PROTECTIVE METALLOTHIONEIN ANALOG COMPOUNDS, THEIR COMPOSITIONS AND USE THEREOF IN THE TREATMENT OF PATHOGENIC DISEASES

Abstract

Embodiments of the present invention relate generally the use of certain compositions, e.g., compositions comprising a glutathione precursor and a selenium source, in the therapy of viral diseases and/or reducing the incidence of viral diseases. Related embodiments of the present invention relate to treatment and/or reducing the incidence of respiratory ailments caused by respiratory syncytial virus (RSV) or hemorrhagic fever (EHF) caused by Ebola viruses (EBV) or Marburg virus. Yet in other embodiments, the invention relates to reducing metal toxicity in a biological system, which involves contacting the biological system with a composition comprising a glutathione precursor and a selenium source, optionally together with a chelating agent, an antioxidant, a metallothionein protein or a fragment of metallothionein.

Claims

1. A method for the treatment or reducing the incidence of a viral disease in a subject in need thereof, comprising administering to said subject, a composition comprising a glutathione precursor and a selenium compound optionally together with a metallothionein or a fragment thereof.

2. The method of claim 2, wherein the glutathione precursor comprises glycine, L-cysteine, and a glutamate source.

3. The method of claim 3, wherein the glutathione precursor comprises glycine, L-cysteine, and glutamine.

4. The method of claim 2, wherein the glutamate source is glutamine or glutamic acid.

5. The method of claim 1, wherein the selenium compound is selenomethionine, selenite, methylselenocysteine or selenium nanoparticles.

6. The method of claim 1, further comprising use of an Fe.sup.3+ chelator, a Zn.sup.2+ chelator, an Ni.sup.2+ chelator, or a combination thereof.

7. The method of claim 6, wherein the chelator is N,N,N,N-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), DPESA, TPESA, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), ethylenediamine-N,N-diacetic-N,N-di-P-propionic (EDPA), diethylene triamine pentaacetic acid (DETAPAC), dipyridyl, pyridoxal isonicotinoyl hydrazone (PIH), desferrioxamine (DFO), deferiprone (DFP), deferasirox (DFS) or a combination of any of these.

8. The method of claim 1, further comprising an antiviral agent selected from the group consisting of abacavir, aciclovir, acyclovir, adefovir, amantadine, amprenavir, arbidol, atazanavir, atripla, brivudine, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, entry inhibitors, famciclovir, fixed dose combinations, fomivirsen, fosamprenavir, foscarnet, fosfonet, fusion inhibitors, ganciclovir, gardasil, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitors, interferon type III, interferon type II, interferon type I, interferon, lamivudine, lopinavir, loviride, MK-0518, maraviroc, moroxydine, nelfinavir, nevirapine, nexavir, nucleoside analogues, oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitors, reverse transcriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir, stavudine, synergistic enhancers, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and zidovudine.

9. The method of claim 1, wherein the viral disease is caused by an Arenaviridae, Reoviridae, Rotaviridae, Retroviridae, Papillomaviridae, Influenza, Adenoviridae, Flaviviridae, Herpesviridae, Filoviridae, Pneumoviridae or Orthomyxoviridae virus.

10. The method of claim 1, wherein the viral disease is caused by a Pneumoviridae virus.

11. The method of claim 1, wherein the viral disease is caused by respiratory syncytial virus.

12. The method of claim 1, wherein the viral disease is caused by a Flaviviridae virus.

Description

EMBODIMENTS OF THE INVENTION

[0054] Accordingly, embodiments of the instant invention provide means for increasing the intracellular glutathione, can be effective competitively and physiologically extracting the metals and the co-factors (e.g., zinc) necessary for the propagation of viruses such as the Ebola virus. Without said elemental zinc, the virus cannot replicate, proliferate or survive.

[0055] In vitro chemical analyses have revealed that GSH is capable of binding to Zn.sup.2+ and Ni.sup.2+ with high affinity. See, Krezel et al. (Studies of Zinc(II) and Nickel(II) complexes of GSH, GSSG and their analogs shed more light on their biological relevance, Bioinorganic Chemistry & Applications; 2(3-4): 293-305, 2004), which is incorporated by reference herein in parts pertinent thereto. GSH is also capable of binding and thus sequestering Fe.sup.3+ ions. See Khan et al., Canadian Journal of Chemistry, 54(20): 3192-3199, 1976.

[0056] Although free glutathione might have sequestration capability in cell-free systems such as those described in Krezel and Khan, a variety of challenges are imposed in biological systems. For example, transition metals are not present in free states but rather bound to proteins in the form of complexes. Thus, glutathione is in direct competition with these proteins, e.g., ZFP or RR. Secondly, cellular absorption of glutathione is inefficient and thus intracellular glutathione levels are not appreciably increased by providing cells with free glutathione. Also, in the case of in vivo supplementation via the oral route, provision of free GSH is ineffective as the antioxidant is broken down in the gastrointestinal system of animals. Recognizing these and other limitations, the inventor of the instant application have contemplated novel ways to provide and ameliorate intracellular levels of glutathione. In accordance with the present invention, embodiments described herein provide compositions and means for using the physiological glutathione synthesis pathway for introducing intact glutathione into the cell and replenishing the cytosolic and other cellular compartments with glutathione. Herein, a distinction is drawn in this invention between using the step-by-step physiological synthesis of glutathione pathway as distinguished from other methods that would bypass the step-by-step physiological synthesis of glutathione. If the step-by-step pathway is bypassed, the bypassing process can eventually result in a weakened immune system and thus be counter-productive, by throwing the vital substrate-specific enzymes, which catalyze each step of the synthesis, into the vestigiality of disuse. In addition, if the physiological step-by-step synthesis pathway is avoided by the importation of intact glutathione, then the physiological regulatory feedback and shut-down mechanisms can be thrown into dysregulation. The biomarker of glutathione quantification would be lost, and its physiological regulation would become an uncertainty. The compositions and methods for increasing intracellular glutathione levels described herein avoid many of the aforementioned issues.

[0057] In protecting the host, the sulfhydryl moiety of physiologically synthesized glutathione competitively and effectively conjugates with the elemental metal such as zinc and copper and deprives the pathogenic virus of those metals, which the pathogenic viruses need to replicate and proliferate.

[0058] Accordingly, in an embodiment of the instant invention, the sulfhydryl group (SH group) of the physiological cytosolic glutathione, if not compromised by the risk of vestigiality, has the biochemical and physiological efficiency to outmaneuver a pathologic virus such as Ebola in order to deprive that pathogen of zinc which viruses such as Ebola virus need to foster budding and survival. If glutathione is not physiologically synthesized, e.g., if the glutathione is imported as an intact molecule into the cytosol avoiding the step-by-step synthesis process, such a procedure can eventually weaken the immune system and thus fail to achieve the goal of glutathione therapy.

[0059] In accordance with the instant invention, embodiments described herein relate to increasing intracellular GSH levels by providing the individual components of glutathione, e.g., glycine, glutamate source (glutamine or glutamate) and cystine (a source of cysteine) optionally together with a selenium source. In this context, one skilled in the art understands that L-cystine is a metabolite amino acid in the catabolism of protein. It is found in certain protein foods, such as lean beef, clams, veal, turkey, chicken, fish, crabs, lobster, et al. L-cystine is a compound of two amino acids, L-cysteine and L-cysteine, which have auto-oxidized into a unity via a disulfide bond which unites these two L-cysteines into the new chemical molecule. L-cystine has radically different properties from the L-cysteine molecules from which it is formed. L-cystine can also be anabolized from L-methionine. It has a vital role in the metabolism of Vitamin B6. It was previously thought that because L-cystine is relatively stable, by virtue of its disulfide bond, that it was inactive, effete, oxidized or used-up. See, Emory University Public Press Release Apr. 4, 2011 Measuring oxidative stress can predict risk of atrial fibrillation.

