WO2000038654A1

WO2000038654A1 - Synthetic and therapeutic methods for the alpha and beta domains of metallothionein

Info

Publication number: WO2000038654A1
Application number: PCT/US1999/030573
Authority: WO
Inventors: Bert L. Vallee
Original assignee: Vallee Bert L
Priority date: 1998-12-23
Filing date: 1999-12-21
Publication date: 2000-07-06
Also published as: AU2591800A; WO2000038654B1

Abstract

The present invention relates to the alpha and beta domains of metallothionein and analogs thereof, their synthesis, and therapeutic applications of them. Purified metal-free and metal containing alpha and beta domains of metallothionein are provided. A high yield method of synthesis and purification is also provided for the metal-free and metal containing alpha and beta domains of metallothionein. Finally, therapeutic methods are provided that use the alpha and beta domains of metallothionein to transport selected metals to specific tissues or to sequester metals from these tissues in order to treat conditions in those tissues that are ameliorated by the addition or sequestration of these metals.

Description

Synthetic and Therapeutic Methods for the Alpha and Beta Domains of Metallothionein

1. INTRODUCTION

The present invention relates to the alpha and beta domains of metallothionein, analogs and derivatives thereof, their synthesis, and therapeutic applications of them. Purified metal-free and metal-containing alpha and beta domains of metallothionein are provided. A high yield method of synthesis and purification is also provided for the metal-free and metal containing alpha and beta domains of metallothionein and their analogs. Finally, therapeutic methods are provided that use the alpha and beta domains of metallothionein to transport selected metals to specific tissues or to remove metals from these tissues in order to treat conditions in those tissues that are ameliorated by the addition or sequestration of these metals.

2. BACKGROUND OF THE INVENTION a. METALLOTHIONEIN

Metallothionein (MT) was discovered in 1957 (Margoshes, M. and Vallee, B. L., 1957, J. Am. Chem. Soc. 79, 4813). By all counts it is a most unusual and unconventional protein (Vallee, B.L., 1979, Experientia Suppl. 34, 19-40; Vallee, B.L., 1987, Experientia Suppl. 52, 5-16; Vallee, B. L., 1991, Meth. Enzymol. 205, 3-7; Vallee, B.L. and Maret, W., 1993, in Metallothionein m, eds. Suzuki, K. T., Imura, N. & Kimura, M. (Birkhauser, Basel), 1; Vallee, 1995, Neurochem. Interntl. 27, 23). One third of its 60+ amino acids are cysteines and eight are lysines. It contains neither aromatic amino acids nor histidine. MT usually binds seven zinc atoms, but it can also contain copper, cadmium, iron and traces of other metals. In an evolutionary sense it is a very old protein. The composition of the two major MT isoproteins has changed only imperceptibly over time. MT-1 and MT-2 are the two prevalent forms, which are expressed, but whose physiological functions are unknown. MT-3 was discovered only recently in brains from patients afflicted with Alzheimer's disease (Uchida, Y., Takio, K., Titani, K., Hiara, Y., and Tomonaga, M. (1991) Neuron 7, 337). Its discovery was based on the fact that it inhibits the growth of neurons. Thus far, it is the only MT that is known to exhibit such a specific biological function. This isoform contains zinc and copper(I), but not cadmium or other metals.

The number of genes that code for human MTs could be as high as 17. Multiple factors (among them members of the nuclear hormone receptor family, interferons, inducers of the acute phase response, and metalloregulatory proteins) affect tissue- and isoprotein- specific gene expression. In addition there are numerous other agents that induce it but whose signaling pathways remain obscure. Thionein, the apoform of MT has never been isolated as such from any biological material. Apparently, upon its formation, it instantaneously combines with zinc, whose "free" concentration in the cell has been reported to be exceedingly low, i.e. in the nanomolar to picomolar range.

b. THE α AND β DOMAINS OF METALLOTHIONEIN

MT was discovered 40 years ago. The 3D structure of MT has been solved both by X-ray crystallography (Robbins, A. H. and Stout, C. D., 1991, Meth. Enzymol. 205, 485) and NMR spectroscopy (Wuthrich, K., 1991, Meth. Enzymol. 205, 502). The protein has the shape of a dumb-bell and envelops the metals which it contains in two separate domains (termed "α" and "β") in a manner which effectively shields them from the environment. The amino acid sequence of the two domains has been reported, as well (Winge et al., J. Biol. Chem., 1984, 259, 11419). It is most remarkable that the metals are arranged in cluster structures unique to biology. In one cluster four atoms are bound to eleven cysteines, five of which bridge the metals, and the other has three metal atoms and nine cysteines with three bridges.

The metal-free domains have previously been prepared by several methods, none of which provide these domains in high yield in pure form. Partial enzymatic digestion of the native protein followed by metal removal has been reported (Winge, D. R.; Miklossy, K.-A., J. Biol. Chem. 1982, 257, 3471; Nielson, K. B.; Winge, D. R., J. Biol. Chem., 1984, 259, 4941.). These domains have also been expressed in E. coli. Solid-phase synthesis of the α and β of MT-2 has been reported, which synthesis provided the domains in 90 to 95 percent purity in 3 to 4 percent yield (Kull, F. A., et al, J. Am. Chem Soc. 1990, 772, 2291).

The metal-binding properties of the individual domains has been studied with respect to Cu(I), Zn(II), Cd(II) Ag(I), and Co(II). See, for example, Nielson, K. B.; Winge, D. R., J. Biol. Chem., 1985, 260, 8698; Good, M.; Vasak, M., Biochemistry, 1986, 25, 3328; George, G. N., et al, J. Inorg. Biochem., 1986, 27, 213; Zelazowski, A. j., et al., J. Biol. Chem., 1984, 259, 12950; Stillman, M. j. et al, J. Biol. Chem., 1987, 226, 4538; Kull, F. A., et al, J. Am. Chem Soc. 1990, 772, 2291.

c THE BIOLOGICAL IMPORTANCE OF TRACE METALS

Iron, copper, and zinc are known to be essential to most or all forms of life. Additionally, vanadium, chromium, manganese, cobalt, nickel, arsenic, selenium, molybdenum and tungsten are known to be essential to some or many life forms. Many of these metals function as part of metalloproteins, either structurally, or in the case of some metalloenzymes, catalytically. Furthermore, metals such as lithium are known to have pharmacological effects.

Catalytic functions of zinc in enzymes and its structural functions in zinc finger proteins have been documented amply (Vallee, B. L. and Falchuk, K. F., 1993, Physiol. Rev. 73, 79-118; Vallee, B. L. and Auld, D. S., 1993, Ace. Chem. Res. 26, 543). Zinc in MT is bound extremely tightly (K_D about 10^"13 M) (Kagi, J. H. R., 1993, in Metallothionein III, eds. Suzuki, K. T., Imura, N. & Kimura, M. (Birkhauser, Basel), pp. 29).

d. OBESITY

MT-1/-2 knock-out mice become obese, demonstrating the involvement of MT in energy metabolism (Beattie, J. H, Wood, A. M., Newman, A. M., Bremner, I., Choo, K. H. A., Michalska, A. E., Duncan, J. S., and Trayhurn, P., 1998, Proc. Natl. Acad. Sci. USA 95, 358).

e. DRUG AND ALCOHOL ADDICTION

Drug addiction or dependence involves repeated, compulsive use of a drug in order to receive its chemical rewarding effects or to avoid the punishing effects of drug withdrawal (C. Page and M. J. Curtis, 1997, "Integrated Pharmacology", Mosby International, London, ch. 30; A.J. Roberts and G.F. Koob, 1997, Alcohol Health and Research World, 21, 101-106; J. Blundell, 1991, TiPS, 12, 147-157). All drugs that produce dependence have chemical effects within the brain and provide pleasurable sensations or rewards, such as anti-fatigue, relaxation, or euphoria. Such rewarding sensations (hereinafter "reward system") are due to potentiation of neurotransmitters in the brain; neurotransmitters implicated in this process are γ-aminobutyric acid (hereinafter, "GABA"), dopamine, norepinephrine, acetylcholine, glutamate, endorphins, and serotonin (id.).

The diligent search for promising alternative pharmacological and therapeutic approaches to alcohol abuse has been extensively documented in a Symposium of the Nobel Foundation (B. Jansson, H. Rydberg, L. Terenius and B. L. Vallee, Toward a Molecular Basis of Alcohol , Birkhauser Verlag, Basel, 1993). The study of the mode of action of addictive drugs proposes a reward system whose existence has not been proven experimentally. Receptors of GABA, dopamine, glutamate, serotonin, opium, heroin, cocaine, and corticotropin-releasing factor, as well as receptors of other neurotransmitters continue to be identified in large numbers and have been shown to play significant roles in the mode of action of addictive drugs, such as heroin. Both their individual and collective role(s) in the mechanisms of action and pharmacology of addictive drugs have been and remain under expanding, intensive investigation. The speculation that ethanol acts in a manner similar or identical to such drugs has received such wide attention that many now consider this a fact. There is, however, no direct experimental evidence for this assumption (A. Goldstein, Addiction, W. H. Freeman and Co., New York, 1994), a disappointment which has both caused much discouragement as well as persistent efforts to re-examine and extend present knowledge (see B. L. Vallee, 1997, Proc. Roy. Instit, 68, 1997).

f. DISORDERS OF THE CENTRAL AND PERIPHERAL NERVOUS

SYSTEMS i. ALZHEIMER'S DISEASE

The cerebral cortexes of Alzheimer's disease ("AD") patients contain neurofibrillary tangles and senile (amyloid) plaques (D. M. A. Mann et al., 1988, Neuropathol. Appl. Neurol., 14, 177). These structures are associated with the dementia and memory loss that are characteristic of the disease.

ii. PARKINSON'S DISEASE

Parkinson's disease involves degeneration of pigmented neuronal systems in the brain stem that leads to neuromediator dysfunction. The principal cytoskeletal pathology associated with Parkinson's disease is the Lewy body which occurs predominately in aminergic and other subcortical, spinal cord, and sympathetic ganglia neurons, and also to a lesser extent in the cerebral cortex. Lewy bodies in Parkinson's disease lead to a degeneration of the dopaminergic pathway of the pigmented neuronal systems as well as to a degeneration of other neuronal systems, and this degeneration leads to a complex set of functional deficits (M. Ebadi et al, 1996, Progr. Neurobiol., 48, 1, and references therein).

