WO2009071905A2 - Use of thiolatocobalamins as regulators/selective promoters of inducible and constitutive nitric oxide synthases - Google Patents

Abstract

The present invention provides methods of treatment and uses of thiolatocobalamin compounds for diseases characterised by NOS depression, malfunction, or dysregulation, for example inflammation, the inflammatory phase of the immune response, SIRS/sepsis/septic, traumatic, anaphylactic shock, a bacterial, viral or parasitidal disease, cancer, spinal injury or cardiovascular disease.

Description

USE OP A TfflOLATOCOBALAMlN AS A REGULATOR/ SELECT] VB PROMOTER OP _ INDUCJBJf j AND CONSTITUTIVE N1TR1C_ OX] DB

The current invention relates to pharmaceutical compositions comprising Vital niπ B 12 as hydroxocobalamin, glutathionylcobalamin or" other thiolatαcobalamin, uid their use as medicaments and in method of treatment of diseases and disori bra associated with reduced nitric oxide synthase activity.

Cobalaxnin, Q_3-6JHa₈Oj₄Ni₄PCo, vitamin B12 (CbI), is a red crystalline, water soli ble substance, (molecular weight 1357 kD), comprising various polycyclic compoui ids, with a central cobalt atom set within a planar, tetrapyπole (coπin) ring. The uppi t β axial cobalt Hgand is variable and can combine with HiO, OH, CN, GSH and ύ her thiols, and with Me and Ado to form the co-enzymes, methylcobalamin, MeCbI ind S-deoxy-5-adeπosylcoba.aπun, AdoCbt. The latter two have a unique, cova eπt carbon-cobalt bond which gives CbI its remarkable chemical and biological reacti\ ity. and makes it a potent physiological compound, with a daily requirement of only J ιιg. The lower α axial ligand for physiological forms of the vitamin is a dimethylbenseimidazole, 'false', nucleotide base, (DMBO- (Figure 6^*). CbI is synthesised by bacteria both in soil and in the lumen of ruminants. Humans derive 2bl from their diet, chiefly liver, kidneys, red meat, oysters, egg yolk and yeast extract Its gastrointestinal absorption is complex, and involves binding of food CbI to the DbI transport protein, transcobalamin I, in saliva, gastric acid to separate CbI from prot sin, and intrinsic factor (JF) in the ileum, There are 3 transport proteins in the circulaj ion (TCS I. H₁ and IH), with separate functions. CbI also, assumes different for ns. principally methylcobalamin (MeCbI) and 5_'deoχyadenosylcobalamin (AdoC Jl). TCEn appears to remove inactive CbI analogues or corrinoids, and cyanocobalai itin (CNCbI), from the tissues and circulation and take them to the liver for excretioi in bile. Such corrinoids seem to interfere with CbI function, and CNCbI, found mostlj in the lungs of smokers, is probably an excretory, detoxification product ancj is functionally inert. CbI, which enters the circulation as OHCbIZH₂QCbI, is transpoi ted by Ten, via .he TCH endocytosis ion channel receptor, into all tissues and cells. Following TC II degradation in the lysosomes, it is converted to MeCbI and AdoCbl, and largely retained for use intracellularly, although some is exported on TCII and TCiπ. High concentrations of TCI are found in the reticuloendothelial system, in neutrophils, and in the liver. TCI is largely confined to the circulation, perhaps as a mobile store of CbI (to complement the larger, long-term storage of hepatic and renal CbI). TCI does not have a specialist receptor, but is taken into the cell via a multipurpose receptor, the asialoglycoprotein receptor. There is clearly some form of communication and flexibility between the TCS: TCII carries the larger fraction of CbI present in portal vein blood than in hepatic and axillary vein blood. In disease, TCπ sometimes holds the bulk of CbI present in peripheral blood, suggesting CbI transfer from TCI as needed.

The conversion of aquacobalamin, H₂OCbI, on cell entry to the co-enzymes, MeCbI and AdoCbl, is still not fully understood. Pezacka et al have suggested that it might proceed via the formation of the unusually stable intermediate, glutathionylcobalamin, (GSCbI), the product of H₂OCbI+ and excess reduced glutathione (GSH).

There is a web of complex inter-relationships sustained by the 2 mammalian CbI coenzymes. It is therefore not surprising that a large CbI chemical, biochemical, medical literature supports claims, and increasing evidence, for the efficacy of CbI in everything from cancer, heart disease, Alzheimers and other neurological conditions, ADDS, SIRS/sepsis/septic and traumatic shock, infertility, depression, circadian rhythm disorders, auto-immune disease, chronic fatigue syndrome, eczema and other skin conditions, allergies, and, not least, growth and megaloblastic anaemia, as CbI is critical to haemopoiesis.

Strictly speaking Vitamin B 12 means cyanocobalamin, although the term is sometimes also loosely used to describe other cobalamins such as methylcobalamin, adenosylcobalamin and hydroxocobalamin. Thiolatocobalamins are derivatives that are characterised by a cobalt-sulphur bond in the upper axial ligand of the molecule. Thiolatocobalamins include the naturally occurring glutathionylcobalamin, and various synthetic thiolatocobalamins, including N-acetyl-cysteine-cobalamin (NAC- CbI).

The upregulation of TC and its membrane receptor in diseases associated with inflammation suggests a central role for cobalamin (CbI) in the inflammatory response. (Wheatley C,. Med Hypotheses, 2006;67: 124-142)). CbI deficiency deficiency, chronic, functional or "compartmental' may contribute to the aetiology of

SIRS/sepsis/septic shock, as well as auto-immune disease, cancer, in particular haematological malignancy (Wheatley C,. J of Orthomol Med, 2002; 17 (1): 7-16), and the progression of AIDS.

In theory, CbI may be essential for the timely promotion of inducible nitric oxide synthase, (iNOS), and concordant regulation of endothelial and neuronal nitric oxide synthases, (eNOS/nNOS).