[0060] In contrast to solo cysteine, which has little bodily physiological and biochemical functions, L-cystine, in addition to the above, is vital to the formation of insulin, sperm cells, skeletal muscle, connective tissues, hair and certain enzymes. Further, the disulfide bond serves many vital bodily biochemical and physiological functions (see list below). The use and role of the auto-oxidation in L-cystine is an evolutionary adaptation of major significance. However, the scientific literature has only peripherally touched upon its importance. Rather than emphasizing its significance, the resulting auto-oxidized molecule, L-cystine, has often been classified as used-up cysteine or classified exclusively as a biomarker of oxidative stress, and as an indication of a pathological oxidized state. See Dhawan et al. (above); Patel et al.'s article entitled Oxidative stress is associated with impaired arterial elasticity.

[0061] Research has recently demonstrated that L-cystine exemplifies a pleiotropic paradox, and its role is vital in the synthesis of glutathione and certain other concomitant but unexpected results, such as the activation of the vital gene Nrf2. See the aforementioned publications by Sinha et al. On closer examination and upon extensive university research, other dimensions to L-cystine have been verified. It has been documented in the literature and in university research that L-cystine is stable and neutral and water insoluble, as compared to L-cysteine, which is highly oxidizable and somewhat toxic to the body. See Janaky et al. (Mechanisms of L-cysteine Neurotoxicity, Neurochemical Research. Vol. 25. Nos. 9/10, 2000, pp 1397-1405); Dilger et al. Excess dietary L-Cysteine, but not L-cystine, is lethal for chicks but not for rats or pigs, Journal of Nutrition, 2007 February; 137(2):331-8); Crum et al. Presentation before American Chemical Society, Aug. 21, 2007 entitled Sulfenic acid, sulfinic acid, sulfonic acid.

[0062] Although L-cysteine is the crucial and most valuable functional detox moiety of glutathione (considered the body's master antioxidant), getting the L-cysteine into the intracellular space, where it could enter into the glutathione synthesis chain, was for a long time considered a scientific enigma. When a highly oxidizable molecule such as L-cysteine, which has toxic features, is also vital for the physiological synthesis of glutathione, it can be comprehended that nature has adapted an evolutionary advantage to auto-oxidation of that molecule (L-cysteine) for its safe carriage to the intracellular milieu where it can be utilized for the physiological synthesis of glutathione. There have been other methods tried to get the highly-oxidizable L-cysteine into the cytosol, but with limited results. The synthetic ester, N-acetyl cysteine, has been used by scientists to reduce the high reactivity and high oxidizability of the solo L-cysteine, so as to enable it to reach the intracellular glutathione synthesis chain with less reactivity and less oxidizability. Large protein molecules from non-denatured whey have also been used in an effort to keep the highly reactive, highly oxidizable but rate-limiting L-cysteine in check until it could enter the intracellular space of the glutathione synthesis milieu.

[0063] The inventor of the instant application utilized the advantage of L-cystine's disulfide bond as the safe physiological carrier of L-cysteine as the method to accomplish this vital L-cysteine delivery role. Upon arrival at the cell wall, substrate-specific enzymes, oxidoreductase, and thioltransferase at the cell membranes and in the cytosolic milieu decouple the tenacious disulfide bond of L-cystine. The decoupling of the disulfide bond permits the released, free form L-cysteine to be available for incorporation into the reducing cytosolic media of the intracellular environment. Also present in the intracellular space is the substrate specific gamma-glutamylcysteine synthetase, readily available to catalyze a unity or L-cysteine to L-glutamic acid. Given the wide-spread perception that the disulfide bond were essentially fixed or irreversible, scientists had not realized or formulated the diverse physiological and biochemical potential of L-cystine in glutathione synthesis. A typical comment or conclusion was that Cystine is not suitable as an intracellular delivery agent (for L-cysteine) because of its marked insolubility. (P. 317 Methods in Enzymology, Volume 143.) Misconceptions have been made in limiting the functions of L-cystine to only a measurement or biomarker for oxidative stress. Attempts have been made to force a parallel interpretation of intracellular glutathione to extracellular L-cystine, because they both contain the sulfhydryl radical and are active in various redox functions. The sulfhydryl group in free form L-cysteine functions with different properties when it is in a solo amino acid as compared to when its sulfhydryl group is a moiety of glutathione.

[0064] In summary, solo L-cysteine has different, complex and paradoxical functions for its sulfhydryl that distinguish it from the sulfhydryl functions when it is a moiety of glutathione. A recent study has interpreted results that need further clarification. See, Patel et al., (Oxidative Stress is associated with impaired arterial elasticity. Atherosclerosis. 2011), which is incorporated by reference. Patel states Non-free radical oxidative stress was assessed as plasma oxidized and reduced amino-thiol levels (cysteine/cysteine, glutathione/GSSG) and their ratios (redox, potentials), and free radical oxidative stress as derivatives of reactive oxygen metabolites (dROMs).

[0065] In accordance with the foregoing analysis, the inventor herein have recognized that if physiologically synthesized by a step-by-step physiological synthesis pathway is followed, the resulting glutathione with only three amino acids and a cofactor (e.g., a selenium source such as selenomethionine or selenocysteine or a combination thereof in any ratio) can outmaneuver the other antioxidant systems. The human clinical trial for this adaptation has been received favorably. See, for example, National Clinical Trials with the accession No. NCTO1251315 and references related thereto.

[0066] In accordance with the present invention, the physiologically active intracellular glutathione described hereinbefore, in order to be immunologically protective in the long term, is synthesized in a step-by-step process. This involves provision of the three amino acid components (either simultaneously or separately), which are then taken up by the respective transporters and synthesized intracellularly. The cysteine component of GSH is preferably provided in a reduced, dipeptide (cystine) form.

[0067] Embodiments of the instant invention indicate that the physiological glutathione synthesized by the aforementioned step-by-step process is better than whole glutathione molecule, e.g., with regard to chelation (and sequestration) of metal ions and the concomitant inhibition of viral replication. In contrast, whole glutathione is less effective because the provision thereof can throw the substrate specific enzymes into a vestigiality of disuse and further result in the dysregulation of the regulatory mechanism of physiological glutathione quantification levels. In fact, credible evidence suggests that the composition of the instant invention comprising the three component amino acids (glutamine or glutamate, cystine and glycine) and a cofactor (e.g., a selenium source such as selenomethionine or selenocysteine or a combination thereof in any ratio) is superior to other cellular thiol-antioxidants such as N-acetyl cysteine, -lipoic acid, etc.

[0068] If the immune system is weakened and the glutathione is low or vestigially compromised, then the host's protective edge is impaired, unable to take molecular control of the zinc in the case of Ebola, and conceding the advantage to the pathogenic virus. If the immune system is robust with physiologically constituted glutathione, the glutathione will provide metallothionein-like protection and the host triumphs against pathogenic viruses biochemically and physiologically. The sulfhydryl of physiologically constituted glutathione is more effective for the conjugation of zinc, than the pathogenic virus for the adherence of this vital metal.

