG-GG


 


grp 78
GAT-AAT-CAA-CCA-ACT-GTT-AC
GTA-TCC-TCT-TCA-CCA-GTT-GG


 


gp 96
TGC-CAA-GGA-AGG-AGT-GAA-GT
GTT-GCC-AGA-CCA-TCC-GTA-CT


 


BACH-1
GGA-CAC-TCC-TTG-CCA-AAT-GCA
TGA-CCT-GGT-TCT-GGG-CTC-TCA









    
    
        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-qenome.wi.mit.edu/cqi-bin/primer/primer3_www.cgi).

Experimental Methods

    
    


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
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
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
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 cm2 or 75 cm2 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% CO2 humidified Heracell incubator.
Determination of Cell Counts and Viability
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
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's instructions.
Freezing, Storage and Thawing of SK-HEP-1 Cells
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 cm2 cell culture flask containing 9 ml fresh complete MegaCell MEM media. Cells were grown at 37° C. in a 5% CO2 humidified Heracell incubator.
Sterilization of Equipment
Filter units containing 0.2 μm filters were autoclaved for filter sterilization of all reagents used under experimental conditions in culture media.
Cell Treatments
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
MTS® assay is a standard measure of cell activity. The CellTiter 96® Aqueous One Solution Cell Proliferation Assay was used according to the manufacturer'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
Cells were plated into 6-, 12-, or 24-well cell culture clusters, at 2×103, 2×105, or 1×105 cells per well and incubated until 90% confluent. Treatments were applied as above.
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
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.
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.
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.
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.
This HIGH salt washing step was carried out a further four times to remove cell debris.
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.
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.
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
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
Microcentrifuge tubes from puReTaqTMReady-To-GoTM 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
Agarose (0.56 g) and 0.56 ml TAE buffer (50×) was added to 27.44 ml ddH2O. 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.
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.
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 PSPTM v10 running under Windows XP®.
Caspase-3 Activity (Apoptosis Assay)
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, H2O2 and etoposide were added to negative control wells. The assay was performed according to the manufacturer'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
Propidium iodide is a standard assay for necrosis. H2O2 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, H2O2 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
siRNAs for human HO-1 were synthesised 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 synthesised by Dharmacon Research Inc.
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.
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 >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.
Transfection was optimised using a standard siControl Tox protocol. All transfection experiments included non-targeted siRNA.
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:
Tube 1—17.5 μl of 2 μM siRNA was added to 17.5 μl OptiMEM media. Total volume 35 μl.
Tube 2—4.8 μl Lipofectamine was added to 30.2 μl OptiMEM media. Total volume 35 μl.
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
Cells were trypsinized and plated into 12- or 96-well plates at cell density of 2×105, 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
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 2×105. Plates were incubated at 37° C. for 32.5 hours for mRNA gene analysis experiments or 72 hours for protein analysis by Western Blot.
Statistics
GraphPad PrismTM 4.0 running under Microsoft Windows XP®. All calculations n=6. For single variable comparisons, Student'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<0.05 (95% confidence interval) or p<0.01 (99% confidence interval) was considered significant.
Results
The effects of homocysteine and H2O2 on endothelial cell viability were assessed first. SK-HEP-1 cells were exposed to increasing concentrations of homocysteine or H2O2 (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 H2O2 on endothelial cell viability. SK-HEP-1 cells were exposed to increasing concentrations of homocysteine or H2O2 (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 H2O2 were sufficient to kill cultured endothelial cells (P<0.001). Greater than 90% (<10% survival) death was achieved by 12.5 μM H2O2. Concentrations of 5 μM and higher of homocysteine killed cells (p<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
H2O2
H2O2
H2O2
H2O2
H2O2
H2O2





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









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 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 (>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<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.