CbI may ensure controlled high output of NO, and its safe deployment, because:

1. CbI is ultimately responsible for synthesis or availability of the NOS substrates and cofactors, heme, arginine, BH₄, FAD/FMN, and NADPH, via far-reaching effects of the two CbI dependent coenzymes in, or on, the folate, glutathione, TCA and urea cycles, oxidative phosphorylation, glycolysis and the pentose phosphate pathway. Deficiency of any of the NOS substrates and cofactors results in "uncoupled' NOS reactions, decreased NO production and increased or excessive O₂ , H₂O₂, ONOO^" and other ROS/RNIS, leading to pathology.

2. CbI is also the overlooked ultimate determinant of positive GSH status, which favours formation of more benign NO species, s-nitrosothiols, the predominant form in which NO is safely deployed. Adequate CbI status may consequently ensure the active status of antioxidant systems, as well as reversing and modulating the effects of nitrosylation in cell signal transduction. The current invention resides in the suprising discovery that hydroxocobalamU and the thiolatocobalamin glutathionylcobalamin may be used to regulate Nitric <j xidβ Synthases, to selectively promote iNOS and eNOS in the early pro-inflaπun ttciy phase, of the immune response. This discovery has profound implications ft* the treatment of diseases characterized by de-regulation of NOS since selective regull ition of NOS during the immune response, entails consequent regulation of key inui iune factors, (such as TNFα), that can optimise outcome and resolve pathologie s of unxesolvabie inflammation. Such NOS regulation will also increase the efficaβ y of any additional therapies, for disease. Such diseases include, without Kmitή ion, SIRS/sepsis/septic, traumatic and anaphylactic shock; cancer; parasite diseases, iuch as malaria where enhanced promotion of iNOS confers some protection, viral dig sasc such as Flu, types A,, (including Avian Flu, HSNl)₁ B, and C; and microbial dls< ase, such as Tuberculosis, and endothelial dysfunction?.

The invention provides a pharmaceutical composition comprising Vitamin BIj I as OHCbI, but preferably glutathionylcobalamin (GSCbI) or other tniolatocobalamta that may be used alone, or in any combination with (a) transcobalamins; (b) I OS substrates, cofactors or precursors; (c) interferons, to promote expression and assembly of NOS; (d) anti-microbia] drugs; (e) anti-parasitic drugs: anti-viral dn igs; aήti-cancer drags; to be used as regulators/selective promoters of constitutive and inducible Nitric Oxide Synthases in the treatment of bacterial, viral, parasitic, fuj gal disease, cancer, inflammation and immune dysregulatiαn,

A second embodiment of this invention provides a pharmaceutical composition¹ for the treatment of inflammation and immune dysregulation, including but with out limitation, SIRS/sepsis/septic* traumatic and anaphylactic shock, or lupus; or emphysema; or spinal injury, comprising Vitamin B12, preferably as QSCbI alonm or in a combination with transcobalamins; NOS substrates, cofactois, precuxsnrs; interferons; antibiotics; anti-parasitic drugs; antifungal drugs, anti-viral drugs; a Ui- cancer drugs. A third embodiment of this invention provides a pharmaceutical composition for the treatment of parasitic disease, including but without limitation, malaria, Schistosomiasis (Bilharzia), Giardiasis, Elephantiasis, Sleeping sickness (African Trypanosomiasis), comprising Vitamin B 12, preferably as GSCbI, alone or in a combination with transcobalamins; NOS substrates, cofactors, precursors; interferons; anti-parasitic drugs.

A fourth embodiment of this invention provides a pharmaceutical composition for the treatment of bacterial disease, including but without limitation, Tuberculosis, Meningitis and MRSA, comprising Vitamin B 12, preferably as GSCbI, alone, or in a combination with transcobalamins; NOS substrates, cofactors, precursors; interferons; anti-microbial drugs.

A fifth embodiment of this invention provides a pharmaceutical composition for the treatment of cancer, and pre-cancerous conditions, including but without limitation,

MGUS, Multiple Myeloma, Leukaemias, and Lymphomas, comprising Vitamin B 12, preferably as GSCbI, alone, or in a combination with transcobalamins; NOS substrates, cofactors, precursors; interferons; anti-microbial drugs; anti-viral drugs; anti-cancer drugs.

A sixth embodiment of this invention provides a pharmaceutical composition for the treatment of viral disease including but without limitation, Flu Types A, (including Avian, H5N1), B, and C, and HIV/ AIDS, comprising Vitamin B 12, preferably as GSCbI, alone, or in a combination with transcobalamins; NOS substrates, cofactors, precursors; interferons; anti-microbial drugs; anti-viral drugs; anti-cancer drugs.

A seventh embodiment of the invention provides a pharmaceutical composition for the treatment of endothelial dysfunction. Endothelial dysfunction is a physiological dysfunction of normal biochemical processes carried out by the endothelium, the cells that line the inner surface of all blood vessels including arteries and veins (as well as the innermost lining of the heart and lymphatic system). A reduction of normal function of endothelial cells is characteristic of endothelial dysfunction. Normal functions of endothelial cells include mediation of blood coagulation, platelet adhesion, immune function. Endothelial dysfunction can result from disease processes, as occurs in septic shock, hypertension, hypercholesterolaemia, diabetes as well as from environmental factors, such as from smoking tobacco products.

Endothelial dysfunction is thought to be a key event in the development of athersclerosis and predates clinically obvious vascular pathology by many years. Endothelial dysfunction has also been shown to be of prognostic significance in predicting vascular events including stroke and heart attacks. A key feature of endothelial dysfunction is the inability of arteries and arterioles to dilate fully in response to an appropriate stimulus. This can be tested by a variety of methods including iontophoresis of acetylcholine, intra-arterial administration of various vasoactive agents, localised heating of the skin and temporary arterial occlusion by inflating a blood pressure cuff to high pressures.