Reducing Metal Toxicity

[0069] In related embodiments, the instant invention provides novel and inventive means for reducing the toxicity caused by metal ions (e.g., due to dysregulation of iron, nickel and/or zinc homeostasis or due to pathogenic conditions) on biological systems. The methods involving contacting the afflicted biological system, which is a cell, a tissue, an organ, or an organism (e.g., a human or a non-human animal) with the aforementioned compositions. Preferably, the compositions comprise glycine, glutamate source (glutamine or glutamic acid) and L-cystine, optionally together with a selenium source (e.g., selenomethionine, selenocysteine, or selenium particles). Further optionally, the compositions may contain additional chelator of Zn.sup.2+, Fe.sup.2+ or Ni.sup.2+, or a combination of such chelators. Preferably, the chelators are bio-compatible and have dissociation constants that are lower than those of proteins which bind to the metal ions (e.g., RR or ZFP). Representative examples of such chelators include, for example, zinc chelators such as N,N,N,N-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), DPESA, TPESA, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), and ethylenediamine-N,N-diacetic-N,N-di--propionic (EDPA), etc. and iron chelators include diethylene triamine pentaacetic acid (DETAPAC), dipyridyl, pyridoxal isonicotinoyl hydrazone (PIH), desferrioxamine (DFO), deferiprone (DFP) or deferasirox (DFS). A combination of such chelators may also be employed.

Definitions

[0070] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0071] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3.sup.rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N. Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5.sup.th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3.sup.rd edition (Cold Spring Harbor Laboratory Press (2002)).

[0072] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or () by increments of 0.1 or 1.0, where appropriate. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term about. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0073] As used in the specification and claims, the singular forms a, an and the include plural references unless the context clearly dictates otherwise. For example, the term a cell includes a plurality of cells, including mixtures thereof.

[0074] As used herein, the term comprising or comprises is intended to mean that the compositions and methods include the recited elements, but not excluding others. Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this invention.

[0075] As is known to those of skill in the art, there are six classes of viruses. The DNA viruses constitute classes I and II. The RNA viruses and retroviruses make up the remaining classes. Class III viruses have a double-stranded RNA genome. Class IV viruses have a positive single-stranded RNA genome, the genome itself acting as mRNA Class V viruses have a negative single-stranded RNA genome used as a template for mRNA synthesis. Class VI viruses have a positive single-stranded RNA genome but with a DNA intermediate not only in replication but also in mRNA synthesis. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.

[0076] Virus includes any infectious agent that relies on a host for replication. Included in this definition are virions, viral particles, and mature viruses, which are either naturally-occurring or synthetic in nature. Representative examples include members of Arenaviridae, Reoviridae, Rotaviridae, Retroviridae, Papillomavirinae, Influenza, Adenoviridae, Flaviviridae (Hepatitis C), Herpesviridae, Filoviridae (e.g., Ebola virus and Marburg virus), Pneumovirinae (e.g., RSV), Orthomyxoviridae (Influenza viruses), etc. In this context, it should be recognized that Ebola virus is a member of the Filovirus family. Others include, but are not limited to Marburg viruses, Cuevavirus and the like.

[0077] The infectivity of a virus intends the ability of the virus to infect the host. Viral infection is affected by the infectivity, replicative fitness, and the ability of the virus to evade the host's immune response and develop resistance to antivirals.

[0078] Chelation intends the formation or presence of two or more separate bindings between a polydentate ligand and a single central atom. A chelant or chelator refers to a chemical that form a soluble and complex molecule with certain metal ions, inactivating the ions so that they cannot normally react with other elements or ions to produce precipitates or scale.

[0079] A zinc chelator refers to a chelator that chelates with zinc ions, e.g., Zn.sup.2+. An iron chelator refers to a chelator that chelates with iron ions, e.g., Fe.sup.2+/Fe.sup.3+. Non-limiting examples of zinc chelators include N,N,N,N-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), DPESA, TPESA, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), and ethylenediamine-N,N-diacetic-N,N-di--propionic (EDPA), etc. Non-limiting examples of iron chelators include diethylene triamine pentaacetic acid (DETAPAC), dipyridyl, pyridoxal isonicotinoyl hydrazone (PIH), desferrioxamine (DFO), deferiprone (DFP) or deferasirox (DFS) which chelates iron and inhibits metal-catalyzed reactions that produce free radical and non-radical reactive species.

[0080] The terms polynucleotide and oligonucleotide are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0081] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics.

[0082] A gene refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated. Any of the polynucleotide or polypeptide sequences described herein may be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art.

[0083] The term express refers to the production of a gene product. As used herein, expression refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA.

[0084] A gene product or alternatively a gene expression product refers to the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.

[0085] The term encode as it is applied to polynucleotides refers to a polynucleotide which is said to encode a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof.

[0086] A probe when used in the context of polynucleotide manipulation refers to an oligonucleotide that is provided as a reagent to detect a target potentially present in a sample of interest by hybridizing with the target. Usually, a probe will comprise a detectable label or a means by which a label can be attached, either before or subsequent to the hybridization reaction. Alternatively, a probe can be a biological compound such as a polypeptide, antibody, or fragments thereof that is capable of binding to the target potentially present in a sample of interest. Detectable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins (e.g., enzymes).

[0087] The term propagate means to grow a cell or population of cells. The term growing also refers to the proliferation of cells in the presence of supporting media, nutrients, growth factors, support cells, or any chemical or biological compound necessary for obtaining the desired number of cells.

[0088] The term culturing refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell.

[0089] A composition is intended to mean a combination of an active ingredient (e.g., individual components of the aforementioned metallothionein analogs) and another compound or composition, wherein the second component may be inert (e.g., a carrier) or active (e.g., another metal chelator).

[0090] For convenience, the term selenium is sometimes used hereinafter to include any of the various water-soluble selenium products which can be transported through the mucosal membrane in the practice of this invention. It will be understood, however, that the particular forms of selenium compounds set forth herein are not to be considered limitative. Other selenium compounds, which exhibit the desired activity and are compatible with the other components in the mixture and are non-toxic, can be used in the practice of the invention. Many of them are available commercially.

[0091] An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which cause oxidative stress and start chain reactions that damage cells. Oxidative stress is caused by an imbalance between the production of reactive oxygen and a biological system's ability to readily detoxify the reactive intermediates or easily repair the resulting damage. All forms of life maintain a reducing environment within their cells. This reducing environment is preserved by enzymes that maintain the reduced state through a constant input of metabolic energy. Disturbances in this normal redox state can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions by being oxidized themselves. Examples of antioxidants include, but are not limited to, glutathione, N-acetylcysteine, ascorbic acid, vitamin E, beta-carotene, a polyphenol, flavonoid and an agent that decreases the generation of free radical and non-radical reactive species, including, for example, a CYP2E1 inhibitor, an NAD(P)H oxidase inhibitor or a nitric oxide synthase inhibitor.

[0092] Ascorbic acid or vitamin C refers a monosaccharide antioxidant found in both animals and plants. As one of the enzymes needed to make ascorbic acid has been lost by mutation during human evolution, it must be obtained from the diet and is a vitamin. Most other animals are able to produce this compound in their bodies and do not require it in their diets. In cells, it is maintained in its reduced form by reaction with glutathione, which can be catalyzed by protein disulfide isomerase and glutaredoxins. Ascorbic acid is a reducing agent and can reduce, and thereby neutralize, reactive oxygen species such as hydrogen peroxide. In addition to its direct antioxidant effects, ascorbic acid is also a substrate for the antioxidant enzyme ascorbate peroxidase, a function that is particularly important in stress resistance in plants. Ascorbic acid is present at high levels in all parts of plants and can reach concentrations of 20 millimolar in chloroplasts. Ascorbic acid can be used in combination with iron chelator because it can act as a pro-oxidant in the presence of iron by reducing iron to Fe2+, which would increase the generation of potent oxidants that would damage the nucleic acids.