iii. EPILEPSY

Epilepsy, as a term, is broadly used to describe a group of disorders that are characterized by transient, recurrent, spontaneous paroxysms of a hyperactive brain resulting in seizures. The interictal (between seizures) state of the epileptogenic cortex displays brief, high-amplitude electrical impulses. Seizures are characterized by loss of inhibitory signals so as to foster hyperexcitability and hyperactivity. g. APOPTOSIS AND INJURY FROM RADIATION OR

CHEMOTHERAPY Apoptosis is a physiological process whereby cells die without swelling, necrosis, or inflammation (J. F. R. Kerr et al., 1972, Brit. J. Cancer, 26, 239). Apoptosis, in a non-pathological context, is considered responsible for the selective deletion of cells during embryogenesis and for homeostasis in continuously renewing tissues (S. Sen, 1992, Biol. Rev., 67, 287). In a pathological context, apoptosis is exhibited by cells in response to irradiation, oxidative stress, and to various chemical stimuli such as glucocorticoids (M. J. Arends, A. H. Wyllie. 1991, Int. Rev. Exper. Pathol., 32, 223). Apoptosis has also been implicated as a process leading to aging (Z. Zakeri and R. A. Lockshin, 1994, Ann. Rev. NY Acad. Sci., 719, 212) and cancer (J. F. R. Kerr et al, 1994, Cancer, 73, 2013). Biophysical and morpho logical indicators of apoptosis include fragmentation of endonuclear and chromatin DNA (Arends et al, 1990, Amer. J. Pathol., 136, 593) and compaction of cytoplasmic organelles into so-called "apoptotic bodies" (A. H. Wyllie et al, 1980, Int. Rev. Cytol., 68, 251).

h. CARCLNOGENESIS

A neoplasm, or tumor, is a cellular mass resulting from abnormal uncontrolled cell growth, which may cause swelling on the body surface, and which can be benign or malignant. Benign tumors generally remain localized. Malignant tumors are collectively termed cancers. The term "malignant" generally means that the tumor can invade and destroy neighboring body structures and spread to distant sites to cause death (for review, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, 68).

Effective treatment and prevention of cancer remains a long- felt need, and a major goal of biomedical research.

i. AUTOIMMUNE DISEASES Autoimmune diseases are estimated to affect from 1 to 2 percent of the human population. Such diseases fall into two broad categories: organ-specific, and systemic diseases. Organ-specific autoimmune diseases include myasthenia gravis, Grave's disease, juvenile insulin-dependent diabetes, Addison's disease, and a host of others. Systemic autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus, scleroderma, rheumatic fever, as well as others. Such diseases result from a breakdown of immune tolerance to self-antigens. Such breakdown may be due to any of one or several mechanisms: lack of elimination of self-reactive clones, activation of anergic self-reactive cells, or the release of sequestered self-antigens that previously were inaccessible to the immune system.

i. SYSTEMIC LUPUS ERYTHEMATOSUS Systemic lupus erythematosus is characterized by inflammation in many different organ systems, as well as by excessive production of antibodies to nuclear, cytoplasmic and membrane antigens.

j. INFECTIOUS DISEASES Viruses are obligate intracellular parasites whose replication in a host cell is at the molecular level (A. K. Field, 1994, Encyclopedia of Virology, R. G. Webster and A. Granoff, Eds., Academic Press, 42). Viruses are dependent on the host cell energy for metabolism and macromolecular synthesis, particularly with respect to reproduction of their genomes in the hostile environment of the host cell (id.). The cycle of viral replication involves attachment of a virion to a host cell, penetration of the host cell, initiation of expression of viral genes, and use of the host cell biosynthetic apparatus to generate viral proteins and nucleic acids and packaging so as to generate and release progeny virions. Antiviral agents for general and specific viral diseases have been proposed that target each of the stages in the viral replication cycle (id.).

i. AIDS AND THE HUMAN IMMUNODEFICIENCY VIRUS

Human immunodeficiency virus (HIV) induces a persistent and progressive infection leading, in the vast majority of cases, to the development of the acquired immunodeficiency syndrome (AIDS) (Barre-Sinoussi et al., 1983, Science 220, 868; Gallo et al., 1984, Science 224, 500). There are at least two distinct types of HIV: HF/-1 (Barre- Sinoussi et al., 1983, Science 220, 868; Gallo et al., 1984, Science 224, 500) and HTV-2 (Clavel et al., 1986, Science 233, 343; Guyader et al., 1987, Nature 326, 662). In humans, HIV replication occurs prominently in CD4⁺ T lymphocyte populations, and HIV infection leads to depletion of this cell type and eventually to immune incompetence, opportunistic infections, neurological dysfunctions, neoplastic growth, and ultimately death.

HIV is a member of the lentivirus family of retroviruses (Teich et al., 1984, RNA Tumor Viruses, Weiss et al., eds., CSH-press, 949). Retroviruses are small enveloped viruses that contain a single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus, H., 1988, Science 240, 1427). Other retroviruses include, for example, oncogenic viruses such as human T-cell leukemia viruses (HTLV-l,-π,-ITJ), and feline leukemia virus. The first isolates of HIV were of the HIV-1 subtype; this subtype is now pandemic. HIV-1 infects T lymphocytes, monocyte-macrophages, dendritic cells, and glia within the central nervous system (e.g., microglia, astrocytes) (Gartner et al, 1986, Science 233, 215; Koenig et al, 1986, Science 233, 1089; Pope et al, 1994, Cell 78, 389; Weissman et al, 1995, Proc. Natl. Acad. Sci. USA 92, 826; Schmidtmayerova et al. 1996, Proc Natl. Acad. Sci. USA, 93, 700). All these cell types express the CD4 glycoprotein, which serves as a receptor for HIV-1 and HIV-2 (Dalgleish et al, 1984, Nature 312, 763; Klatzmann et al, 1984, Nature 312, 767; Maddon et al, 1986, Cell 47, 333).

HIN, like other enveloped viruses, introduces viral genetic material into the host cell through a viral-envelope mediated fusion of viral and target membranes. HIV-1 infection is mediated through the binding of the virus to the CD4 glycoprotein and other co- receptors. The HΓV-1 envelope glycoproteins gp41 (a transmembrane protein) and gpl20 (a cell surface protein) direct this binding. gpl20 is non-covalently attached to gp41, which is anchored in the viral lipid bilayer. HIV-1 entry is mediated by the high-affinity binding of gpl20 to the amino-terminal domain of the CD4 glycoprotein, causing conformational changes in gpl20 (McDougal et al, 1986, Science 231, 382; Helseth et al, 1990, J. Virol. 64, 2416; Wain-Hobson, 1996, Nature 384, 117) and subsequent binding of gpl20 to co- receptors, such as CXC-CKR4 and CC-CKR5 (Wu et al, 1996, Nature 384, 179; Trkola et al, 1996, Nature, 384, 184; Wain-Hobson, 1996, Nature 384, 117). Individuals afflicted with AIDS exhibit progressive loss of CD4+ T lymphocytes, the major cell target of the virus (Fauci et al, 1984, Ann. Int. Med., 100, 92) and slow deterioration of the immune system. In consequence, these individuals suffer from a variety of opportunistic infections and certain types of cancers (Levy, 1989, J. Am. Med. Assoc, 261, 2997) that ultimately prove fatal in the vast majority of cases. HIV infection is pandemic and HIV-associated diseases represent a major world health problem. Although considerable effort is being put into the design of effective compounds, currently no curative anti-retroviral drugs against AIDS exist. In attempts to develop such drugs, several stages of the HIV life cycle have been considered as targets for therapeutic intervention (Mitsuya, H. et al, 1991, FASEB J., 5, 2369-2381). Many viral targets for intervention with HIV life cycle have been suggested, as the prevailing view is that interference with a host cell protein would have deleterious side effects. For example, virally encoded reverse transcriptase has been one focus of drug development. A number of reverse-transcriptase-targeted drugs, including 2',3'-dideoxynucleoside analogs such as AZT, ddl, ddC, and ddT have been developed which have been shown to be active against HIV (Mitsuya, H. et al, 1991, Science, 249, 1533). While beneficial, these nucleoside analogs are not curative, probably due to the rapid appearance of drug resistant HIV mutants (Lander, B. et al, 1989, Science, 243, 1731). In addition, the drugs often cause toxic" side effects such as bone marrow suppression, vomiting, and liver function abnormalities.

Attempts are also being made to develop drugs which can inhibit viral entry into the cell, the earliest stage of HIN infection. Here, the focus has thus far been on CD4, the cell surface receptor for HIV. Recombinant soluble CD4, for example, has been shown to inhibit infection of CD4⁺ T cells by some HTV-1 strains (Smith, D.H. et al, 1987, Science, 238, 1704). Certain primary HIN-1 isolates, however, are relatively less sensitive to inhibition by recombinant CD4 (Daar, E. et al, 1990, Proc. Νatl. Acad. Sci. USA, 87, 6574). In addition, recombinant soluble CD4 clinical trials have produced inconclusive results (Schooley, R. et al, 1990, Ann. Int. Med., 112, 247; Kahn, J.O. et al, 1990, Ann. Int. Med., 112, 254; Yarchoan, R. et al, 1989, Proc. Vth Int. Conf. on AIDS, p. 564, MCP 137).

The late stages of HIV replication, which involve crucial virus-specific processing of certain viral encoded proteins, have also been suggested as possible anti-HIV drug targets. Late stage processing is dependent on the activity of a viral protease, and drugs are being developed which inhibit this protease (Erickson, J., 1990, Science, 249, 527). The clinical outcome of these candidate drugs is still in question.