Embodiments of the invention as described above relating to pharmaceutical compositions therefore also equally relate to methods of treatment of disease as defined above. The methods of treatment comprise the administration of said composition to a subject in need thereof.

Where the composition of the invention comprises more than active substance, the component substances within the composition may be for simultaneous, separate or subsequent administration of the active substances. The composition may therefore be provided in the form of a kit with instructions for use in accordance with the various embodiments and aspects described above.

The present invention therefore also provides for the use of substances as defined above in the manufacture of compositions for the treatment of disease as described herein.

In addition, the compositions may include polymers of these analogues or vitamin B 12 conjugated to other molecules or encapsulated. For clarification the following abbreviations are used:, CN-CbI means vitamin B 12 as cyanocobalamin, H₂OCbI means vitamin B 12 as aquacobalamin, AdoCbl means vitamin B 12 as adenosylcobalamin, MeCbI means vitamin B 12 as methylcobalamin, OHCbI means vitamin B 12 as hydroxycobalamin, GSCbI means vitamin B 12 as glutathionylcobalamin, NOS means Nitric Oxide Synthases, iNOS means inducible nitric oxide synthase, eNOS means endothelial nitric oxide synthase, nNOS means neuronal nitric oxide synthase, TNFα means Tumour Necrosis Factor alpha, IL-6 means Interleukin 6, GSH means glutathione, NOS substrates means arginine, N^G- Hydroxyl-arginine, (NHA) and arginine analogues, NOS cofactors means iron, Fe[III]+, Zinc (Zn⁺), tetrahydrobiopterin, (BH₄), and other pterins, reduced nicotinamide adenine dinucleotide phosphate (NADPH), guanosine triphosphate (GTP), adenosine triphosphate (ATP), folic cid and folate analogues, glutathione, (GSH), S-adenosyl-methionine (SAM). GSPDH means Glyceraldehyde Phosphate Dehydrogenase, Anxa means Annexin-1, PMN means Polymorphonuclear Neutrophils, RT-PCR means Reverse Transcriptase Polymerase Chain Reaction assay, ELISA means Enzyme-Linked Immuno-Sorbent Assay, i.p. injection means intra-peritoneal injection, PBS or phosphate buffer saline means an injectable solution that serves as a negative control because it does not have any physiological or therapeutic effects. SIRS means Systemic Inflammatory Response Syndrome

Interferons means "interferon compounds' which means interferon-alpha, interferon- alpha analogues, derivatives, and conjugates, interferon- beta, interferon-beta analogues, derivatives, conjugates, interferon-gamma, interferon-gamma analogues, derivatives, and conjugates, and mixtures thereof. Preferred interferon compounds include Roferon®, Intron®, Alferon®, Infergen®, Onmiferon®, Alfacon-1®, interferon-alpha, interferon-alpha analogues, pegylated interferon-alpha, polymerized interferon-alpha, dimerized interferon-alpha, interferon-alpha conjugated to carriers, interferon-alpha as oral inhalant, interferon-alpha injectable composition, interferon- alpha as a topical compostion, Roferon® analogues, Intron® analogues, Alferon® analogues, and Infergen® analogues, Omniferon® analogues, Alfacon-1® analogues, interferon beta, Avonex™, Betaseron™, Betaferon™, Rebif™, interferon-beta analogues, pegylated interferon-beta, polymerized interferon-beta, dimerized interferon-beta, interferon-beta conjugated to carriers, interferon-beta as oral inhalant, interferon-beta as a topical composition, Avonex™ analogues, Betaseron™, Betaferon™ analogues, and Rebif™ analogues. Preferred interferon compounds include Actimmune™, interferon-gamma, interferon-gamma analogues, pegylated interferon-gamma, polymerized interferon-gamma, dimerized interferon-gamma, interferon-gamma conjugated to carriers, interferon-gamma as oral inhalant, interferon-gamma injectable composition, interferon-gamma as a topical compostion, Actimmune™ analogues. Alternatively, agents that induce interferon-alpha or interferon-beta or interferon-gamma may also be employed. The singular form, interferon', may mean any one or more compounds from the class of interferon compounds, without limitation.

Cancer means a class of diverse diseases and disorders characterized by a lack of control or poorly controlled cell division or proliferation as a result of deregulation of Nitric Oxide Synthases, (NOS). Cancer diseases include:

Acute lymphoblastic leukaemia, acute myeloid leukaemia, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, basal cell carcinoma, bile duct cancer, bladder cancer, brain tumour, breast cancer, cerebella astrocytoma, cerebral astrocytoma, cervical cancer, chronic lymphocytic leukaemia, chronic myeloid leukaemia, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, Ewing's sarcoma, gallbladder cancer, gestational trophoblastic tumour, glioma, hairy cell leukaemia, hemangiomas, Hodgkin's lymphoma, hypopharyngeal cancer, islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, liver cancer, sarcomas, malignant melanoma, malignant mesothelioma, malignant thymoma, medulloblastoma, MGUS (Monoclonal gammopathy of unknown significance), multiple myeloma, mycosis funoides, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung carcinoma, oesophageal cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian epithelial cancer, ovarian germ cell tumour, pancreatic cancer, parathyroid cancer, penile cancer, pituitary tumour proliferative diabetic retinopathy, prostate cancer, rectal cancer, renal cell carcinoma, restenosis, retinoblastoma, salivary gland cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thyroid cancer, uterine cancer/sarcoma, vaginal cancer, vulvar cancer, WiIm' s tumour. The singular form 'cancer' includes any one or more diseases selected from the class of cancer diseases, and includes any compound or complex disease state wherein a component of the disease state includes a disease selected from the class of cancer diseases.