[0093] Glutathione intends a cysteine-containing peptide found in most forms of aerobic life. It is not required in the diet and is instead synthesized in cells from its constituent amino acids. Glutathione has antioxidant properties since the thiol group in its cysteine moiety is a reducing agent and can be reversibly oxidized and reduced. In cells, glutathione is maintained in the reduced form by the enzyme glutathione reductase and in turn reduces other metabolites and enzyme systems, such as ascorbate in the glutathione-ascorbate cycle, glutathione peroxidases and glutaredoxins, as well as reacting directly with oxidants. In some organisms glutathione is replaced by other thiols, such as by mycothiol in the Actinomycetes, or by trypanothione in the kinetoplastids. Plasma and liver glutathione concentrations can be raised by oral administration of S-adenosylmethionine (SAMe). Glutathione precursors rich in cysteine include N-acetylcysteine (NAC) and undenatured whey protein, and these supplements have been shown to increase glutathione content within the cell. N-Acetylcysteine, is available both as a drug and as a generic supplement. Alpha Lipoic Acid has also been shown to restore intracellular glutathione. Melatonin has been shown to stimulate a related enzyme, glutathione peroxidase, and silymarin or milk thistle has also demonstrated an ability to replenish glutathione levels. Of all of these methods, the two methods that are the most thoroughly researched for efficacy in raising intracellular glutathione are variants of cysteine. N-acetyl-cysteine, which is a pharmaceutical over the counter drug, and bonded cysteine as is found in the undenatured whey protein nutraceutical, are both proven to be efficacious in raising glutathione values. Also, glutathione can be supplied in the form of glutathione esters.

[0094] Melatonin, known chemically as N-acetyl-5-methoxytryptamine, refers to a naturally occurring hormone found in animals and in some other living organisms, including algae.

[0095] Vitamin E is the collective name for a set of eight related tocopherols and tocotrienols, which are fat-soluble vitamins with antioxidant properties. A non-limiting example, .alpha.-tocopherol has been most studied as it has the highest bioavailability, with the body preferentially absorbing and metabolizing this form. .alpha.-tocopherol protects membranes from oxidation by reacting with lipid radicals produced in the lipid peroxidation chain reaction. This removes the free radical intermediates and prevents the propagation reaction from continuing. This reaction produces oxidized .alpha.-tocopheroxyl radicals that can be recycled back to the active reduced form through reduction by other antioxidants, such as ascorbate, retinol or ubiquinol. This is in line with findings showing that .alpha.-tocopherol, but not water-soluble antioxidants, efficiently protects glutathione peroxidase 4 (GPX4)-deficient cells from cell death. Vitamin E is available from dietary sources such as asparagus, avocado, egg, milk, nuts, seeds, spinach, unheated vegetable oil, wheat germ or wholegrain foods.

[0096] A pharmaceutical composition is intended to include the combination of an active polypeptide, polynucleotide or antibody with a carrier, inert or active such as a solid support, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0097] As used herein, the term pharmaceutically acceptable carrier encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin (1975) Remington's Pharm. Sci., 15th Ed. (Mack Publ. Co., Easton).

[0098] A subject, individual or patient is used interchangeably herein, and refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, rats, rabbit, simians, bovines, ovine, porcine, canines, feline, farm animals, sport animals, pets, equine, and primate, particularly human. Besides being useful for human treatment, the present invention is also useful for veterinary treatment of companion mammals, exotic animals and domesticated animals, including mammals, rodents, and the like which is susceptible to viral infection. In one embodiment, the mammals include horses, dogs, and cats. In another embodiment of the present invention, the human is an adolescent or infant under the age of eighteen years of age.

[0099] The terms disease, disorder, and condition are used inclusively and refer to any condition mediated at least in part by infection by a pathogenic agent such as viruses, bacteria or the like.

[0100] As used herein, the term treatment is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. Treating or treatment of a disease includes: (1) preventing the disease, i.e., causing the clinical symptoms of the disease not to develop in a patient that may be predisposed to the disease but does not yet experience or display symptoms of the disease; (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; or (3) relieving the disease, i.e., regression of the disease or its clinical symptoms.

[0101] The term suffering as it related to the term treatment refers to a patient or individual who has been diagnosed with or is predisposed to infection or a disease incident to infection. A patient may also be referred to being at risk of suffering from a disease because of active or latent infection. This patient has not yet developed characteristic disease pathology.

[0102] An effective amount is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents of the present invention for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. Treatment dosages generally may be titrated to optimize safety and efficacy. Typically, dosage-effect relationships from in vitro and/or in vivo tests initially can provide useful guidance on the proper doses for patient administration. In general, one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with the concentrations found to be effective in vitro. Determination of these parameters is well within the skill of the art. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks. Consistent with this definition, as used herein, the term therapeutically effective amount is an amount sufficient to inhibit RNA virus replication in vitro or in vivo. Prophylactically effective as used herein means the amount of the composition which is sufficient to achieve the desired result, for example, to reduce the incidence of viral infection in a particular subject or a subject population.

[0103] As used herein, the term reduced intends a lower level as compared to a control or a prior measurement or value. In one aspect, a reduced mutation rate of an RNA virus in a cell treated with an iron chelator or an antioxidant refers to a level of mutation rate that is lower than the level of mutation rate of the RNA virus in a cell not treated with the iron chelator or the antioxidant or alternatively, prior to such treatment. In another aspect, it is a lower mutation rate as compared to treatment with another, different agent, alone or in combination with the iron chelator or the antioxidant. Reduced intends a reduction by at least about 5%, or alternatively about 10%, or alternatively about 15%, or alternatively about 20%, or alternatively about 25%, or alternatively about 30%, or alternatively about 35%, or alternatively about 40%, or alternatively about 45%, or alternatively about 50%, or alternatively about 55%, or alternatively about 60%, or alternatively about 65%, or alternatively about 70%, or alternatively about 75%, or alternatively about 80%, or alternatively about 85%, or alternatively about 90%, or alternatively about 95%, or alternatively or about 100% as compared to a control or prior measurement or value.

[0104] As used herein, the term enhanced intends a higher level as compared to a control or a prior measurement or value. In one aspect, an enhanced efficacy of an agent or a therapy to reduce or prevent infection of a cell by an RNA virus, which cell is treated with an iron chelator or an antioxidant, is a higher efficacy as compared to the agent or therapy to reduce or prevent infection of the cell by the RNA virus, which cell is not treated with the iron chelator or the antioxidant. In another aspect, it is a higher efficacy as compared to treatment with another, different agent, alone or in combination with the iron chelator or the antioxidant. Enhanced intends an increase by at least about 5%, or alternatively about 10%, or alternatively about 15%, or alternatively about 20%, or alternatively about 25%, or alternatively about 30%, or alternatively about 35%, or alternatively about 40%, or alternatively about 45%, or alternatively about 50%, or alternatively about 55%, or alternatively about 60%, or alternatively about 65%, or alternatively about 70%, or alternatively about 75%, or alternatively about 80%, or alternatively about 85%, or alternatively about 90%, or alternatively about 95%, or alternatively or about 100%, as compared to a control or prior measurement or value.

[0105] Pharmaceutically acceptable means one that is generally recognized as safe, approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0106] The term administration shall include without limitation, administration by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant), by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration. The invention is not limited by the route of administration, the formulation or dosing schedule.