Attention is also being given to the development of vaccines for the treatment of HIN infection. The HIV-1 envelope proteins (gplόO, gpl20, gp41) have been shown to be the major antigens for anti-HIV antibodies present in AIDS patients (Barin, et al, 1985, Science, 228, 1094). Thus far, therefore, these proteins seem to be the most promising candidates to act as antigens for anti-HIV vaccine development. Several groups have begun to use various portions of gplόO, gpl20, and/or gp41 as immunogenic targets for the host immune system. See for example, Ivanoff, L. et al, U.S. Pat. No. 5,141,867; Saith, G. et al, PCT Publ. No. WO 92/22,654; Shafferman, A., PCT Publ. No. WO 91/09,872; Formoso, C. et al, PCT Publ. No. WO 90/07,119. To this end, vaccines directed against HIV proteins are problematic in that the virus mutates rapidly rendering many of these vaccines ineffective. Clinical results concerning these candidate vaccines, however, still remain far in the future. Thus, although a great deal of effort is being directed to the design and testing of anti-retroviral drugs, effective, non-toxic treatments are still needed.

ii. HUMAN PAPILLOMA VIRAL CARCINOGENESIS

Human papilloma viruses (HPV) are DNA viruses, most of which merely cause benign warts. Of the 60 known HPVs, several are known to be involved in invasive cancer of the uterine cervix. One distinguishing feature of such cancer-involved HPVs, is that E7 proteins are known to be expressed in them. iii. SEMLIKI FOREST VIRUS

Semliki Forest Virus (SFV) is a mosquito-borne pathogen that in humans, can cause arthralgia, rash and fever. Along with Sindbis virus, it is a member of the Togaviridae Alphavirus genus. It has been isolated in Africa, India and Southeast Asia, and is primarily found in small wild animals, birds and subhuman primates.

iv. HEPATITIS C

Hepatitis C is the major etiological agent of human parenterally and community-acquired non-A and non-B hepatitis. Chronic hepatitis C is estimated to be carried by 300 million humans worldwide, and is more prevalent in Europe and Japan than either Hepatitis A or B. Protective vaccination is not available for Hepatitis C, and no truly effective treatment is available for the disease.

v. MEASLES Measles viral infection in non-immune humans causes fever, cough, conjunctivitis and a rash. Complications of the disease can lead to pneumonia and immunodepression that leads to bacterial invasion of the lungs; occasionally, post-infectious encephalitis may result and can lead to demyelination with risk of mortality or loss of intellectual function. There are no known chemotherapeutics effective against measles infection, although hyperimmune serum globulin can prevent or modify the disease if given within 5 days of exposure.

vi. ECHOVIRUSES AND RHINOVrRUSES

Echoviruses (enteric cytopathogenic human orphan viruses; lately, classified simply as enteroviruses) and rhinoviruses cause such diseases as the common cold (with over 100 known variants of that disease know to infect humanity) and can cause a wide range of clinical syndromes involving many of the body systems. They grow well in the throat and intestinal tract, and spread among body systems chiefly via the bloodstream. Rhinoviruses tend to stay localized in the upper respiratory tract, causing inflammation, edema and copious exudation, following a short (2-3 day) incubation period. The average person contracts 2 to 4 of these viral diseases per year, typically through contact with respiratory secretions.

There have been only limited successes to date in combating and treating these viruses, with only marginal success having been achieved using interferon and certain flavin compounds.

3. DEFINITIONS As used herein, the following terms shall have the meanings indicated: MT = metallothionein; GSH = glutathione; T = thionein; EDTA = ethylenediaminetetraacetic acid;

HIV-1 = human immunodeficiency virus type I; frnoc = 9-fluorenylmethoxycarbonyl; t-boc or boc = tertiary-butoxycarbonyl; CBZ = carbobenzoxy. As used herein, the term compound means any molecule, salt, metal, or any other combination of one or more atoms, including but not limited to covalently bonded molecules, ionic materials, metallic materials, crystalline materials, atoms or molecules or ions in solution, atoms or molecules or ions in the gas phases, and combinations of any of the preceding. As used herein, (unless otherwise specified) the term metallothionein includes all of the isoforms of metallothionein- 1, metallothionein-2, metallothionein-3, and metallothionein-4; furthermore the primary sequence of such metallothionein can correspond to that of any species known to produce metallothionein.

As used herein, the term domain means a synthetic polypeptide synthesized by the procedures of the present invention, that (1) has the same primary structure as the alpha domain of metallothionein, (2) has the same primary structure as the beta domain of metallothionein, or (3) is an analogous polypeptide (hereinafter "analog") having a closely related primary structure to either the alpha or beta domain of metallothionein which analogous polypeptide is capable of binding metals in a cluster arrangement similar to the arrangements of the alpha or beta domain of metallothionein. The differences in primary sequences of the analogs as compared to the alpha or beta domains of metallothionein are (a) substitution of one or more non-cysteine residues with different amino acids (either naturally occurring or non-naturally occurring amino acids, including but not limited to side-chain modified amino acids), (b) repetition of, repetitions of, or combination of, the primary sequences of alpha and/or beta domains of metallothionein (with or without spacer sequences to separate the repeated or combined sequences) to provide larger polypeptides with more metal binding sites, (c) one or more additional peptide residues grafted to the N-terminal and/or C- terminal end or ends of the alpha or beta domains of metallothionein, or (d) a combination of two or more of the substitution of residues, repetition/combination of sequences, and additional N-terminal and C- terminal residues described in the preceding subparagraphs (a), (b), and (c). As used herein, the term domain also includes derivatives of domains. Unless otherwise specified, the term domain shall include metal-free and metal-containing domains, and for purposes of the present invention, selenium shall be considered to be included in the term metal.

As used herein, the term derivative means a molecule (including but not limited to amino acids and peptides) chemically modified, for example by binding to that molecule another atom, molecule or ion, where such other molecule or ion is either monomeric or polymeric.

As used herein, the term sequence means two or more amino acids, and where there is more than one amino acid, the amino acids are covalently bonded through one or more peptide bonds, with the number of peptide bonds being one less than the number of amino acids.

4. SUMMARY OF THE INVENTION

In a first embodiment, the present invention provides a method of synthesis and purification of a domain having a primary amino acid sequence comprising the following steps:

(a) synthesizing the domain using a solid support and at least two alpha amino acids having alpha amino groups, wherein the alpha amino acids are selected from the group consisting of amino acids with aliphatic group containing side chains wherein said aliphatic group is hydrogen or alkyl, amino acids with aromatic group containing side chains, amino acids with sulfur group containing side chains wherein said sulfur group is a thiol or a thiol ether, amino acids with hydroxyl group containing side chains, amino acids with amine group containing side chains, amino acids with guanidinium group containing side chains, amino acids with carboxylate group containing side chains, and amino acids with amide group containing side chains, wherein the alpha amino groups are protected with a protecting group selected from the group consisting of Fmoc, t-Boc, and CBZ, the carboxylate groups are protected with a protecting group selected from the group consisting of t-butyl ester and benzyl ester, the hydroxyl groups are protected with a protecting group selected from the group consisting of t-butyl ethers, and dimethylphosphate esters, the amine groups are protected with a protecting group selected from the group consisting of t-Boc and CBZ, and the thiol groups are protected with an acetimidomethyl group;

(b) cleaving the peptide synthesized in step (a) from the solid support and removing the non-acetimidomethyl protecting groups;

(c) purifying the peptide obtained from step (b);

(d) precipitating the peptide obtained from step (c); and

(e) removing the acetimidomethyl protecting group with a solution comprising a silver(I) salt; wherein the domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of a metallothionein, an analog of the alpha domain of metallothionein, and an analog of the beta domain of metallothionein; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of

(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, (iii) one or more primary sequences of the beta domain of metallothionein are fused to the N- terminal end of the alpha domain of metallothionein, wherein the the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (v) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vi) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (vii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v) and (vi); and wherein the primary amino acid sequence of the analog of the beta domain of metallothionein differs from the primary amino acid sequence of the beta domain of metallothionein in a way selected from the group consisting of

(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) one or more repetitions of the primary sequence of the beta domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, (iii) one or more primary sequences of the alpha domain of metallothionein are fused to the N-terminal end of the beta domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (iv) one or more primary sequences of the alpha domain of metallothionein are fused to the C-terminal end of the beta domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (v) one or more additional amino acid residues fused to the N-terminal end of the beta domain of metallothionein, (vi) one or more additional amino acid residues fused to the C-terminal end of the beta domain of metallothionein, and (vii) a combination of two or more of the differences in (i), (ii), (iii), (iv),

(v) and (vi). Preferably, the method step (a) is accomplished using an automated solid- phase synthesizer.

Also, preferably, the domain is the alpha domain of metallothionein or the beta domain of metallothionein.

In a preferred embodiment of the method, the alpha amino groups are protected with a Fmoc protecting group, the carboxylate groups are protected with a t-Butyl ester protecting group, the hydroxyl groups are protected with a t-butyl ether protecting group, and the amine groups are protected with a t-Boc protecting group. In another preferred embodiment of the method, the cleaving step (b)'is accomplished using a solution comprising about 75 parts by weight phenol, about 28 parts by weight ethanedithiol, about 53 parts by weight thioanisole, about 50 parts by weight water, and about 142 parts by weight trifluoroacetic acid; and the purifying step (c) is accomplished by gel filtration chromatography using a gel prepared from beads comprising dextran that has been cross linked with epichlorohydrin under alkaline conditions wherein the dry beads have a diameter in a range from about 20 micrometers to about 150 micrometers, and wherein the gel is prepared and eluted with an aqueous solution comprising 0.1 % trifluoroacetic acid. In yet another preferred embodiment of the method, the removing step (e) is accomplished with a solution comprising silver(I) nitrate in acetic acid.

Most preferably, the domain synthesized according to the method is the alpha domain of metallothionein or the beta domain of metallothionein; and wherein the alpha amino groups are protected with an Fmoc protecting group, the carboxylate groups are protected with a t-Butyl ester protecting group, the hydroxyl groups are protected with a t-butyl ether protecting group, and the amine groups are protected with a t-Boc protecting group; wherein the synthesizing step (a) is accomplished using a solid-phase synthesizer wherein the cleaving step (b) is accomplished using a solution comprising about 75 parts by weight phenol, about 28 parts by weight ethanedithiol, about 53 parts by weight thioanisole, about 50 parts by weight water, and about 142 parts by weight trifluoroacetic acid; wherein the purifying step (c) is accomplished by gel filtration chromatography using a gel prepared from beads comprising dextran that has been cross linked with epichlorohydrin under alkaline conditions wherein the dry beads have a diameter in a range from about 20 micrometers to about 150 micrometers, and wherein the gel is prepared and eluted with an aqueous solution comprising 0.1 % trifluoroacetic acid; and wherein the removing step (e) is accomplished with a solution comprising silver(I) nitrate in acetic acid.

Preferably, all the alpha amino groups are protected with an Fmoc protecting group, all the side chain carboxylic acid groups are protected with a t-Butyl ester protecting group, all side chain hydroxyl groups are protected with a t-butyl ether protecting group, and all side chain amine groups are protected with a t-Boc protecting group. Also, preferably, the cleavage of the peptide from the solid support and removal of the non-acetimidomethyl protecting groups is accomplished using a solution comprising about 75 parts by weight phenol, about 28 parts by weight ethanedithiol, about 53 parts by weight thioanisole, about 50 parts by weight water, and about 142 parts by weight trifluoroacetic acid!^' Also; preferably, the purification by gel filtration chromatography is accomplished using a gel prepared from Sephadex-25 which comprises beads comprising dextran that has been cross linked with epichlorohydrin under alkaline conditions wherein the dry beads have a diameter in a range from about 20 micrometers to about 150 micrometers, and preferably, the Sephadex gel is prepared and eluted with an aqueous solution comprising 0.1 % trifluoroacetic acid. Also, preferably, the removal of the acetimidomethyl protecting group is accomplished with a solution comprising silver(I) nitrate in acetic acid.