Anti-cancer therapeutics include: anastrozole (Arimidex®), Apo2LTRAIL, amsacrine (AMSA), L-asparaginase (Elspar®), bleomycin, bleomycin sulfate (Blenoxane®), busulfan (Myleran®), carboplatin (Paraplatin®), carmustine (BCNU®, Gliadel®, BiCNU®), celecoxib (Celebrex®), cetuximab (IMC-C225 or Erbitux™)), cisplatin (platinum analogs, Platinol®), chlorambucil (Leukeran®), cladribine (2-chlorodeoxyadenosine; "2-CDA"; Leustatin®), cyclophosphamide (Cytoxan®), cytarabine (ara-C;Cytosar-U®), daunorubicin (Cerubidine®), dexamethasone, docetaxel, doxorubicin (Doxil®, Rubex® or Adriamycin®), eloposide, epirubicin (Pharmorubicin®), estramustine, estramustine phosphate (Emcyt®), etoposide (VePesid® or VP- 16®), exemestane (Aromasin®), floxuridine (FUDR®), 5-fluorouracil, fludarabine phosphate (Fludara®), flutamide, gemcitabine HCL (Gemzar®), hydroxyurea (Hydrea®), hexamethylmelamine, idarubicin (Idamycin®), ifosamide (Ifex®), imatinib, alfa-2a interferon, interferon β and γ interferon, irinotecan (CPT-Il, or Camptosar®), Interleukins, Iressa, letrozole (Femara®), leucovorin, leuprolide, lomustine, mechlorethamine, megestrol (Megace®), methotrexate, methotrexate sodium plus 6-mercaptopurine (6-MP; Purinethol®), mitomycin C, mitoxantrone (Novantrone®), nitrosureas, oxaliplatin, oxorubicin, paclitaxel (Taxol®, Taxotere®, Tamoxifen®, Nolvadex®), pentostatin (2-deoxycoformycin; "DCF"; Nipent®), prednisone, procarbazine, raloxifene (Evista®), raltitrexed (Tomudex®), retinoic acid (ATRA), revimid, temozolomide (Temodar®), 6-thioguanine (Tabloid®), thiotepa (Thioplex®), topotecan (Hycamtin®), toremifene (Fareston®), trastuzumab (Herceptin®), velcade, valrubicin (Valstar™), vinblastine, vincristine, vincristine sulfate (Oncovin®), vinorelbine, and/or vinblastine sulfate (Velban®). Anti-microbial compounds means any known clinically acceptable antibiotic including, for example, but without limitation: Azithromycin, amphotericin B, bacitracin, clarithromycin, cephalosporin, ciprofloxacin, doxyclycline, erythromycin, gentamicin, griseofulvin, levofloxacin, metronidazole, neomycin, ofloxain, penicillin, polymyxin B, rifamycin, streptomycin, tetracycline, tobramycin, trimethroprim vancomycin.

Anti-parasitic compounds means any known clinically accepted anti-parasitic, including, for example, but without limitation: albendazole, diethylcarbamazepine citrate, chloroquine, doxycycline, malarone, mebendazole, mefloquine, metronidazole, niclosamide, nitazoxanide, oxamniquine, permethrin, praziquantel, proguanil, pyrantel, pyrethrin, pyantel pamoate, thiabendazole.

The pharmaceutical compositions of this invention for use in any preceding claim, preferably contain a pharmaceutically acceptable excipient or carrier, which may include, without limitations, excipients selected from the group consisting of carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may be present as an excipient. Specific carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof. The preparation may also include an antimicrobial agent for preventing or deterring microbial growth. Nonlimiting examples of antimicrobial agents suitable for the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of the conjugate or other components of the preparation. Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

A surfactant may be present as an excipient. Exemplary surfactants include: polysorbates, such as "Tween 20" and "Tween 80," and pluronics such as F68 and F88 (both of which are available from BASF, Mount Olive, New Jersey); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; and chelating agents, such as EDTA, zinc and other such suitable cations.

Acids or bases may be present as an excipient in the preparation. Nonlimiting examples of acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, without limitation, bases selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate, and combinations thereof. The pharmaceutical medicament or compounds of the present invention may be administered by any medically accepted route: oral, intranasal, by inhaler, by subcutaneous, intra-muscular or intraperitoneal injection, transdermal, or sublingual. Preferably the medicament or compounds are for administration by the intramuscular, or intravenous route. Preferably, the invention will be given in the form of Vitamin B 12 as OHCbI, more preferably as GSCbI.

The dose of these two cobalamins as a regulator/selective promoter of the NOS, as alone or in combination with any, or all, NOS precursors, substrates and cofactors, may be determined by the condition of the individual patient, disease, and its severity, as assessed by those skilled in the art, and medical knowledge. The dose may range within the known parameters of safety and use in the pre-clinical and clinical studies, and as used in orthodox medical treatment of pernicious anaemia, subacute degeneration of the spinal cord for nearly a century and as an antidote to cyanide in France and other countries for nearly 50 years. Vitamin B 12 has a superb pharmacological safety profile at the doses used for such treatments which range between lOOOmcg i.m. to 5 grams i.v.. The present invention of Vitamin B 12, as OHCbI, but preferably GSCbI, without limitation, will use doses in the range of lOmcg to 5g daily. In our experimental pre-clinical studies we have found a dose of 40mg/kg to be safe, and this is within the limits known in the safety literature. When used in combination with interferons, preferably interferon α or β, to enhance expression and assembly of the NOS, the dose of interferons will be determined by the pharmacological safety data of interferons, and will be guided by clinical use. Interferons may be given before or after the pharmaceutical compound/s of this invention. Preferably they will be administered after.

The current invention provides methods and compositions to resolve inflammation caused by depression or dysregulation of the NOS, and thus to enhance and resolve the immune response. This means it can be applied without limitation to a wide spectrum of diseases, bacterial, viral, fungal or parasitidal disease, inflammation, cancer and immune dysregulation. Those skilled in the art will readily understand that specific diseases and pathologies mentioned herein are not intended to limit the scope of its use, and that its pharmaceutical safety profile should render it suitable for combination with any concurrent therapy for disease, for which such combinations can be expected to enhance the efficacy of therapy.