[0107] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Specific Embodiments

[0108] A few of the many embodiments encompassed by the present description are summarized in the following numbered paragraphs. The numbered paragraphs are self-referential. In particular, the phase in accordance with any of the foregoing or the following used in these paragraphs refers to the other paragraphs. The phrase means in the following paragraphs embodiments herein disclosed include both the subject matter described in the individual paragraphs taken alone and the subject matter described by the paragraphs taken in combination. In this regard, the purpose in setting forth the following paragraphs to describe various aspects and embodiments particularly by the paragraphs taken in combination. That is, the paragraphs are a compact way of setting out and providing explicit written description of all the embodiments encompassed by them individually and in combination with one another. As such, any subject matter set out in any of the following paragraphs, alone or together with any other subject matter of any one or more other paragraphs, including any combination of any values therein set forth taken alone or in any combination with any other value set forth, may be presented.

Formulations/Compositions

[0109] Composition 1. A composition comprising a glutathione (GSH) precursor and a selenium source.

[0110] Composition 2. The composition in accordance with the foregoing or the following, wherein the glutathione precursor comprises glycine, L-cystine and a glutamate source.

[0111] Composition 3. The composition in accordance with the foregoing or the following, wherein the glutathione precursor comprises glycine, L-cystine and glutamate.

[0112] Composition 4. The composition in accordance with the foregoing or the following, wherein the glutamine source is glutamate (Glu) or glutamine (Gln).

[0113] Composition 5. The composition in accordance with the foregoing or the following, which is a pharmaceutical composition comprising a carrier, a solvent, an excipient, a surfactant or an emollient and optionally further comprising an additional pharmaceutical agent.

[0114] Composition 6. The composition in accordance with the foregoing or the following, wherein the selenium source is selenonomethionine, selenite, methylselenocysteine, or selenium nanoparticles.

[0115] Composition 7. The composition in accordance with the foregoing or the following, further comprising an additional pharmaceutical agent which is N-acetylcysteine, vitamin C, vitamin E, -lipoic acid, folic acid, vitamins B6 and B12, silibinin, resveratrol or a combination thereof.

[0116] Composition 8. The composition in accordance with the foregoing or the following, further comprising a metallothionein or a fragment thereof.

[0117] Composition 9. A combination compensating at least two of the aforementioned compositions.

[0118] Composition 10. A composition in accordance with the foregoing or the following, which is a pharmaceutical composition.

[0119] Composition 11. A composition in accordance with the foregoing or the following, further comprises a metal chelator.

[0120] Composition 12. A composition in accordance with the foregoing or the following, which further comprises a Zn.sup.2+ chelator, a Fe.sup.3+ chelator, a Ni.sup.2+ chelator, a combination thereof.

[0121] Composition 13. A composition in accordance with the foregoing or the following, wherein the chelator is N,N,N,N-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), DPESA, TPESA, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), and ethylenediamine-N,N-diacetic-N,N-di--propionic (EDPA), diethylene triamine pentaacetic acid (DETAPAC), dipyridyl, pyridoxal isonicotinoyl hydrazone (PIH), desferrioxamine (DFO), deferiprone (DFP) or deferasirox (DFS) or a combination thereof.

[0122] Composition 14. A composition in accordance with the foregoing or the following, which further comprises an antiviral selected from the group consisting of abacavir, aciclovir, acyclovir, adefovir, amantadine, amprenavir, arbidol, atazanavir, atripla, brivudine, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, entry inhibitors, famciclovir, fixed dose combinations, fomivirsen, fosamprenavir, foscamet, fosfonet, fusion inhibitors, ganciclovir, gardasil, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitors, interferon type III, interferon type II, interferon type I, interferon, lamivudine, lopinavir, loviride, MK-0518, maraviroc, moroxydine, nelfinavir, nevirapine, nexavir, nucleoside analogues, oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitors, reverse transcriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir, stavudine, synergistic enhancers, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and zidovudine.

Kits

[0123] Kit 1. A kit comprising, in one or separate compartments or packages, a glutathione (GSH) precursor and a selenium source, optionally together with an excipient, carrier or oil.

[0124] Kit 2. The kit in accordance with any of the foregoing or the following, comprising the glutathione precursor in one compartment and a selenium source in another compartment.

[0125] Kit 3. The kit in accordance with any of the foregoing or the following, comprising an additional pharmaceutical agent which is N-acetylcysteine, vitamin C, vitamin E, -lipoic acid, folic acid, vitamins B6 and B12, silibinin, resveratrol or a combination at least two of the additional agents.

[0126] Kit 4. The kit in accordance with any of the foregoing or the following, further comprising instructions for formulating a composition comprising said glutathione (GSH) precursor and a selenium source.

[0127] Kit 5. The kit in accordance with any of the foregoing or the following, further comprising instructions for using the components, either individually or together, for the treatment of pathogenic diseases.

[0128] Kit 6. The kit in accordance with any of the foregoing or the following, further comprising instructions for using the components, either individually or together, for the treatment of viral diseases.

[0129] Kit 7. The kit in accordance with any of the foregoing or the following, further comprising instructions for using the components, either individually or together, for reducing the incidence of viral diseases.

[0130] Kit 8. The kit in accordance with any of the foregoing or the following, further comprising a metallothionein or a fragment thereof.

[0131] Kit 9. The kit in accordance with any of the foregoing or following, further comprising an antiviral agent selected from the group consisting of abacavir, aciclovir, acyclovir, adefovir, amantadine, amprenavir, arbidol, atazanavir, atripla, brivudine, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, entry inhibitors, famciclovir, fixed dose combinations, fomivirsen, fosamprenavir, foscamet, fosfonet, fusion inhibitors, ganciclovir, gardasil, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitors, interferon type III, interferon type II, interferon type I, interferon, lamivudine, lopinavir, loviride, MK-0518, maraviroc, moroxydine, nelfinavir, nevirapine, nexavir, nucleoside analogues, oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitors, reverse transcriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir, stavudine, synergistic enhancers, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and zidovudine.

Method of Treating

[0132] Treatment 1. A method of treating a disease associated with viral infection or reducing the incidence of infection associated with viral infection in a subject in need thereof comprising employing the composition in accordance with any of the foregoing or the following.

[0133] Treatment 2. The method for the treatment or reducing the incidence of a viral disease in a subject in accordance with any of the foregoing or the following, which is for the treatment of a viral disease.

[0134] Treatment 3. The method for the treatment or reducing the incidence of a viral disease in a subject in accordance with any of the foregoing or the following, wherein said composition additionally comprises a pharmaceutically acceptable carrier, excipient, emollient, surfactant or solvent.

[0135] Treatment 4. The method for the treatment or reducing the incidence of a viral disease in a subject in accordance with any of the foregoing or the following, wherein said composition is a pharmaceutical composition for oral administration, topical administration, nasal administration, sublingual administration, buccal administration, intravenous administration, surgical administration, anal administration or vaginal administration.

[0136] Treatment 5. The method for the treatment or reducing the incidence of a viral disease in a subject in accordance with any of the foregoing or the following, wherein said subject is a human or a non-human mammal.

[0137] Treatment 6. A method for the treatment or reducing the incidence of a viral disease in a subject in accordance with the foregoing or following, further comprising administering N-acetylcysteine, vitamin C, vitamin E, -lipoic acid, folic acid, vitamins B6 and B12, silibinin, resveratrol or a combination thereof.