In another embodiment, the invention also provides the domains according to method of synthesis and purification. These domains may be metal containing or metal- free. Where metal containing, the metal may be selected from the group consisting of main group metals, transition metals, lanthanides, and actinides. Preferred metals of the invention are zinc, copper, gold, cadmium, iron, cobalt, calcium, selenium, manganese, nickel, silver, arsenic, molybdenum, tungsten, aluminum, barium, strontium, bismuth, hafnium, technicium, lanthanum, and combinations thereof. Most preferably, the metal is zinc.

The invention also provides compositions comprising a domain, wherein said domain comprises no more than 5 percent impurities arising from the synthesis and isolation of the domain, and wherein said domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, and analogs thereof; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of

(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids,

(iii) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids,

(iv) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (v) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein,

(vi) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (vii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v) and (vi); and wherein the primary amino acid sequence of the analog of the beta domain of metallothionein differs from the primary amino acid sequence of the beta domain of metallothionein in a way selected from the group consisting of

(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) one or more repetitions of the primary sequence of the beta domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, (iii) one or more primary sequences of the alpha domain of metallothionein are fused to the N-terminal end of the beta domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (iv) one or more primary sequences of the alpha domain of metallothionein are fused to the C-terminal end of the beta domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (v) one or more additional amino acid residues fused to the N-terminal end of the beta domain of metallothionein, (vi) one or more additional amino acid residues fused to the C-terminal end of the beta domain of metallothionein, and (vii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v) and (vi).

Preferably, the domain of this composition comprises no more than 2 percent impurities arising from the synthesis and isolation of the domain. The invention further provides a composition comprising a domain, wherein said domain comprises at least 95 % of the total protein in said composition, and wherein said domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, and analogs thereof; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of

(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, (iii) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (v) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vi) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (vii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v) and (vi); and wherein the primary amino acid sequence of the analog of the beta domain of metallothionein differs from the primary amino acid sequence of the beta domain of metallothionein in a way selected from the group consisting of

(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) one or more repetitions of the primary sequence of the beta domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, (iii) one or more primary sequences of the alpha domain of metallothionein are fused to the N-terminal end of the beta domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (iv) one or more primary sequences of the alpha domain of metallothionein are fused to the C-terminal end of the beta domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (v) one or more additional amino acid residues fused to the N-terminal end of the beta domain of metallothionein,

(vi) one or more additional amino acid residues fused to the C-terminal end of the beta domain of metallothionein, and (vii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v) and (vi). Preferably the domain of this composition comprises at least 98 % of the total protein in said composition.

(iii) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (v) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein,

(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) one or more repetitions of the primary sequence of the beta domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, (iii) one or more primary sequences of the alpha domain of metallothionein are fused to the N-terminal end of the beta domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (iv) one or more primary sequences of the alpha domain of metallothionein are fused to the C-terminal end of the beta domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (v) one or more additional amino acid residues fused to the N-terminal end of the beta domain of metallothionein, (vi) one or more additional amino acid residues fused to the C-terminal end of the beta domain of metallothionein, and (vii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v) and (vi). Preferably, the domain of this composition comprises no more than 2 percent impurities arising from the synthesis and isolation of the domain.

The invention further provides a composition comprising a domain, wherein said domain comprises at least 95 % of the total protein in said composition, and wherein said domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, and analogs thereof; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of

(iv) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids,

(v) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vi) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (vii) a combination of two or more of the differences in (i), (ii), (iii), (iv),

(v) and (vi); and wherein the primary amino acid sequence of the analog of the beta domain of metallothionein differs from the primary amino acid sequence of the beta domain of metallothionein in a way selected from the group consisting of

(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) one or more repetitions of the primary sequence of the beta domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids,

(iii) one or more primary sequences of the alpha domain of metallothionein are fused to the N-terminal end of the beta domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids,

(iv) one or more primary sequences of the alpha domain of metallothionein are fused to the C-terminal end of the beta domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids,

(v) one or more additional amino acid residues fused to the N-terminal end of the beta domain of metallothionein, (vi) one or more additional amino acid residues fused to the C-terminal end of the beta domain of metallothionein, and (vii) a combination of two or more of the differences in (i), (ii), (iii), (iv),

(v) and (vi). Preferably the domain of this composition comprises at least 98 % of the total protein in said composition.

In one embodiment of the present invention, a domain of the present invention includes one or more probe characteristics. Non-limiting examples of probe characteristics useful in the invention are incorporation of one or more radioactive metals into a domain, isotopic substitution of one or more atoms in the domain, fusion to a domain of one or more of a fluorophore, an NMR contrast reagent, or a Foerster radiation-less energy transfer reagent, and arsenoazo modification of amino acid residues such as tyrosine.

The domains may be formulated as pharmaceutical compositions. Such pharmaceutical compositions comprise one or more domains and further comprise one or more ingredients selected from the group consisting of a pharmaceutically acceptabl carrier, a pharmaceutically acceptable excipient, a wetting agent, a buffering agent, an emulsifying agent, and a binding agent. These compositions may also use domains wherein the domains are immobilized on a biologically-inert non-polymeric or polymeric support. This support may comprise, but is not limited to, a material selected from the group consisting of a polymeric support in a form selected from a bead, a fiber, and a sheet; paper; and cotton thread. Preferably, the polymeric support comprises a functionalized polystyrene.

In a third embodiment, the invention provides for a method of treatment of pathological conditions, where the method involves administration of an effective amount of a domain which is metal-containing or metal free. The disclosed treatment method is applicable to classes of conditions including but not limited to disorders of the central nervous system, inflammatory diseases, disorders of the endocrine system, pathological apoptosis, injury from radiation, injury from chemotherapy, carcinogenesis, immune diseases, infectious diseases, skeletal diseases, circulatory disorders, visual disorders, skin diseases, gastrointestinal disorders, immunologic phenomena, metal deficiencies, and metal overload diseases.

The disclosed treatment method, employing metal-free domains, is applicable to conditions including but not limited to: Alzheimer's disease, neurodegenerative disease, epilepsy, bulimia, obesity, prostate cancer, prostate hypertrophy, a disorder of the thyroid, a disorder of the parathyroid, a disorder of the ovaries, a disorder of the adrenal gland, cancer, AIDS, HIV infection, hepatitis C, measles, osteoarthritis, and cardiomyopathy.

The disclosed treatment method, employing zinc-containing domains, is applicable to conditions including but not limited to Parkinson's disease, addiction, severe mental illness, anorexia nervosa, neuropathy, colitis, Crohn's disease, asthma, cancer, systemic lupus erythematosus, altered immune function, papilloma viral carcinogenesis, Semliki Forest virus, complication of kidney dialysis, retinitis pigmentosa, acrodermatitis enteropathica, baldness, parakeratosis, Wilson's disease, hemochromatosis, diarrhea, rhinoviruses, and echoviruses.

The disclosed treatment method, employing gold-containing domains, is applicable to but not limited to rheumatoid arthritis.

The disclosed treatment method, employing copper-containing domains, is applicable to but not limited to peripheral vascular disease. The disclosed treatment method, employing iron-containing domains, is applicable to but not limited to hemochromatosis. The disclosed treatment method, employing selenium-containing domains, is applicable to but not limited to white muscle disease in lambs.

The method also includes co-administration of a cofactor. Cofactors include but are not limited to ATP, GTP and GSH. The invention also discloses pharmaceutical compositions and kits comprising the domains of the invention, and includes but is not limited to suppositories and intravenous solutions.

5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates to the alpha and beta domains of metallothionein and analogs thereof, their synthesis, and therapeutic applications of them. Purified metal-free and metal-containing alpha and beta domains of metallothionein and analogs thereof are provided as described below. A high yield method of synthesis and purification is also provided for the metal-free and metal containing alpha and beta domains of metallothionein and analogs thereof. Finally, therapeutic methods are provided that use the alpha and beta domains of metallothionein and analogs thereof to transport selected metals to specific tissues or to remove metals from these tissues in order to treat conditions in those tissues that are ameliorated by the addition or sequestration of these metals.

a. THE α AND β DOMAINS OF METALLOTHIONEIN. ANALOGS

THEREOF. AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM i. THE q AND β DOMAINS OF METALLOTHIONEIN AND

ANALOGS THEREOF The present invention is directed in part to polypeptides synthesized by the procedures described below. Specifically, the polypeptides of the present invention include purified synthetic polypeptides with the same primary structure (i.e. amino acid sequence) as the alpha or beta domains of metallothionein, where these polypeptides have been synthesized according to the synthetic procedures described below. Preferably, the sequences of the polypeptides correspond to the sequences of the alpha and beta domains of human metallothionein. More preferably, the sequences of the polypeptides correspond to the sequences of the alpha and beta domains of human metallothionein-2. The primary sequence of human metallothionein-2 alpha domain is

Ks£ls&PVGOAK(! AQG(5( KGASDκ sicA.

^{" "} I I \ I \ I u The primary sequence of human metallothionein-2 beta domain is

MDPNCSCAAGDSCTCAGSCKCKECKCTSCKK.

\ I I I L I I I The present invention is also directed to analogs of the alpha or beta domains of metallothionein, where these analogs are also synthesized according to the synthetic procedures described below. The primary sequences of these analogs are related to but not identical to those of the alpha or beta domains of metallothionein. The tertiary structures of these analogs maintain similar three-dimensional arrangements of the cysteine residues to those in the alpha or beta domains of metallothionein. Thus, these analogs are capable of binding metal atoms in an analogous fashion to the metal binding of the alpha or beta domains of metallothionein.