The effect of increasing concentrations of NACCbl and GSCbl to protect endothelial cells from the effect of H2O2 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 H2O2 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 H2O2. SK-HEP-1 cells were exposed to increasing concentrations of NACCbl (▪) or GSCbl (▴) for 2 hours prior to exposure to 25 μM H2O2 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 H2O2-induced cell death. There was no difference between the protection afforded by NACCbl and that by GSCbl. Both required 100 μM to achieve >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 H2O2 for 24 hours.






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.






Protection of endothelial cells by NACCbl (at a constant concentration of 30 μM) was also observed when cells were exposed to variable concentrations of H2O2, however, the protection decreased below 60% survival above 25 μM H2O2 (FIG. 5). FIG. 5 illustrates that NACCbl (▪) and GSCbl (▴) protect endothelial cells from the effect of H2O2. Cells were pre-treated with NACCbl or GSCbl (30 μM) for two hours and then exposed to variable concentrations of H2O2 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 H2O2 (also FIG. 5).







TABLE 11*





n = 6





















H2O2








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
















H2O2








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
















H2O2








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 H2O2.






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.
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<0.05 or p<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<0.05 or ** p<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: H2O2 25 μM, 2 hr; lane 3: folate 30 μM, 2 hr+H2O2 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+H2O2 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.
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).
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.
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 Hsp32 or 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<0.001, FIGS. 11 and 12). Inhibition of Hsp70 by both methods (chemically and directly) reduced the protection by NACCbl by 20-40% (p<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
















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









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









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. 14a 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



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.










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
















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
















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
















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









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
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 H2O2-induced oxidative stress.
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.

## Claims

1. A method of reducing serum homocysteine levels or treating one or more of cardiovascular disease, cerebrovascular disease, peripheral vascular disease, glaucoma, Alzheimer's disease, and dementia, in an animal selected from the group of livestock, a domestic animal or a human comprising administering an effective amount of N-acetyl-L-cysteinylcobalamin.

2. The method according to claim 1 further comprising administering said N-acetyl-L-cysteinylcobalamin in combination with one or more of a folate compound and vitamin B6.

3. The method according to claim 2 wherein said folate compound is selected from the group consisting of folate, (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.

4. The method according to claim 2 in which N-acetyl-L-cysteinylcobalamin is administered with vitamin B6.

5. A method of reducing high homocysteine levels or of reducing free radical formation levels in a subject animal comprising livestock, a domestic animal or a human comprising administering an effective amount of N-acetyl-L-cysteinylcobalamin to the subject animal.

6. A method according to claim 5 further comprising administering said N-acetyl-L-cysteinylcobalamin in combination with one or more of a folate compound and vitamin B6.

7. The method according to claim 6 wherein said folate compound is selected from the group consisting of folate, (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.

8. A pharmaceutical composition comprising N-acetyl-L-cysteinylcobalamin and one or more of a folate compound and vitamin B6.

9. A pharmaceutical composition according to claim 8 wherein said N-acetyl-L-cysteinylcobalarnin comprises a crystalline salt of N-acetyl-L-cysteinylcobalamin.

10. The pharmaceutical composition according to claim 9 wherein said crystalline salt is a sodium salt of said N-acetyl-L-cysteinylcobalamin.

11. The pharmaceutical composition according to claim 8 wherein the folate compound is folate or a natural isomer of reduced folate.

12. The pharmaceutical composition according to claim 11 wherein the natural isomer of reduced folate is one or more of (6S)-tetrahydrofolic acid, 5-methyl-(68)-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.

13. The pharmaceutical composition according to claim 8 further comprising vitamin B6.

14. A dietary supplement comprising N-acetyl-L-cysteinylcobalamin and one of more of a folate compound and vitamin B6.

15. The dietary supplement according to claim 14 further comprising a crystalline salt of N-acetyl-L-cysteinylcobalamin.

16. The dietary supplement according to claim 15 wherein said crystalline salt is a potassium or sodium salt of said N-acetyl-L-cysteinylcobalamin.

17. The pharmaceutical composition according to claim 14 wherein the folate compound is selected from the group consisting of folate, (6S)-tetrahydrofolic acid, 5-methyl-(6S)-tetrahyclrofolic 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.