The first method of treatment is the administration of a pharmaceutical composition including Vitamin B 12 as OHCbI, but preferably as GSCbI.

The second method of treatment is the administration of a pharmaceutical composition including Vitamin B 12 as OHCbI, but preferably as GSCbI in combination with transcobalamins.

The third method of treatment is the administration of a pharmaceutical composition including Vitamin B 12 as OHCbI, but preferably as GSCbI, in combination with any or all of the NOS substrates, cofactors and precursors.

The fourth method of treatment is the administration of a pharmaceutical composition including Vitamin B 12 as OHCbI, but preferably as GSCbI, in combination with interferons, to promote expression and assembly of NOS.

The fifth method of treatment includes the step of the administration of a pharmaceutical composition including Vitamin B 12 as OHCbI, but preferably as GSCbI, followed by administration of interferons to promote expression and assembly of NOS.

The sixth method of treatment includes any or all of methods described above in combination with other medical therapies, such as antibiotic, anti-parasitidals, anti- virals, anti-fungals, immunotherapies, chemotherapy, radio-therapy, phototherapy, hormonal or other therapy, without limitation. The methods and compositions of this invention may be applied before, concurrently, or after, said therapies. Preferably they are applied before. The seventh method of treatment of endothelial dysfunction includes the step of administration of a pharmaceutical composition including Vitamin B 12 as OHCbI, but preferably as GSCbI.

In some alternative embodiments of the invention there is provided a thiolatocobalamin compound for use as a promoter of endothelial nitric oxide synthase (eNOS) activity and/or inducible nitric oxide synthase (iNOS) in the inflammatory phase of the immune response. The compound can be used in the early- pro-inflammatory phase of the immune response or in states of unresolved inflammation.

A thiolatocobalamin is a compound characterised by a cobalt-sulphur bond in the upper axial ligand of cobalamin. Examples include N-acetylcysteinylcobalamin and cysteinylcobalamin.

Suitable thiolatocobalamins may include the preferred compound glutathionylcobalamin (CAS: 129128-04-7) and related sulphur-containing cobalamins having the generic form Co alpha- [alpha-(5, 6-Dimethylbenzimidazolyl)]-Co beta- ligandyl) cobamide, in which the upper beta-axial ligand group is coordinated to the cobamide by a sulphur-cobalt bond, such as (but not limited to) sulphitocobalamin (syn-sulfitocobalamin; CAS: 15671-27-9), cysteinylcobalamin (CAS:60659-91-8), cyclohexylthiolatocobalamin and pentafluorophenylthiolatocobalamin

Examples of suitable thiolatocobalamin compounds includes: N-acetyl-L- cysteinylcobalamin, cysteinylcobalamin, glutathionylcobalamin (GSCbI), captopril- cobalamin, gamma-glutamylcysteinylCbl (gamma-GluCys-Cbl) and cysteinylglycinylcobalamin (CysGly-Cbl).

The thiolatocobalamin compound used according to the present invention can therefore be N-acetyl-L-cysteinylcobalamin, cysteinylcobalamin, glutathionylcobalamin (GSCbI), captopril-cobalamin, gamma-glutamylcysteinylCbl (gamma-GluCys-Cbl) and cysteinylglycinylcobalamin (CysGly-Cbl) The invention also provides the use of a thiolatocobalamin compound in the manufacture of a medicament for the treatment of inflammation, cardiovascular disease, or endothelial dysfunction.

As described herein, the invention also provides for methods of treatment. Further embodiments of the invention relating to such therapeutic embodiments are as follows.

A method of treatment for the inflammatory phase of the immune response comprising administering a composition comprising a thiolatocobalamin compound to a patient in need thereof.

A method of treatment for inflammation comprising administering a composition comprising a thiolatocobalamin compound to a patient in need thereof.

A method of treatment for a disease characterised by NOS depression, malfunction, or dysregulation comprising administering a composition comprising a thiolatocobalamin compound to a patient in need thereof. Such diseases characterised by NOS depression, malfunction, or dysregulation can be SIRS/sepsis/septic, traumatic or anaphylactic shock.

A method of treatment for a bacterial, viral or parasitidal disease characterised by NOS depression, malfunction, or dysregulation comprising administering a composition comprising a thiolatocobalamin compound to a patient in need thereof.

The bacterial disease can be Tuberculosis, meningitis, MRSA, or other antibiotic resistant disease. The viral disease is influenza type A, B or C, including influenza subtype H5N1. The parasitidal disease is malaria.

The disease characterised by NOS depression, malfunction, or dysregulation may also be cancer, spinal injury, cardiovascular disease, or endothelial dysfunction. Examples of acceptable protocols may be as follows:

I. Hourly, daily, weekly, more than once monthly, or monthly, administration of Vitamin B 12 as OHCbI, but preferably GSCbI, for the effective treatment of disease characterized by depression or dysregulation of the NOS, and to enhance and resolve the immune response.

II. Hourly, daily, weekly, more than once monthly, or monthly, administration of Vitamin B 12 as OHCbI, but preferably GSCbI, in combination with Transcobalamin for the effective treatment of disease characterized by depression or dysregulation of the NOS, and to enhance and resolve the immune response.

III. Hourly, daily, weekly, more than once monthly, or monthly, Vitamin B 12 as OHCbI, but preferably GSCbI, in combination with any or all of the NOS substrates, cofactors and precursors, for the effective treatment of disease characterized by depression or dysregulation of the NOS, and to enhance and resolve the immune response.

IV. Hourly, daily, weekly, more than once monthly, or monthly, Vitamin B 12 as OHCbI, but preferably GSCbI, in combination with interferons, to promote expression and assembly of NOS, for the effective treatment of disease characterized by depression or dysregulation of the NOS, and to enhance and resolve the immune response.