[0138] Treatment 7. A method for the treatment or reducing the incidence of a viral disease in a subject in need thereof, comprising administering to said subject a composition comprising a glutathione precursor and a selenium source.

[0139] Treatment 8. The method for the treatment or reducing the incidence of a viral disease in accordance with the foregoing or the following, wherein the glutathione precursor comprises glycine, L-cystine and a glutamate source.

[0140] Treatment 9. The method for the treatment or reducing the incidence of a viral disease in accordance with the foregoing or the following, wherein the selenium source is selenocysteine or selenomethionine.

[0141] Treatment 10. The method for the treatment or reducing the incidence of a viral disease in accordance with the foregoing or the following, wherein the composition further comprises a metallothionein or a fragment thereof.

[0142] Treatment 11. The method for the treatment or reducing the incidence of a viral disease in accordance with the foregoing or the following, wherein the virus is of the family Arenaviridae, Reoviridae, Rotaviridae, Retroviridae, Papillomavirinae, Influenza, Adenoviridae, Flaviviridae (Hepatitis C), Herpesviridae, Filoviridae, Pneumovirinae, or Orthomyxoviridae.

[0143] Treatment 12. The method for the treatment or reducing the incidence of a viral disease in accordance with the foregoing or the following, wherein the virus is Ebola virus, Marburg virus, influenza virus, or respiratory syncytial virus (RSV).

[0144] Treatment 13. The method for the treatment or reducing the incidence of a viral disease in accordance with the foregoing or the following, further comprising administering, to a subject in need thereof, a metal chelator.

[0145] Treatment 14. The method for the treatment or reducing the incidence of a viral disease in accordance with the foregoing or the following, further comprising administering, to a subject in need thereof, an Fe.sup.3+ chelator, a Zn.sup.2+ chelator, an Ni.sup.2+ chelator, or a combination thereof.

[0146] Treatment 15. The method for the treatment or reducing the incidence of a viral disease in accordance with the foregoing or the following, wherein the chelator is N,N,N,N-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), DPESA, TPESA, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), and ethylenediamine-N,N-diacetic-N,N-di--propionic (EDPA), diethylene triamine pentaacetic acid (DETAPAC), dipyridyl, pyridoxal isonicotinoyl hydrazone (PIH), desferrioxamine (DFO), deferiprone (DFP) or deferasirox (DFS) or a combination thereof.

[0147] Treatment 16. The method for the treatment or reducing the incidence of a viral disease in accordance with the foregoing or the following, further comprising an antiviral agent selected from the group consisting of abacavir, aciclovir, acyclovir, adefovir, amantadine, amprenavir, arbidol, atazanavir, atripla, brivudine, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, entry inhibitors, famciclovir, fixed dose combinations, fomivirsen, fosamprenavir, foscamet, fosfonet, fusion inhibitors, ganciclovir, gardasil, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitors, interferon type III, interferon type II, interferon type I, interferon, lamivudine, lopinavir, loviride, MK-0518, maraviroc, moroxydine, nelfinavir, nevirapine, nexavir, nucleoside analogues, oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitors, reverse transcriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir, stavudine, synergistic enhancers, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and zidovudine.

[0148] Treatment 17. The method for the treatment or reducing the incidence of a viral disease in accordance with the foregoing or the following, wherein the viral disease is cancer.

[0149] Treatment 18. The method for the treatment or reducing the incidence of a viral disease in accordance with the foregoing or the following, wherein the cancer is Kaposi's sarcoma, Burkett's lymphoma, adult T-cell leukemia, Merkel cell carcinoma, papilloma-virus induced cancers of cervix, vulva, vagina, penis, anus, and nasopharyngeal carcinoma.

Methods of Reducing Toxicity

[0150] Toxicity 1. A method for reducing iron, nickel or zinc toxicity in a biological system, comprising contacting said biological system with the composition in accordance with the foregoing or following.

[0151] Toxicity 2. A method for reducing iron, nickel or zinc toxicity in a biological system, comprising administering to said subject a composition comprising a glutathione precursor and a selenium source.

[0152] Toxicity 3. The method for reducing iron, nickel or zinc toxicity in a biological system in accordance with the foregoing or the following, wherein the glutathione precursor comprises glycine, L-cystine and a glutamate source.

[0153] Toxicity 4. The method for reducing iron, nickel or zinc toxicity in a biological system in accordance with the foregoing or the following, wherein the selenium source is selenocysteine or selenomethionine.

[0154] Toxicity 5. The method for reducing iron, nickel or zinc toxicity in a biological system in accordance with the foregoing or the following, wherein the composition further comprises a metallothionein or a fragment thereof.

[0155] Toxicity 6. The method for reducing iron, nickel or zinc toxicity in a biological system in accordance with the foregoing or the following, wherein the system is a cellular system, a tissue system, an organ system, or an organism.

[0156] Toxicity 7. The method for reducing iron, nickel or zinc toxicity in a biological system in accordance with the foregoing or the following, wherein the toxicity is due to dysregulated iron, nickel or zinc homeostasis.

[0157] Toxicity 8. The method for reducing iron, nickel or zinc toxicity in a biological system in accordance with the foregoing or the following, further comprising administering, to a subject in need thereof, a metal chelator.

[0158] Toxicity 9. The method for reducing iron, nickel or zinc toxicity in a biological system in accordance with the foregoing or the following, further comprising administering, to a subject in need thereof, an Fe.sup.3+ chelator, a Zn.sup.2+ chelator, an Ni.sup.2+ chelator, or a combination thereof.

[0159] Toxicity 10. The method for reducing iron, nickel or zinc toxicity in a biological system in accordance with the foregoing or the following, wherein the chelator is N,N,N,N-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN), DPESA, TPESA, ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), and ethylenediamine-N,N-diacetic-N,N-di--propionic (EDPA), diethylene triamine pentaacetic acid (DETAPAC), dipyridyl, pyridoxal isonicotinoyl hydrazone (PIH), desferrioxamine (DFO), deferiprone (DFP) or deferasirox (DFS) or a combination thereof.

[0160] Toxicity 10. A method for reducing iron, nickel or zinc toxicity in a biological system in accordance with the foregoing or following, further comprising administering N-acetylcysteine, vitamin C, vitamin E, -lipoic acid, folic acid, vitamins B6 and B12, silibinin, resveratrol or a combination thereof.

[0161] According to one embodiment of the present invention there is provided a method of treatment of viral diseases comprising administering to a subject in need of such treatment an effective amount of a composition comprising components for increasing intracellular glutathione (GSH [reduced form] or GSSG [oxidized form]) and a selenium source. The individual components of the composition are disclosed in detail in Crum et al. (US patent app. pub. No. 2012-0029082), which is incorporated by reference herein in its entirety.

[0162] As detailed in the aforementioned Crum et al., the individual components of the compositions include: (1) three amino acids which serve as precursors of glutathione, i.e., glycine, L-cysteine (as L-cystine) and glutamate (which can, in turn, be provided in the form of glutamic acid or glutamine). These components are the precursors of the metallothionein analogs described herein.

[0163] The glutathione precursor includes, individual components, e.g., glutamic acid, cystine (as the cysteine source) and glycine, or one or more biological precursors thereof (e.g., glutamate [Glu] or glutamine [Gln] as a precursor of glutamic acid; cysteine [Cys], including modified cysteine derivatives such as N-acteylcysteine [NAC], as a source of cysteine for the cystine, etc.). Other usable forms of the GSH component compounds include, for example, salts, esters, anhydrides, tautomers or analogs of glutamic acid, cystine and glycine. The aforementioned components of the compositions of the instant invention can be administered simultaneously, sequentially or separately to a subject in need of such treatment.