The differences in primary sequences of the analogs as compared to the alpha or beta domains of metallothionein are (a) substitution of individual residues with different amino acids (either naturally occurring or non-naturally occurring amino acids, including but not limited to side-chain modified amino acids),

(b) one or more repetitions of, or combinations of, the primary sequences of alpha and/or beta domains of metallothionein (with or without spacer sequences to separate the repeated or combined sequences) to provide larger polypeptides with more binding sites,

(c) one or more additional peptide residues grafted to the N-terminal and/or C-terminal end or ends of the alpha or beta domains of metallothionein, or (d) a combination of two or more of the substitution of residues, repetition/combination of sequences, and addition of N-terminal and/or C-terminal residues described in the preceding subparagraphs (a), (b), and (c). Synthesis of the analogs is accomplished using the methods described below. With the analogs, the change in primary structure will accomplish one or more of the following desired effects: (1) it will change the metal binding constants so as to facilitate metal release or so as to inhibit release of metals picked up through administration of the metal-free domains; (2) it will provide suitable functionality for derivatizing and/or solubilizing the domains; and (3) it will provide additional metal binding sites to increase the quantity of metal atoms released or picked up through administration of metal containing and/or metal-free domains. Alteration of the primary structures to form analogs may also alter the' cofactor binding sites of the domains. Alteration of the cofactor binding sites enhances or diminishes the effects of the co factors on the metal uptake and release of the analogs. The domains of the present invention then can be tuned so as to optimize the metal binding to suit a particular therapeutic need.

The domains of the present invention also include derivatives, where such derivatives are domains that are bound to another atom, molecule or ion, where such other molecule or ion is either monomeric or polymeric, and where this other molecule or ion is not itself a domain. The domains of the present invention may be metal-free or may contain one or more metal atoms.

ii. SITE SPECIFIC TARGETING

In one embodiment of the present invention, a domain of the present invention is derivatized so as to be bound to an antibody to a cellular antigen to target particular sites for release or uptake of metal ions at that site. Such derivatization is well known to those skilled in the art.

iii. DOMAINS INCLUDING PROBE CHARACTERISTICS In one embodiment of the present invention, a domain of the present invention includes one or more probe characteristics. Non-limiting examples of probe characteristics useful in the invention are incorporation of one or more radioactive metals into a domain, isotopic substitution of one or more atoms in the domain, fusion to a domain of one or more of a fluorophore, an NMR contrast reagent, or a Foerster radiation-less energy transfer reagent, and arsenoazo modification of amino acid residues such as tyrosine (B. L. Vallee et al., Biochem. 11, 2584, 1972.). These and other probe characteristics and methods for their incorporation into proteins are well known to those skilled in the art.

iv. THERAPEUTIC/PROPHYLACTIC COMPOSITIONS

As described below, the invention provides methods of treatment (and prophylaxis) by administration to a subject of an effective amount of a domain described above. The subject is preferably an animal, including but not limited to animals such as cows, pigs, chickens, primates, etc., and is preferably a mammal, and most preferably human.

Various delivery systems are known and can be used to administer a domain according to the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, and oral routes. The domains may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce domains of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection maybe facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.

In a specific embodiment, it may be desirable to administer the domains of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non- porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.

Thus, the present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of one or more domains and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, and combinations thereof. The carrier and composition can be sterile. The formulation should suit the mode of administration.

The pharmaceutical composition, if desired, can also contain wetting or emulsifying agents, or pH buffering agents. The pharmaceutical composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. The pharmaceutical composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc., and should include a means (e.g. coating, inclusion in a liposome, etc.) that prevents digestion of the domains by stomach enzymes.

In a preferred embodiment, the pharmaceutical composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, pharmaceutical compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the pharmaceutical composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The domains according to the invention may also be derivatized so as to immobilize them, for example, on a biologically-inert non-polymeric or polymeric support. Non-limiting examples of such supports include functionalized polystyrene or other polymeric beads, fibers, sheets, etc, and functionalized cellulosic materials such as paper, cotton thread, and the like, immobilization may involve covalent attachment to the support, or inclusion, adsorption or absoφtion on or into a porous, non-porous or swellable support. Such immobilization permits easy introduction of the compounds to a specific site by administering the compound on the support, and also provides for easy later removal by removal of the support. The support may be the same support used in the solid-state synthesis described above, where the cleavage step has been omitted. Preferably, the support is a functionalized polystyrene. The domains of the invention may also be formulated for transdermal and transmucosal administration. One of ordinary skill would understand that there are numerous technologies available for carrying out such transdermal and transmucosal administration.

The amount of the domains of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.

Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

b. SYNTHESIS OF THE α AND β DOMAINS OF METALLOTHIONEIN AND ANALOGS THEREOF

Another embodiment of the present invention is a method for synthesis of the purified domains of the present invention in high yield and purity.

Prior syntheses of the α and β domains of metallothionein and other cysteine- rich polypeptides have employed N-alpha-t-boc-S-(4-methylbenzyl)-L-cysteine and N-alpha- fmoc-S-benzyl-L-cysteine. These syntheses have provided the desired polypeptides in very low yield (3 percent) and only 90 percent purity.

It has now been discovered that suφrisingly and unexpectedly, substituting N-alpha-fmoc-S-acetimidomethyl-L-cysteine ("Fmoc-Cys(Acm)") for the N-alpha-t-boc-S- (4-methylbenzyl)-L-cysteine or N-alpha- fmoc-S-benzyl-L-cysteine permits solid-phase synthesis of the desired polypeptides in about 90 percent yield, and because of the large quantity so obtained, in as high a purity as is desired for a given puφose. Fmoc-Cys(Acm) is available commercially from Calbiochem-Novabiochem Coφ. (San Diego, California).

Using synthetic methods well known in the art, and using Fmoc-Cys(Acm) as the protected source of cysteine, the problems of low yield and inability to purify synthetic cysteine-rich polypeptides has been overcome. Standard procedures for deprotecting the other amino acid side groups are employed, and the removal of the Acm group is accomplished in a separate step using a Ag(I) salt (e.g. AgBF₄).

Thus, in this embodiment of the invention, a method is provided for synthesis and purification of a domain having a primary amino acid sequence comprising the following steps:

(c) purifying the peptide obtained from step (b);

(d) precipitating the peptide obtained from step (c); and

(e) removing the acetimidomethyl protecting group with a solution comprising a silver(I) salt; wherein the domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, an analog of the alpha domain of metallothionein, and an analog of the beta domain of metallothionein; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of

(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, (iii) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (v) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vi) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (vii) a combination of two or more of the differences in (i), (ii), (iii), (iv),

(v) and (vi); and wherein the primary amino acid sequence of the analog of the beta domain of metallothionein differs from the primary amino acid sequence of the beta domain of metallothionein in a way selected from the group consisting of (i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) one or more repetitions of the primary sequence of the beta domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, (iii) one or more primary sequences of the alpha domain of metallothionein are fused to the N-terminal end of the beta domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (iv) one or more primary sequences of the alpha domain of metallothionein are fused to the C-terminal end of the beta domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (v) one or more additional amino acid residues fused to the N-terminal end of the beta domain of metallothionein, (vi) one or more additional amino acid residues fused to the C-terminal end of the beta domain of metallothionein, and

(vii) a combination of two or more of the differences in (i), (ii), (iii), (iv),

(v) and (vi). Preferably, the method step (a) is accomplished using an automated solid- phase synthesizer. Also, preferably, the domain is the alpha domain of metallothionein or the beta domain of metallothionein. In a preferred embodiment of the method, the alpha amino groups are protected with an Fmoc protecting group, the carboxylate groups are protected with a t-Butyl ester protecting group, the hydroxyl groups are protected with a t-butyl ether protecting group, and the amine groups are protected with a t-Boc protecting group. In another preferred embodiment of the method, the cleaving step (b) is accomplished using a solution comprising about 75 parts by weight phenol, about 28 parts by weight ethanedithiol, about 53 parts by weight thioanisole, about 50 parts by weight water, and about 142 parts by weight trifluoroacetic acid; and the purifying step (c) is accomplished by gel filtration chromatography using a gel prepared from beads comprising dextran that has been cross linked with epichlorohydrin under alkaline conditions wherein the dry beads have a diameter in a range from about 20 micrometers to about 150 micrometers, and wherein the gel is prepared and eluted with an aqueous solution comprising 0.1 % trifluoroacetic acid. An example of such a gel is Sephadex 25.

In yet another preferred embodiment of the method, the removing step (e) is accomplished with a solution comprising silver(I) nitrate in acetic acid.

Most preferably, the domain synthesized according to the method is the alpha domain of metallothionein or the beta domain of metallothionein; and wherein the alpha amino groups are protected with an Fmoc protecting group, the carboxylate groups are protected with a t-Butyl ester protecting group, the hydroxyl groups are protected with a t-butyl ether protecting group, and the amine groups are protected with a t-Boc protecting group; wherein the synthesizing step (a) is accomplished using a solid-phase synthesizer wherein the cleaving step (b) is accomplished using a solution comprising about 75 parts by weight phenol, about 28 parts by weight ethanedithiol, about 53 parts by weight thioanisole, about 50 parts by weight water, and about 142 parts by weight trifluoroacetic acid; wherein the purifying step (c) is accomplished by gel filtration chromatography using a gel prepared from beads comprising dextran that has been cross linked with epichlorohydrin under alkaline conditions wherein the dry beads have a diameter in a range from about 20 micrometers to about 150 micrometers, and wherein the gel is prepared and eluted with an aqueous solution comprising 0.1 % trifluoroacetic acid; and wherein the removing step (e) is accomplished with a solution comprising silver(I) nitrate in acetic acid. Examples of amino acids with aliphatic group containing side chains wherein said aliphatic group is hydrogen or alkyl include but are not limited to glycine (Gly, G), alanine (Ala, A), Valine (Val, V), Leucine (Leu, L), isoleucine (He, I), and proline (Pro, P). Examples of amino acids with aromatic group containing side chains include but are not limited to phenylalanine (Phe, F), tryptophan (Tφ, W), and histidine (His, H). Examples of amino acids with sulfur group containing side chains wherein said sulfur group is a thiol or a thiol ether include but are not limited to Cysteine (Cys, C) and methionine (Met, M). Examples of amino acids with hydroxyl group containing side chains include but are not limited to tyrosine (Tyr, Y), serine (Ser, S) and threonine (Thr, T). An example of an amino acid with an amine group containing side chain includes but is not limited to lysine (Lys, K). An example of an amino acid with a guanidinium group containing side chain includes but is not limited to arginine (Arg, R). Examples of amino acids with carboxylate group containing side chains include but are not limited to aspartic acid (Asp, D) and glutamic acid (Glu, E). Examples of amino acids with amide group containing side chains include but are not limited to asparagine (Asn, N) and glutamine (Gin, Q). One skilled in the art realizes that there are many other possible non-naturally occurring amino acids that are known and useful in the synthesis of peptides, and the method and domains of the present invention may employ these other amino acids, as well.