V. Hourly, daily, weekly, more than once monthly, or monthly, Vitamin B 12 as OHCbI, but preferably GSCbI, followed by the step of administration of interferons to promote expression and assembly of NOS, for the effective treatment of disease characterized by depression or dysregulation of the NOS, and to enhance and resolve the immune response.

VI. Hourly, daily, weekly, more than once monthly, or monthly, Vitamin B 12 as OHCbI, but preferably GSCbI, in combination with any of protocols I to V and with other medical therapies, such as antibiotic, anti-parasitidals, anti-virals, anti-fungals, immunotherapies, chemotherapy, radio-therapy, phototherapy, hormonal or other therapy, without limitation. The methods and compositions of this invention may be applied before, concurrently, or after, said therapies. Preferably they are applied before, for the effective treatment of disease characterized by depression or dysregulation of the NOS, and to enhance and resolve the immune response.

Preferred features for the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis.

The invention will now be further described by way of reference to the following Examples and Figures which are provided for the purposes of illustration only and are not to be construed as being limiting on the invention. Abreviations: iNOS = inducible Nitric Oxide Synthase; GAPDH =Glyceraldehyde Phosphate Dehydrogenase; ANXA - Annexin-1; LPS =

Lipopolysaccharide; GSH = Glutathionylcobalamin

Reference is made to a number of Figures in which:

FIGURE l(a) shows CbI Promotion of iNOS at 2 hrs post LPS: Results for the RT PCR on the cDNA obtained from the Lungs. FIGURE l(b) shows CbI Promotion of TNF at 2 hrs post LPS: Results for the RT PCR on the cDNA obtained from the Lungs.

A - Control

B - LPS 0114:B4 (10 me/kg i.p.; time 0

C - OHCbI (40mg/kg i.p., - 1 h) + LPS (timeO)

D - GSCbI (40mg/kg i.p., - 1 h) + LPS (timeO)

FIGURE 2 shows CbI Promotion of iNOS 6 hrs post-LPS FIGURE 3 shows CbI promotion of iNOS/eNOS, and attenuation of eNOS depression, and CbI regulation of TNF alpha, 6 hrs post LPS. Inverse relationship of iNOS/Annexin-1 still seen.

FIGURE 4 shows CbI regulation of Interleukin 6 and Tumour Necrosis Factor

Alpha 6 hrs post LPS. Lower graphs for BL-6 and TNFα are the mean result of 5 assays. CbI maintains IL-6 whist suppressing excessive expression of TNFα.

FIGURE 5 shows Effect of Vitamin B12, as OHCbI, and GSCbI, on cDllb quantity and expression in circulating PMN, 6 hours post LPS

FIGURE 6 shows Structure of vitamin Bi₂ and its derivatives

FIGURE 7 shows Human nNOS, eNOS and iNOS domain structure. (PDZ

Domain, named after homologous domains in 3 proteins, PSD-95, DH/g, ZO- 1.)

FIGURE 8 shows a model of NOSox dimmer with approximate scheme for hypothetical GSCbI links in relation to the two hemes ZnS4, BH4, Arginiπe.

Ser-104 is in the loop with the Cys ligands and H-bonds to the C-6 side chain of BH4.

EXAMPLES General Aims of all Examples.

To demonstrate that the positive outcomes from Vitamin B12/thiolatocobalamin treatment of a range of inflammatory auto-immune, viral and microbial disease, known in the literature, are not as has been hitherto thought, the result of Vitamin B12/thiolatocobalamin simply "mopping up' excess Nitric Oxide, but rather of Vitamin B12/thiolatocobalamin regulating Nitric Oxide Synthases, by promoting high NO production via inducible NOS, whilst maintaining normal constitutive NOS function, and consequently producing well-regulated immune responses. A) Examples Demonstrating a Strong Promotional Effect of hydroxocobalamin and glutathionylcobalamin, on inducible and constitutive Nitric Oxide Synthases, and Consequent Modulation of the Immune System.

Example 1

Effect of vitamin B 12, as Hydroxocobalamin, OHCbI, and Glutathionylcobalamin, GSCbI, on inducible Nitric Oxide Synthase in the early Pro-inflammatory Stage of the Immune Response, with consequent effects on anti-inflammatory Annexin-1.

Aims:

To show that hydroxocobalamin, but particularly GSCbI, determines a strong immune response by promoting inducible Nitric Oxide Synthase, and Nitric Oxide, levels of which control expression of the anti-inflammatory immune cascade.

Experimental Protocol:

An acute endotoxemia model was studied using Swiss-balb mice, (5 animals per group.) Except for group A which was left untreated, mice were pretreated 60 minutes before LPS with: saline, injected i.p. for control group B; OHCbI 40mg/kg for group C; GSCbI 40mg/kg for group D. LPS, E. coli serotype 0114:B4 (Sigma Ltd Code L2630), lOmg/kg was then administered i.p. to groups B,C,D, at time zero. Mice were sacrificed at 2 hours post LPS, plasma collected by cardiac puncture, then organs harvested, and stored.

Lungs were then homogenized and assayed for iNOS cDNA and ANXA-I expression using real time. Reverse Transcriptase Polymerase Chain Reaction assay RT-PCR, (qualitative). Glyceraldehyde Phosphate Dehydrogenase, GAPDH, was used as a control to gauge accuracy of iNOS/ANXA-1 expression.

Results and Conclusions. Treatment of the acute endotoxaemia model with high dose vitamin B 12 as OHCbI and GSCbI, showed a significantly enhanced promotion of iNOS which appeared to have a direct inverse correlation to expression of Annexin-1. This trend was most pronounced with respect to GSCbI (Figure F)

Example 2 Effect of vitamin B 12, as Hydroxocobalamin, and Glutathionylcobalamin, on inducible Nitric Oxide Synthase in the early Pro-inflammatory Stage of the Immune Response, with consequent effects on anti-inflammatory Annexin-1.