[0164] All ammo acids employed in this invention, except glycine which does not form optical isomers, are m the natural or L-form. The individual components of the metallothionein analogs may be provided in singularity (e.g., as a mixture of the individual components in the desired ratio) or in one or more separate packages.

Selenium Source

[0165] The compositions of the invention also include a selenium source, which serves as a co-factor in the synthesis of GSH. Selenium is one of numerous trace metals found in many foods. The compositions may optionally comprise a selenium containing amino acid such as selenomethionine or selenocysteine. The composition may also contain other amino acids, such as, for example, methionine, arginine, oxoproline, and the like. These optional components may be provided together with, or separate from, the individual components of GSH, i.e., glycine, cystine, and glutamate.

[0166] In the compositions of this invention, selenium may be employed as one of several non-toxic, water-soluble organic or inorganic selenium compounds capable of being absorbed through the mucosal membrane. Representative examples of the selenium source include, but are not limited to selenomethionine, selenite, methylselenocysteine, selenium nanoparticles, including salts, esters, anhydrides, tautomers or analogs, etc. of the individual selenium sources.

[0167] Representative examples of inorganic selenium compounds are aliphatic selenium metal salts containing selenium in the form of selenite or selenate anions. However, organic selenium compounds are also employable because they are normally less toxic than their inorganic counterparts. Other selenium compounds which may be mentioned by way of example include selenium cystine, selenium methionine, mono- and di-seleno carboxylic acids with about seven to eleven carbon atoms in the chain. Seleno-amino acid chelates are also useful. These selenium compounds may be considered for use in the present invention as selenium particles or salts thereof. Representative examples are known in the art. See Kojouri et al. The Effects of Oral Consumption of Selenium Nanoparticles on Chemotactic and Respiratory Burst Activities of Neutrophils in Comparison with Sodium Selenite in Sheep, Biol Trace Elem Res. May 2012; 146(2): 160-166.

[0168] Although any ratiometric amounts of the individual components of the GSH precursor may be employed, it will be apparent to those skilled in the art that the optimum ratio of glutamic acid to cystine to glycine in the novel compositions described herein is between 0.5:1.0:0.5 (or 1:2:1) to 1:0.5:1 (or 2:1:2), including all ratiometric values in between, e.g., 1.1:2.0:1.1, 1.2:2.0:1.2, 1.3:2.0:1.3, 1.4:2.0:1.4, 1.5:2.0:1.5, 1.6:2.0:1.6, 1.7:2.0:1.7, 1.8:2.0:1.8, 1.9:2.0:1.9, 1.0:1.0:1.0, 1.1:1.0:1.1, 1.2:1.0:1.2, 1.3:1.0:1.3, 1.4:1.0:1.4, 1.5:1.0:1.5, 1.6:1.0:1.6, 1.7:1.0:1.7, 1.8:1.0:1.8, 1.9:1.0:1.9, 2.0:1.0:2.0, etc. If an excess of any acid is used, it will presumably be of nutritional value or may simply be metabolized.

[0169] As will be apparent to the skilled artisan, owing to the toxicity of the selenium compound, the dosage units for mammalian administration by any selected route will cater to avoiding treatment either with single or multiple dosages of the toxic compound and the dosage of the selenium compound will be adjusted so that the total delivery does not reach the toxic limit of 400 g/day for humans (Institute of Medicine, Food and Nutrition Board. Dietary Reference Intakes: Vitamin C, Vitamin E, Selenium, and Carotenoids. National Academy Press, Washington, D C, 2000).

[0170] The recommended daily allowances for selenium as reported in The Pharmacological Basis of Therapeutics, 9.sup.th Ed., The McGraw-Hill Companies, 1996 are shown in Table 1 below:

TABLE-US-00001 TABLE 1 Recommended daily allowances for selenium. Subject Age/Years Dose/g Infants 0.0-0.5 10 0.5-1.0 15 Children 1.0-3.0 20 4.0-6.0 20 7.0-10.0 30 Males 11.0-14.0 40 15.0-18.0 50 19.0-24.0 70 25.0-50.0 70 51+ 70 Females 11.0-14.0 45 15.0-18.0 50 19.0-24.0 55 25.0-50.0 55 51+ 55 Pregnant 65 Lactating 1.sup.st six mo. 75 2.sup.nd six mo. 75

[0171] The recommended daily dosage for humans therefore ranges from 10 to 75 g per day and any range or value in between, including, but not limited to, 15 to 70 g/day, 20 to 60 g/day, 25 to 50 g/day, 30 to 40 g/day, etc. For animals the range may be generally higher but will, of course, depend upon the animal and its size.

[0172] The precise amount of the therapeutically useful compositions of this invention for daily delivery and the duration of the period of such delivery will depend upon the professional judgment of the physician or veterinarian in attendance. Numerous factors will be involved in that judgment such as age, body weight, physical condition of the patient or animal and the ailment or disorder being treated.

[0173] It is important for the practice of this invention that the selenium as employed in the composition be capable of transport through the mucosal membrane of the patient under treatment. For this reason, water insoluble selenium compounds are not generally useful.

[0174] Preferably, the selenium is provided with L-methionine (e.g., selenomethionine) or with L-cystine (e.g., selenocystine). The provision of selenium as the latter allows accomplishment of two vital goals simultaneously, (a) provision of the selenium co-factor; and (b) provision of an additional safe source of L-cysteine.

[0175] In fact, the amount of selenium precursor employed in the novel compositions is only enough to provide a catalytic quantity of the element to activate the glutathione system. The catalytic quantity of selenium precursor utilized in the compositions of this invention is such that it will produce either in one dosage unit or in multiple dosage units sufficient elemental selenium to promote the production and activation of glutathione. Typically, this will be at or near the recommended daily allowance of selenium for the individual mammal under treatment. This amount will be well below the toxicity limit for elemental selenium. By way of non-limiting examples, a representative range of catalytic quantity of selenium is presented in the aforementioned Table 1, as shown to be effective based on the subject's age.

Compositions

[0176] This invention provides pharmaceutical compositions used in the method of the invention. Such compositions comprise a therapeutically effective amount of combined glutamic acid (in the form of glutamate or glutamine), cystine (as the L-Cysteine source), glycine and a selenium precursor in a pharmaceutically acceptable carrier. The individual components may also be provided individually with a common carrier or different carriers.

[0177] The compositions which may be provided in bulk or dosage unit form are prepared in accordance with standard pharmaceutical practice and may contain excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil may also be useful. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, coloring agents or buffering agents.

[0178] Buffering agents are sometimes used in the compositions of the invention to maintain a relatively constant hydrogen ion concentration in the mouth (pH about 7.5) or other point of entry. An appropriate buffering agent may be selected from numerous known reagents including, for example phosphate, carbonate and bicarbonate systems. Alpha-lactalbumin is useful because of its buffering properties. Additionally, it is non-toxic, water-soluble and contains appreciable amounts of the required amino acids.

[0179] The compositions may also contain mucous membrane penetration enhancers such as sodium lauryl sulphate, sodium dodecyl sulphate, cationic surfactants such as palmitoyl DL carnitine chloride, cetylpyridinium chloride, non-ionic surfactants such as polysorbale 80, polyoxyethylene 9-lauryl either, glyceryl monolaurate, polyoxyalkylenes, polyoxyethylene 20 cetyl ether, lipids such as oleic acid, bile salts such as sodium glycocholate, sodium taurocholate and related compounds.