The method of synthesis and purification of the present invention will now be illustrated by means of a non-limiting example of the synthesis of the alpha and beta domains of human metallothionein.

An ABI 433 A solid phase peptide synthesizer was loaded with Fmoc Lys(BOC)-Wang resin (p-benzyloxyybenzyl resin, Bachem, Switzerland) All amino acids used in the synthesis were N-alpha-fmoc protected, and the side-chain functional groups were protected as follows: O-tert-butylaspartic acid, O-tert-butylglutamic acids, O-tert- butylserine, O-tert-butylthreonine, lysine(Boc), and Cys(Acm); all protected amino acids were obtained from Bachem. Each protected amino acid was added to the synthesizer in turn, and an Fmoc/HBTU monitor was used to follow the incoφoration into the peptide. On completion of the synthesis, the peptides were cleaved and the non-Acm protecting groups removed using a solution of 0.75 g. phenol, 0.25 mL ethanedithiol, 0.5 mL thioanisole, 0.5 mL water, and 10 mL trifluoroacetic acid. The peptides were precipitated with t-butyl methyl ether, and then purified by gel filtration chromatography on Sephadex 25 in 0.1 % trifluoroacetic acid. The ACM group was then removed by dissolving the peptide in acetic acid containing silver nitrate and stirred at 0 °C for 1 hour. The peptides were precipitated with ether, isolated by centrifuge, then treated with dithiothreitol in acetic acid at 25 °C for 3 hours. The final peptides were desalted with Sephadex 15 under a nitrogen atmosphere to yield respectively 38 mg of the alpha-domain and 60 mg of the beta-domain. The purity of the peptides was determined by electrospray mass spectrometry and was higher than 93 %. c. THERAPEUTIC METHODS USING THE α AND β DOMAINS OF

METALLOTHIONEIN AND ANALOGS THEREOF

In another embodiment of the present invention, the domains are used as therapeutic agents in the treatment of conditions requiring addition of or sequestration of metals either generally or from a particular site. Such treatment is effected using the domains either as sources of selected metals or as agents for the sequestration of metal ions from the area or areas requiring treatment.

The domains of the present invention are particularly suitable for addition of and sequestration of metals from cellular and extracellular sites. The domains are either synthetic and purified versions of naturally occurring metal transport agents (where the domains are the alpha and beta domains of metallothionein synthesized according to the methods of the present invention) or they are related to these naturally occurring agents. Because the domains are related to natural agents, toxicity and immune response problems found for other agents are lessened. Furthermore, because the domains of the present invention also include analogs, the metal binding properties of the domains may be tuned according to the specific needs of the treatment for which they are designed. For example, the domains may be designed to release metals gradually over an extended period, or they may be designed to release a relatively large number of metal atoms in a relatively short period of time. The metals that may be delivered or taken up by the domains of the present invention include both main group and transition group metals, as well as the lanthanides, and actinides. For puφoses of the present invention, selenium shall be considered to be included in the term metal.

Table 1, below, lists some of the conditions susceptible to treatment with the domains of the present invention. It should be obvious to a practitioner that there are other conditions, not listed in Table 1, that are also susceptible to treatment by the domains of the present invention. Thus, the conditions in Table 1 are meant to be illustrative, and the list is not to be construed as exhaustive.

Table 1. Conditions susceptible to treatment with the domains of the invention.

Table 1. Conditions susceptible to treatment with the domains of the invention.

i. THERAPIES INVOLVING ADDITION OR SEQUESTRATION OF

ZINC

In a preferred embodiment of the present invention, the metal delivered to a site or removed from a site is zinc. Zinc plays a central role in cellular metabolism, and zinc performs regulatory functions. Zinc is a modulator of synaptic transmission, which is an extracellular regulatory function. It is quite likely that zinc also is an intracellular regulator that operates at concentrations different from those of calcium and hence coordinates different sets of biochemical processes.

(1) DISORDERS OF THE CENTRAL NERVOUS SYSTEM Zinc is concentrated in vesicles of the pre-synaptic terminals of certain glutamatergic neurons and is released during high-frequency neuronal firing, and metallothionein-III ("MT-3") is abundant in these neurons (J. C. Erikson et al, 1997, J. Neuroscience, 17, 1271-1281, and references therein). Zinc is known to have a wide variety of neuromodulatory functions, and to be involved in the pathophysiology of several neurological disorders (id.). Zinc has been found to be neurotoxic, and its intracellular accumulation may contribute to nerve death in seizure disorders (id.). In one embodiment of the present invention, the metal-free domain is administered to a patient so as to reduce intracellular accumulation of zinc. Thus in one embodiment, the invention provides a method of treating a subject having a disease of the central nervous system comprising administering to the subject a metal-free domain in an amount effective for treatment of the disease of the central nervous system.

(a) ALZHEIMER'S DISEASE

While the relationship between zinc and the growth of neurofibrillary tangles is not altogether clear, the relationship between zinc and the growth of senile plaques is clear. A major component of senile plaques in the AD brain is Aβ_M0, a 40 residue polypeptide whose precipitation from cerebrospinal fluid leads to the formation of Aβ amyloid plaque. Aβ ₀ has been shown to bind zinc, and the Zn-Aβ_M0 complex has been shown to precipitate out of cerebrospinal fluid at physiological zinc concentrations (A. Bush et al, 1994, Science, 256, 1464-1467; X. Huang et al, 1997, J. Biol. Chem., 272, 26464- 26470).

In one embodiment of the present invention, prevention of accumulation or further accumulation of the amyloid plaques characteristic of Alzheimer's disease is effectuated by treatment with metal-free domains of the present invention. Thus in one embodiment, the invention provides a method of treating a subject having Alzheimer's disease comprising administering to the subject one or more metal-free domains of the present invention in an amount effective for treatment of the Alzheimer's disease.

(b) NEURODEGENERATrVE DISEASES

Various neurodegenerative disorders are associated with free radicals (J. A. Knight, 1997, Annal. Clin. Lab. Sci., 27, 11). For example, amyotrophic lateral sclerosis (Lou Gehrig's disease), Parkinson's disease, Down's syndrome and multiple sclerosis have all been found to be associated with excess free radicals, particularly oxygen-based free radicals (id.).

In one embodiment of the present invention, one or more metal-free domains of the present invention are administered to a cell so as to sequester intracellular zinc, thereby preventing damage from zinc influx. Thus in one embodiment, the invention provides a method of treating a subject having a neurodegenerative disease comprising administering to the subject one or more metal-free domains of the present invention so as to sequester intracellular zinc in an amount effective for treatment of the neurodegenerative disease. The neurodegenerative disease may be selected from the group consisting of Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis, etc. (c) PARKINSON'S DISEASE Oxidative stress is believed to be a cause of Parkinson's disease, (id.) The degeneration of dopaminergic neurons results in increased metabolism of dopamine and a resultant increase in hydroxyl radicals. This same effect can be produced by 6- hydroxydopamme and related substances, the introduction of which can cause Parkinsonism (id.). Further evidence for the involvement of oxidative stress is found with the correlation between the extent of neuronal loss in Parkinson's patients and a depletion in glutathione (id.).

Intracellular zinc generally mitigates damage from oxidative stress because available zinc competes with iron and copper species for reactive oxygen species, and because zinc protects cellular thiols. Without committing ourselves to a particular theory or mechanism, it is believed that inter- and intracellular zinc prevents damage due to oxidizing species, and treatment with zinc-containing domains of the present invention increases concentrations of inter- and intracellular zinc and thereby prevents neuronal death associated with Parkinson's disease. Thus, in one embodiment, the invention provides a method of treating a subject having Parkinson's disease comprising administering to the subject a one or more zinc-containing domains of the present invention in an amount effective for treatment of the Parkinson's disease.

(d) EPILEPSY

Zinc has been found to diminish synaptic inhibition by γ-aminobutyric acid ("GABA") in a rat model for epilepsy. One condition associated with epilepsy is hyperexcitability of synapses in the mammalian forebrain (P. A. Schwartzkroin, Ed., 1993, Epilepsy: Models, Mechanisms, and Concepts, Cambridge Univ. Press). This hyperexcitability is favored by a reduction in synaptic inhibition, and synaptic inhibition is primarily mediated by GABA.

Zinc is associated with synaptic inhibition in epilepsy. The dentate gyms of several experimental models, as well as of humans suffering from temporal lobe epilepsy ("TLE") exhibits a distinctive aberrant sprouting of mossy fibers (S. Otis, et al, 1994, Proc. Natl. Acad. Sci. U.S.A., 91, 7698). For example, the Wistar rat kindling model for epilepsy has enhanced functional inhibition of GABA (id.). The mossy fibers of this and other models, as well as those of humans, are loaded with Zn²⁺ that can be released on stimulation (C. J. Fredrickson and D. W. Moncrief, 1994, Biol. Signals, 3, 127). Furthermore, Zn²⁺ has been shown to inhibit certain types of GABA receptors, particularly during early development (T. G. Smart et al, 1994, Prog. Neurobiol., 42, 393). Zn^2" has been shown reversibly to antagonize epileptic granule cell GABA_A receptors so as to block inhibitory post-synaptic currents (E. H. Buhl, 1996, Science, 271, 369-73). MT-3 is a growth-inhibitory factor and is expressed by zinc-containing neurons. MT-3 has been suggested to be involved in seizure disorders (J. C. Erickson et al, 1997, J. Neurosci., 17, 1271), and MT-3 knockout mice have been found to be more susceptible to seizures induced by treatment with kainic acid (id.). In one embodiment of the present invention, metal-free domains of the present invention are administered to patients suffering from epilepsy so as to sequester synaptic zinc. Sequestration of zinc reduces the hyperexcitability characteristic of epileptic pathology, and thereby mitigates epileptic symptoms. Thus, in one embodiment, the invention provides a method of treating an epileptic subject comprising administering to the subject one or more metal-free domains of the present invention in an amount effective for treatment of the epilepsy.

(e) ADDICTIONS Zinc metabolism has been related to alcoholism. For example, the concentration of zinc has been shown to be altered in the tissues of alcoholics (M. Ebadi et al, 1995, Neurochem. Int., 27, 1-22).

In one embodiment of the present invention, zinc-containing domains of the present invention are administered along with disulfiram so as to increase intra- and intercellular zinc concentration. Without committing to any particular theory or mechanism, it is believed that zinc released from the domains in conjunction with disulfiram leads to improved anti-drinking effects as compared to disulfiram alone.