Aims: To further confirm results obtained in Example 1 by assaying vitamin B 12, as OHCbI, but particularly Glutathionylcobalamin, GSCbI, promotion of iNOS in the acute endotoxaemia model at a later time point in the pro-inflammatory phase, 6 hours was chosen as the known time-point for peak iNOS expression.

Experimental Protocol: An acute endotoxemia model was studied using Swiss-balb mice, (5 animals per group.) Except for group 1 which was left untreated, mice were pretreated 60 minutes before LPS with: saline, injected i.p. for control group B; OHCbI 40mg/kg by i.p. injection for group C; GSCbI 40mg/kg by i.p. injection for group D. LPS, e.coli serotype 0114:B4 (Sigma Ltd Code L2630), 10mg/kg was then administered i.p. to groups B,C,D, at time zero. Mice were sacrificed at 6 hours post LPS, plasma collected by cardiac puncture, organs harvested, and stored.

Lungs were then homogenized and assayed for iNOS and ANXA expression using real time, Reverse Transcriptase Polymerase Chain Reaction assay RT-PCR, (qualitative). Glyceraldehyde Phosphate Dehydrogenase, GAPDH, was used as a control to gauge accuracy of iNOS/ANXA expression.

Results and Conclusions.

Treatment of the acute endotoxaemia model with vitamin B 12 as OHCbI and GSCbI, showed a significantly enhanced promotion of iNOS which appeared to have a direct inverse correlation to expression of Annexin-1. This trend was most pronounced with respect to GSCbI (Figure 2) Example 3

Regulatory Effects of Vitamin B 12, as Hydroxocobalamin and Glutathionylcobalamin, on constitutive and inducible Nitric Oxide Synthases, Tumour Necrosis Factor alpha, and Annexin-1 in the early pro-inflammatory stage of the immune response.

Aims: To show that Vitamin B 12, as Hydroxocobalamin but particularly Glutathionylcobalamin, both decreases suppression of, and promotes expression of, constitutive endothelial Nitric Oxide Synthase, whilst simultaneously promoting inducible Nitric Oxide Synthase in the early pro-inflammatory stage of the immune response. To further show that Vitamin B 12, as OHCbI but particularly GSCbI, regulates levels of Tumour Necrosis Factor alpha by promoting strong expression of inducible Nitric Oxide Synthase, and, that this relationship also determines levels of early anti-inflammatory Annexin-1 expression. If eNOS expression is supported and promoted by Vitamin B 12, as OHCbI but particularly GSCbI, and TNFα is also regulated in a complementary fashion by Nitric Oxide as a result of OHCbI, but particularly GSCbl's strong promotion of iNOS, Vitamin B 12 as OHCbI, but particularly GSCbI, will mitigate against toxicity in the host and maximise toxicity to invading pathogens.

Experimental Protocol:

An acute endotoxemia model was studied using Swiss-balb mice, (5 animals per group.) Except for group A which was left untreated, mice were pretreated 60 minutes before LPS with: saline, injected i.p. for control group B; OHCbI 40mg/kg by i.p. injection for group C; GSCbI 40mg/kg by i.p. injection for group D. LPS, e.coli serotype 0111:B4 (Sigma Ltd Code L2630), 10mg/kg was then administered i.p. to groups B,C,D, at time zero. Mice were sacrificed at 6 hours post LPS, plasma collected by cardiac puncture, organs harvested, and stored.

Lungs were then homogenized and assayed using real time Reverse Transcription Polymerase Chain Reaction, assay RT-PCR, (semi -quantitative) for endothelial NOS, inducible NOS, Tumour Necrosis Factor alpha and Annexin-1. Results and Conclusions.

In the early stages of the pro-inflammatory immune response, (6 hours) Vitamin B 12, as OHCbI, but particularly GSCbI, decreased suppression of eNOS and promoted expression of eNOS, whilst simultaneously promoting expression of iNOS. Promotion of iNOS by OHCbI, but particularly GSCbI, appeared to bear a direct inverse correlation with OHCbI GSCbl/Nitric Oxide regulatory effects on TNFα, and suppressed expression of Annexin-1. With respect to all four markers Vitamin B 12, as GSCbI, had the most consistently pronounced effect. (Figure 3.)

Example 4

Effects of Vitamin B 12, as Hydroxocobalamin, and Glutathionylcobalamin, on regulation of key inflammatory cytokines, Tumour Necrosis Factor alpha, TNFα, Interleukin-6, IL-6, as a result of OHCbl/GSCbl's strong promotion of iNOS in the early pro-inflammatory stage of the immune response.

Aims: To demonstrate that Vitamin B 12 as OHCbI, but particularly GSCbI' s strong promotion of iNOS, and lesser promotion of eNOS, in the early pro-inflammatory stage of the immune response, and consequent high levels of NO production, constitute the mechanism behind OHCbI, but particularly GSCbI' s regulation of EL-6 and regulation/partial suppression of TNFα.

Experimental Protocol:

An acute endotoxemia model was studied using Swiss-balb mice, (5 animals per group.) Except for group A which was left untreated, mice were pretreated 60 minutes before LPS with: saline, injected i.p. for control group B; OHCbI 40mg/kg by i.p. injection for group C; GSCbI 40mg/kg by i.p. injection for group D. LPS, e.coli serotype 0114:B4 (Sigma Ltd Code L2630), lOmg/kg was then administered i.p. to groups B,C,D, at time zero. Mice were sacrificed at 6 hours post LPS, plasma collected by cardiac puncture, organs harvested, and stored. Lungs were then homogenized and assayed using real time Reverse Transcription Polymerase Chain Reaction, assay RT-PCR, (semi-quantitative) for endothelial NOS, inducible NOS, Tumour Necrosis Factor alpha and Annexin-1. Plasma ELISA assays were performed for IL-6 and TNFα and the mean result of 5 assays noted for each.