[0180] Examples of these suitable earners are described in Remington's Pharmaceutical Sciences, Nineteenth Edition (1990), Mack Publishing Company, Easton, Pa. in Handbook of Pharmaceutical Excipients, published by The American Pharmaceutical Association and The Pharmaceutical Society of Great Britain (1986) and the Handbook of Water-Soluble Gums and Resins, Ed. By R. L. Davidson, McGraw-Hill Book Co., New York, N.Y. (1980). Compositions and methods of manufacturing compositions capable of absorption through the mucosal tissues are taught in U.S. Pat. No. 5,288,497. These publications are incorporated by reference herein in their entirety. They can be readily employed by the skilled artisan to devise methods of delivery other than those specifically described in this disclosure.

Dosages

[0181] For compounds, exemplary doses include milligram or microgram amounts of the compound per kilogram of subject or sample weight, for example, about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated, particularly when one delivers the molecule directly to the cell cytosol. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid described herein, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

[0182] A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, nasal, optical, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0183] Oral compositions optionally may include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound need not be but can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0184] Pharmaceutical compositions that are suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CRMPHOR EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, other fluids configured to preserve the integrity of the viral capsid, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride sometimes are included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0185] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0186] The pharmaceutical compositions of the invention are most conveniently utilized in dosage units for oral administration. They may be used alone but are preferably provided as tablets, suitably sublingual tablets. Such tablets may be prepared in one a day form or for intermittent use throughout the day, for example every three hours.

[0187] For example, tablets will typically weigh from about 0.5 to 5.0 grams, including all ranges and values in between, for example, about 0.6 to 4.5 grams, about 0.7 to 4.0 grams, about 0.8 to 3.5 grams, 0.9 to 3.0 grams, 1.0 to 2.5 grams, 1.5 to 2.0 grams. Microtablets that are less than 0.5 grams are also contemplated by the instant invention. The tablets will contain a therapeutically effective amount of the essential ingredients together with the selected vehicle.

[0188] A particular advantage of the compositions of the invention is that they can be provided in a number of different forms and at dosage levels appropriate to the individual mammal being treated. For example, tablets, elixers, solutions, emulsions, powders, capsules and other forms can be provided for one a day treatment or successive treatments on the same day for animals or humans whether male or female, whether infant, adolescent or adult. The defining feature of this advantage is the amount of selenium precursor utilized since the other components are essentially non-toxic.

[0189] Referring to the table above, tablets and other forms of the immunoenhancing compositions can be prepared to provide any quantity of elemental selenium from less than 1.0 g (e.g., 0.9 g, 0.8 g, 0.7 g, 0.6 g, 0.5 g, 0.4 g, 0.2 g, 0.1 g, 0.05 g, 0.01 g or less) to 7.5 g or more (e.g., 8.0 g, 9.0 g, 10.0 g, 15 g, 20.0 g, 40.0 g, 100.0 g, or more) including all values in between, for example, between 1.5 g to 20 g, between 2.0 g to 15 g, between 2.5 g to 10 g, between 1.5 g to 7.5 g, between 2.0 g to 5.0 g, etc. Herein it is understood that a tablet containing 10 g of selenium methionine is capable of delivering 4 g of elemental selenium, and 7.5 g of selenium methionine is capable of delivering 3 g of selenium. Tablets may be given several times per day to achieve the desired immune enhancing effect.

[0190] A one a day tablet weighing two grams may contain 200 mg or more (e.g., up to 200 mg, up to 300 mg, up to 500 mg, up to 1000 mg, up to 2000 mg, or more) of the composition (containing, for example, 5% to 10% by weight of the active ingredient). A similar tablet intended to be used every four hours may contain 50 mg to 100 mg or more of the therapeutically effective composition. Equivalent amounts of carrier and active components will be utilized in other compositions designed for other methods of administration.

Formulations

[0191] The aforementioned compositions and combinations may be formulated to include suitable additives and further pharmaceutical ingredients. Examples of such additives include, but are not limited to, for example, coenzyme Q10 (CoQ10), ubiquinone, 7-keto dehydroepiandosterone (7-keto DHEA), N-acetyl-cysteine, magnesium orotate or a combination thereof. See Hastings et al. (U.S. Pat. No. 6,368,617) and Richardson et al. (U.S. Pat. No. 6,207,190), which are incorporated by reference in parts pertinent thereto.

[0192] The compositions may include antiviral agents known in the art. Suitable antiviral agents include, for example, abacavir, aciclovir, acyclovir, adefovir, amantadine, amprenavir, arbidol, atazanavir, atripla, brivudine, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, entry inhibitors, famciclovir, fixed dose combinations, fomivirsen, fosamprenavir, foscamet, fosfonet, fusion inhibitors, ganciclovir, gardasil, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitors, interferon type III, interferon type II, interferon type I, interferon, lamivudine, lopinavir, loviride, MK-0518, maraviroc, moroxydine, nelfinavir, nevirapine, nexavir, nucleoside analogues, oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitors, reverse transcriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir, stavudine, synergistic enhancers, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, and zidovudine. Exemplary antiviral agents are listed in, for example, U.S. Pat. Nos. 6,093,550 and 6,894,033; and also those listed in Table 2 of Sharma et al. (US patent app. Pub. No. 2010-0081713), the disclosures in which are incorporated by reference herein. Any combination of antiviral agents may also be used.

[0193] Certain biologics can be used for modifying a given biological response, the drug moiety delivered via the viral capsid is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a polypeptide such as tumor necrosis factor, -interferon, -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), or other growth factors. Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate.

[0194] Nucleic acid molecules can be inserted into viral capsids and used in gene therapy methods for treatment, including without limitation, cancer. Gene therapy capsids can be delivered to a subject by, for example, intravenous injection and local administration. Pharmaceutical preparations of gene therapy capsids can include a gene therapy capsid in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.

Delivery Agents

[0195] As indicated above, the presently preferred method of delivery for the compositions is oral, topical, sublingual or buccal. It is convenient to provide dosage units for such delivery in the form of pills, powders, lozenges or tablets such as gelled tablets, which will slowly dissolve in the mouth. Furthermore, for topical delivery, the formulation may be in the form that would be appropriate to the skin, such as lotions, unguents, emollients, creams, etc.

[0196] Sprays or drops will typically accomplish nasal delivery of the agents of the instant invention. Suppositories will be useful for rectal or vaginal delivery.

[0197] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0198] Systemic administration can also be by transmucosal or transdermal means, including nasal and optical. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. Delivery vehicles can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0199] The present composition may include flavorings. Flavors may be based on peppermint oil, parsley, clove oil or a combination of the flavors.

Dosimetry

[0200] In some embodiments oral or parenteral compositions are formulated in a dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0201] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Molecules which exhibit high therapeutic indices often are utilized. While molecules that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0202] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such molecules often lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any molecules used in the methods described herein, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC.sub.50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. Another example of effective dose determination for an individual is the ability to directly assay levels of free and bound compound in the serum of the test subject. Such assays may utilize antibody mimics and/or biosensors.

Kits and Packs

[0203] Pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration. Pharmaceutical compositions of active ingredients can be administered by any of the paths described herein for therapeutic and prophylactic methods for treatment. With regard to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from pharmacogenomic analyses described herein. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes, oligonucleotides, and analgesics.

[0204] Every document cited herein, including any cross-referenced or related patent or application is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference(s), teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

[0205] While particular embodiments of the present invention have been illustrated, it would be well within the skill and expertise of those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.