Zinc metabolism is also related to withdrawal from cocaine (D. J. Ennulat and B. M. Cohen, 1997, Brain Res. Mol. Brain. Res., 49, 299-302). Cocaine-treated rats have been shown to have repressed levels of mRNA coding for a zinc-finger protein transcriptional regulator. After 2 days without cocaine, the level of this mRNA was found to have returned to normal. At the same time mRNA for a different zinc-finger protein transcriptional regulator was found to be induced by cocaine treatment (id.).

In one embodiment of the present invention, zinc-containing domains are administered to maintain high levels of available zinc. Without committing to any particular theory or mechanism of action, it is believed that such high levels of available zinc interferes with the biochemical reward system (C. Page and MJ. Curtis, 1997, "Integrated Pharmacology", Mosby International, London, ch. 30; A.J. Roberts and G.F. Koob, 1997, Alcohol Health and Research World, 21, 101-106; J. Blundell, 1991, TiPS, 12, 147-157) leading to reduced desire for cocaine. Thus, in one embodiment, the invention provides a method of treating a subject having a drug or alcohol addiction comprising administering to the subject one or more zinc-containing domains of the present invention in an amount effective for treatment of the addiction. (f) SEVERE MENTAL ILLNESSES

The concentration of zinc in the brains of schizophrenics is altered (M. Ebadi et al, 1995, Neurochem. Int., 27, 1-22). Furthermore, patients with chronic major depression, particularly treatment-resistant depression have been found to have increased levels of interleukin 6 that correlate with decreased levels of serum zinc (M. Maes et al, 1997, Cytokine, 9, 853-8), and decreased levels of tryptophan that correlates with decreased levels of zinc (M. Maes et al, 1997, Eur. Arch. Psych. Clin. Neurosci., 247, 154-61), suggesting that cell-mediated immune response may be implicated in such depressions.

In one embodiment, the invention provides method of treating a subject with severe mental illness comprising administering to the subject one or more zinc-containing domains of the present invention in an amount effective for treatment of the severe mental illness.

(g) EATING DISORDERS Zinc is associated with energy metabolism and has been implicated in several eating disorders. For example, zinc is believed to be associated with the energy homeostasis of obesity via its interaction with dietary fat consumption (M. D. Chen et al, 1996, Biol. Trace. Elem. Res., 52, 125-32). Support for this belief is found, for example, in the correlation between hair zinc concentration and obesity (S. K. Taneja et al, 1996, Experientia, 52, 31-3).

In one embodiment, the invention provides a method for treating a subject having an eating disorder selected from the group consisting of anorexia nervosa, bulimia and obesity by administering to the subject an amount of a compound effective to treat said condition, wherein the compound is selected from the group consisting of one or more zinc- containing domains of the present invention and one or more metal- free domains of the present invention.

In another embodiment, the invention provides a method of treating a subject with anorexia nervosa comprising administering to the subject one or more zinc-containing domains of the present invention in an amount effective for treatment of the anorexia nervosa.

In another embodiment, the invention provides a method of treating an obese subject comprising administering to the subject one or more metal-free domains of the present invention in an amount effective for treatment of the obesity.

(h) NEUROPATHY

In another embodiment, the invention provides a method of treating a patient suffering from neuropathy comprising administering to the subject one or more zinc- containing domains of the present invention in an amount effective for treatment of the neuropathy.

(2) INFLAMMATORY DISEASES Various disorders exhibiting inflammatory response have been shown to depend on zinc or to respond to zinc therapy. Nowhere, however, has there been a suggestion of the novel therapies of the present invention where a synthetic version of a naturally occurring zinc-containing peptide is administered so as to increase or decrease the amount of inter- and intra-cellular zinc. Such decreases are proposed to be due to zinc blocking the docking of human rhinovirus to intercellular adhesion molecule 1 ("ICAM-1"); because docking to ICAM-1 is also necessary for initiation of the inflammatory response, zinc ion is believed to be an important inhibitory factor to inflammation, as well (id.). Zinc has also been shown to enhance the expression of ICAM-1 in cells actively involved in the inflammatory response (S. Martinotti et al., 1995, Biochem. Biophys. Acta, 1261, 107-14). In one embodiment, the invention provides a method of treating a subject with an inflammatory disease comprising administering to the subject one or more zinc- containing domains of the present invention in an amount effective for treatment of the inflammatory disease.

Colitis and Crohn's disease reveal decreased zinc superoxide dismutase activity (L. Lih-Brody et al, 1996, Digest. Dis. Sci., 41, 2078-86) and colitis has been experimentally successfully treated in rats using a zinc chelate compound (T. Yoshikawa et al, 1997, Digestion, 58, 464-8). Lung epithelial tissue in asthmatics, furthermore, has been shown to have reduced zinc-specific activity, perhaps due to inflammatory responses.

In one embodiment, the invention provides a method for treating a subject with colitis or Crohn's disease comprising administering to the subject one or more zinc- containing domains of the present invention in an amount effective for treatment of the inflammatory disease.

In another embodiment of the present invention, one or more zinc-containing domains of the present invention are administered by inhalation in an amount effective for treatment of asthma.

(3) DISORDERS OF THE ENDOCRINE SYSTEM Prostate glands of most mammals are known to contain among the highest levels of zinc of any tissues. In benign prostate hypeφlasia, zinc levels are increased over normal prostate levels and are known to inhibit androgen metabolism (S. Dutkiewicz, 1995, Materia Medica Polona, 27, 15). Antioxidant enzyme expression, furthermore, is reduced in malignant (neoplastic) prostate glands (A. M. Baker et al, 1997, Prostate, 32, 229). In one embodiment, the invention provides a method of treating a subject suffering from a disorder of the endocrine system comprising administering to the subject one or more metal-free domains of the present invention in an amount effective for treatment of the disorder of the endocrine system. Disorders of the endocrine system that may be so treated include but are not limited to prostate cancer, prostate hypertrophy, a disorder of the thyroid, a disorder of the parathyroid, a disorder of the ovaries, and a disorder of the adrenal gland.

In one embodiment of the present invention, one or more metal-free domains of the present invention are supplied to hypeφlastic or neoplastic prostate glands so as to sequester zinc released from MT.

(4) APOPTOSIS AND INJURY FROM RADIATION OR CHEMOTHERAPY Zinc is believed to play a role in pathological apoptosis. Where mice or rats have been fed a zinc-deficient diet, "massive" increase in apoptotic bodies has been observed in mucosal cells of their small intestines (M. Elmes, 1977, J. Pathol., 123, 219-23), and thymic atrophy and defective T helper cell function have also been observed (P. J. Fraker et al, 1977, J. Nutr., 107, 1889-95). Furthermore, zinc-deprived mice also have been found to exhibit skin lesions and stunted growth that are linked to apoptosis (G. Fernandes et a , 1979, Proc. Natl. Acad. Sci., 76, 457-61).

While treatment with zinc salts has been shown to inhibit apoptosis, there has never before been a suggestion of administration of the zinc-containing domains of the present invention for treatment of pathological apoptosis. In cultured cells, addition of zinc salts to the culture medium has been shown to prevent cells from undergoing apoptosis. For example, following γ-irradiation (J. J. Cohen and R. C. Duke, 1984, J. Immunol., 132, 38- 42; P. Beletsky et al, 1989, Gen. Physiol. Biophys., 8, 381-98) or exposure to methasone (K. S. Sellins and J. J. Cohen, 1987, J. Immunol., 139, 3199-3206), murine thymocytes undergo DNA or chromatin degradation that is prevented by addition of zinc salts to the culture medium. Furthermore, in vivo protection by zinc against apoptosis has been seen in mice, where pretreatment with zinc acetate three days prior to injection of S. typhimurium lipopolysaccharide reduced internucleosomal cleavage of thymic DNA as compared to non- pretreated mice so injected (D. J. Thomas and T. C. Caffrey, 1991, Toxicology, 68, 327-37). These and various other studies have shown that "a chelatable pool of intra-cellular Zn²⁺ influences apoptosis, since influx of Zn²⁺ prevented apoptosis, while chelation of Zn²⁺ induced apoptosis" (F. W. Sunderman Jr., 1995, Annal. Clinic. Lab. Sci., 25, 134-42). In one embodiment of the present invention, zinc-containing domains are administered in order to combat pathological apoptosis. Thus in one embodiment, the invention provides a method of treating a subject with pathological apoptosis comprising administering to the subject one or more zinc-containing domains of the present invention in an amount effective for treatment of the pathological apoptosis.

(5) CARCINOGENESIS

In one embodiment, one or more domains of the invention is administered to treat a cancerous condition, or to prevent progression from a pre-neoplastic or non- malignant state (e.g., metaplastic condition) into a neoplastic or a malignant state.

In another embodiment of the present invention, one or more metal-free domains of the present invention are administered, thereby limiting the bioavailability of zinc as would be required for cellular proliferation, and thus reducing the proliferation of selected cells. Thus, in one embodiment, the invention provides a method of treating a subject with cancer, said method comprising administering to the subject one or more metal- free domains of the present invention in an amount effective for inhibiting proliferation of the cells of the cancer.

(6) IMMUNE DISEASES Dietary zinc deficiency is linked to autoimmune diseases in mice (R. S. Beach et al, 1982, J. Immunol., 129, 2686). In one embodiment of the present invention, one or more zinc-containing domains of the present invention are administered to subjects at risk for or experiencing autoimmune diseases so as to ameliorate the disease. Thus in one embodiment, the invention provides a method of treating a subject with an autoimmune disease comprising administering to the subject one or more zinc-containing domains of the present invention in an amount effective for treatment of the autoimmune disease.

(a) SYSTEMIC LUPUS ERYTHEMATOSUS Systemic lupus erythematosus has been associated with dietary zinc deficiency (id.). In one embodiment of the present invention, one or more zinc-containing domains of the present invention are administered to subjects at risk for or experiencing systemic lupus erythematosus so as to ameliorate the disease. Thus, in one embodiment, the invention provides a method of treating a subject with systemic lupus erythematosus comprising administering to the subject one or more zinc-containing domains of the present invention in an amount effective for treatment of the lupus erythematosus.

(b) ALTERED IMMUNE FUNCTION

In one embodiment, the invention provides a method of treating a subject with altered immune function comprising administering to the subject one or more zinc- containing domains of the present invention in an amount effective for treatment of the altered immune function.