Results and Conclusions.

Whilst levels of IL-6 were maintained at a high reading which is desirable in a well- regulated immune response, the high levels of iNOS appeared to have a direct inverse regulatory effect on levels of TNFα. Such regulation of TNFα, in spite of high NO production, is desirable in a good immune response, to minimise damage to host and maximise damage to pathogens. Such regulation was concluded to be the result of OHCbI, but particularly GSCbI, strong promotion of iNOS, and consequently Nitric Oxide, in the early stage of the pro-inflammatory immune response. Regulation of EL- 6 and TNFα by Vitamin B 12 was consistently most pronounced in its GSCbI form, as seen in the previous example 3. (Figures 3 and 4.)

Example 5

Effect of Vitamin B 12, as Hydroxocobalamin, but particularly as Glutathionylcobalamin, on the integrin CDl Ib in Polymorphonuclear Neutrophils, PMN.

Aims: To demonstrate that Vitamin B 12, as OHCbI, but particularly as GSCbI, by regulating iNOS, has consequent regulatory effects on the plasma cell membrane integrin, CDlIb in PMN. CDl Ib is a major adhesion molecule in endothelium, involved in phagocytosis, the respiratory burst of the degranulation in PMN. It is also a marker of chemotaxis. Excessive expression of CDl Ib contributes to pathology of unresolvable inflammation.

Experimental Protocol: An acute endotoxemia model was studied using Swiss-balb mice, (5 animals per group.) Except for group A which was left untreated, mice were pretreated 60 minutes before LPS with: saline, injected i.p. for control group B; OHCbI 40mg/kg by i.p. injection for group C; GSCbI 40mg/kg by i.p. injection for group D. LPS, e.coli serotype 0111:B4 (Sigma Ltd Code L2630), 10mg/kg was then administered i.p. to groups B,C,D, at time zero. Mice were sacrificed at 6 hours post LPS, plasma collected by cardiac puncture, organs harvested, and stored. Blood aliquots were then used to measure CDlIb+ cells, as well as extent of expression. Plasma was then prepared for ELISA assay, whilst RT-PCR was performed on lung extracts with analyses of eNOS; iNOS; Annexin-1 and TNFα.

Results and Conclusions. Vitamin B 12, as OHCbI, but particularly GSCbI, and supported/promoted eNOS, whilst modulating TNFα, and Annexin-1, in the early pro-inflammatory stage of the immune response, Vitamin B 12, as OHCbI and GSCbI, had no statistically significant effect on CDl Ib expression. However, in spite of this, Vitamin B 12 as GSCbI did have a marked modulatory effect on the quantity of CDl Ib+ present in blood PMN. {Figure 5.)

Claims

1. A thiolatocobalamin compound for use as a promoter of endothelial nitric oxide synthase (eNOS) activity and/or inducible nitric oxide synthase (iNOS) in the inflammatory phase of the immune response.

2. A thiolatocobalamin compound for use as claimed in claim 1, in which the thiolatocobalamin is N-acetyl-L-cysteinylcobalamin, cysteinylcobalamin, glutathionylcobalamin (GSCbI), captopril-cobalamin, gamma-glutamylcysteinylCbl (gamma-GluCys-Cbl) and cysteinylglycinylcobalamin (CysGly-Cbl).

3. The use of a thiolatocobalamin compound in the manufacture of a medicament for the treatment of inflammation, cardiovascular disease, or endothelial dysfunction.

4. A use as claimed in claim 3, in which the thiolatocobalamin is N-acetyl-L- cysteinylcobalamin, cysteinylcobalamin, glutathionylcobalamin (GSCbI), captopril- cobalamin, gamma-glutamylcysteinylCbl (gamma-GluCys-Cbl) and cysteinylglycinylcobalamin (CysGly-Cbl).

5. A method of treatment for the inflammatory phase of the immune response comprising administering a composition comprising a thiolatocobalamin compound to a patient in need thereof.

6. A method of treatment for inflammation comprising administering a composition comprising a thiolatocobalamin compound to a patient in need thereof.

7. A method of treatment for a disease characterised by NOS depression, malfunction, or dysregulation comprising administering a composition comprising a thiolatocobalamin compound to a patient in need thereof.

8. A method as claimed in claim 7 in which the disease characterised by NOS depression, malfunction, or dysregulation is SIRS/sepsis/septic, traumatic or anaphylactic shock.

9. A method of treatment for a bacterial, viral or parasitidal disease characterised by NOS depression, malfunction, or dysregulation comprising administering a composition comprising a thiolatocobalamin compound to a patient in need thereof.

10. A method as claimed in claim 9 in which the bacterial disease is Tuberculosis, meningitis, MRSA, or other antibiotic resistant disease.

11. A method as claimed in claim 9 in which the viral disease is influenza type A, B or C.

12. A method as claimed in claim 11 in which the influenza is subtype H5N1.

13. A method as claimed in claim 9 in which the parasitidal disease is malaria.

14. A method as claimed in claim 7 in which the disease characterised by NOS depression, malfunction, or dysregulation is cancer.

15. A method as claimed in claim 7 in which the disease characterised by NOS depression, malfunction, or dysregulation is spinal injury.

16. A method of treatment for cardiovascular disease comprising administering a composition comprising a thiolatocobalamin compound to a patient in need thereof.

17. A method for the treatment of endothelial dysfunction comprising administering a composition comprising a thiolatocobalamin compound to a patient in need thereof.

18. A method as claimed in any one of claims 5 to 17 in which the thiolatocobalamin compound is N-acetyl-L-cysteinylcobalamin, cysteinylcobalamin, glutathionylcobalamin (GSCbI), captopril-cobalamin, gamma-glutamylcysteinylCbl (gamma-GluCys-Cbl) and cysteinylglycinylcobalamin (CysGly-Cbl).