(7) INFECTIOUS DISEASES (a) AIDS AND HIV INFECTION

Zinc plays an important role in several of the steps in the life-cycle of H1N-1. For example, integration of HIV-1 DΝA into host-cell genome is an essential step in the HIV-1 life cycle, thus interruption of this integration is a means of combating HIV-1 infection. HIV-1 integration requires cleavage of two nucleotides from the 3' ends of viral DΝA by HIV-1 integrase. HIV-1 integrase contains three distinct domains (J. Kukosky and A. M. Skalka, 1994, Pharmacol. Ther., 61, 185), one of which (the Ν-terminal domain) is known to bind zinc (M. S. Johnson et al, 1986, Proc. Νatl. Acad. Sci. USA, 83, 7468). Zinc binding has been shown to play important structural and functional roles in integrase activity (S.P. Lee et al, 1997, Biochemistry, 36, 173). Additionally, mature HIV-l nucleocapsid protein (ΝCP) requires a zinc atom to reach its folded conformation and removal of zinc from ΝCP prevents the operation on it by viral protease (E. W. Wondrak et al, 1994, J. Biol. Chem., 269, 21948).

Furthermore, dietary intake of zinc has been associated with poorer survival of AIDS patients (A. M. Tang et al, 1996, Am. J. Epidem., 143, 1244). In one embodiment of the present invention, metal-free domains of the present invention are administered to a patient infected with HIN so as to reduce zinc transfer to HIV-l integrase and thus to inhibit HIV-1 integrase activity. Thus in one embodiment, the invention provides a method of treating a subject suffering from an infection with HIV comprising administering to the subject metal-free domains of the present invention in an amount effective for treatment of the infection with HIN.

(b) PAPILLOMA VIRAL CARCINOGENESIS E7 proteins of cancer- involved HPVs are cysteine-rich zinc-binding proteins. (E. J. Roth et al, 1992, J. Biol. Chem., 267, 16390). In one embodiment of the present invention, reducing compounds of the present invention are administered to a patient infected with or in danger of infection with cancer-involved HPVs so as to prevent transfer of zinc from MT to the HPV E7 protein and thereby to prevent or mitigate cervical cancer. Thus, in one embodiment, the invention provides a method of treating a subject infected with papilloma virus, said method comprising administering to the subject one or more zinc- containing domains of the present invention in an amount effective for treatment of the papilloma virus. (c) SEMLfKI FOREST VIRUS

Zn²⁺ has been shown to in vitro to inhibit SFV-liposome fusion due to interference with El protein trimer formation (J. Corver et al, 1997, Virology, 238, 14). In one embodiment of the present invention, an animal in need of treatment for SFV infection is treated with one or more zinc-containing domains of the present invention to inhibit access of SFV to the host cytosol. Thus, in one embodiment, the invention provides a method of treating a subject infected with Semliki Forest virus comprising administering to the subject one or more zinc-containing domains of the present invention in an amount effective for treatment of the Semliki Forest virus.

(d) HEPATITIS C

Several Hepatitis C (HCV) non-structural proteinases (NSP) exhibit zinc- dependent activity (A. Grakoui et al, 1993, J. Virol., 67, 2832; M. Hijikata et al, 1993, J. Virol., 67, 4665). Activity of these proteinases is necessary for the replication of the HCV genome. At least one of the NSP, NS3 has a zinc-binding site (R. A. Love et al, 1996, Cell, 87, 332).

In one embodiment of the present invention, metal-free domains are administered to a patient infected with HCV so as to prevent transfer of zinc to HCV NS3, and thereby to inhibit further replication of the HCV genome. Thus, in one embodiment, the invention provides a method of treating a subject infected with hepatitis C virus comprising administering to the subject one or more metal-free domains of the present invention in an amount effective for treatment of the hepatitis C virus.

(e) MEASLES One result of measles virus (MV) infection is production of MV V protein and diffuse cytoplasmic distribution (E. A. Wardrop and D. J. Briedis, 1991, J. Virol., 65, 3421). MV V protein is believed to play a role in transcription and/or replication of MV genome. V protein binds zinc (P. Liston and D. J. Briedis, 1994, Virol., 198, 399).

In one embodiment of the present invention, one or more metal-free domains of the present invention are administered to a patient who has been exposed to measles so as to prevent the transfer of zinc to MV V protein. Thus, in one embodiment, the invention provides a method of treating a subject infected with measles virus comprising administering to the subject one or more metal-free domains of the present invention in an amount effective for treatment of the measles virus. (f) ECHOVIRUSES AND RHINOVIRUSES

Free ionic zinc in saliva has been shown to marginally decrease the duration and severity of the common cold (S. G. Novick et al, 1997, Med. Hypoth., 49, 347-57, and references therein). Nowhere, however, has there been a suggestion of using zinc- containing domains of the present invention to treat the common cold. Thus, in one embodiment, the invention provides a method of treating a subject a subject suffering from the common cold comprising administering to the subject one or more zinc-containing domains of the present invention in an amount effective for treatment of the common cold.

(8) SKELETAL DISEASES

(a) OSTEOARTHRITIS

Osteoarthritis is believed to be due, in part, to matrix metalloproteins that are zinc enzymes (e.g. coUagenases). Thus, in one embodiment of the present invention, one or more metal-free domains of the present invention are administered to a patient that is suffering from osteoarthritis in order to sequester inter and intracellular zinc.

(9) CIRCULATORY DISORDERS (a) CARDIOMYOPATHIES

Chronic cardiac failure leads to remodeling of the ventricle by matrix metalloproteins that are regulated by zinc. Thus, in one embodiment of the present invention, one or more metal-free domains of the present invention are administered to a patient that is suffering from cardiomyopathy in amounts effective to treat the cardiomyopathy.

(b) KIDNEY DIALYSIS

Kidney dialysis leads to a loss of zinc. Thus, in one embodiment of the present invention, one or more zinc-containing domains of the present invention are administered to a patient that is undergoing kidney dialysis in amounts effective to treat any resultant zinc deficiency.

(10) VISUAL DISORDERS

(a) RETINITIS PIGMENTOSA Retinitis pigmentosa and macular degeneration is responsive to zinc therapies. Thus, in one embodiment of the present invention, one or more zinc-containing domains of the present invention are administered to a patient suffering from retinitis pigmentosa in amounts effective to treat the retinitis pigmentosa.

(11) SKIN DISEASES

(a) ACRODERMATITIS ENTEROPATHICA AND

BALDNESS Thus, in one embodiment of the present invention, one or more zinc- containing or metal-free domains of the present invention are administered to a patient that is suffering from acrodermatitis and baldness in amounts effective to treat the acrodermatitis and baldness .

(12) GASTROINTESTINAL DISORDERS

(a) PARAKERATOSIS

Parakeratosis leads to a keratotic change in the esophagus and is believed due to excess calcium. Calcium regulation is effected by the presence and absence of zinc, thus in one embodiment, one or more zinc-containing or metal-free domains of the present invention are administered to a patient that is suffering from parakeratosis in amounts effective to treat the parakeratosis.

(b) DIARRHEA Certain forms of diarrhea are known to be due to induction of certain hormones by zinc. Thus, in one embodiment, one or more metal-free domains of the present invention are administered to a patient that is suffering from diarrhea in amounts effective to treat the diaπhea.

ii. THERAPIES INVOLVING METALS OTHER THAN ZINC

(1) RHEUMATOID ARTHRITIS

Rheumatoid arthritis is susceptible to treatment with gold.. Thus, in one embodiment of the present invention, one or more gold-containing domains of the present invention are administered to a patient that is suffering from rheumatoid arthritis in an amount effective to treat the osteoarthritis.

(2) PERIPHERAL VASCULAR DISEASE

Peripheral vascular disease is known to be caused in part by an excess of copper. Thus, in one embodiment of the present invention, one or more metal- free domains of the present invention are administered to a patient that is suffering from peripheral vascular disease in an amount effective to treat the peripheral vascular disease.

(3) WILSON'S DISEASE

Wilson's disease is known to be caused in part by a copper imbalance. Thus, in one embodiment of the present invention, one or more copper-containing and/or metal- free domains of the present invention are administered to a patient that is suffering from Wilson's disease in an amount effective to treat the Wilson's disease. (4) HEMOCHROMATOSIS

Hemochromatosis is known to be caused in part by an iron imbalance. Thus, in one embodiment of the present invention, one or more iron-containing and/or metal-free domains of the present invention are administered to a patient that is suffering from hemochromatosis in an amount effective to treat the hemochromatosis.

(5) WHITE MUSCLE DISEASE IN LAMBS

In one embodiment of the present invention, one or more selenium- containing domains of the present invention are administered to a lamb that is suffering from white muscle disease in an amount effective to treat the white muscle disease.

iii. COFACTOR ADDITION

In any of the embodiments above, cofactors may be co-administered conc rently with or within a time period just prior to administration of the compound in order to provide target specificity to the zinc released. Preferably, the cofactors are administered from 0.01 second to 24 hours prior to administration of the compound. Cofactors useful for co-administration with the compounds of the present invention include but are not limited to ATP, GTP and GSH.

Table 2. Preferred forms of administration of compounds as a function of condition

Various publications are cited herein, the disclosures of which are incoφorated by reference in their entireties.

The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications, as would be obvious to a person skilled in the art, are intended to be included in the scope of the following claims.

Claims

WE CLAIM:

1. A method of synthesis and purification of a domain having a primary amino acid sequence, said method comprising the following steps: (a) synthesizing the domain using a solid support and at least two alpha amino acids having alpha amino groups, wherein the alpha amino acids are selected from the group consisting of amino acids with aliphatic group containing side chains wherein said aliphatic group is hydrogen or alkyl, amino acids with aromatic group containing side chains, amino acids with sulfur group containing side chains wherein said sulfur group is a thiol or a thiol ether, amino acids with hydroxyl group containing side chains, amino acids with amine group containing side chains, amino acids with guanidinium group containing side chains, amino acids with carboxylate group containing side chains, and amino acids with amide group containing side chains, wherein the alpha amino groups are protected with a protecting group selected from the group consisting of Fmoc, t-Boc, and CBZ, the carboxylate groups are protected with a protecting group selected from the group consisting of t-butyl ester and benzyl ester, the hydroxyl groups are protected with a protecting group selected from the group consisting of t-butyl ethers, and dimethylphosphate esters, the amine groups are protected with a protecting group selected from the group consisting of t-Boc and CBZ, and the thiol groups are protected with an acetimidomethyl group; (b) cleaving the peptide synthesized in step (a) from the solid support and removing the non-acetimidomethyl protecting groups;

(c) purifying the peptide obtained from step (b);

(d) precipitating the peptide obtained from step (c); and

(e) removing the acetimidomethyl protecting group with a solution comprising a silver(I) salt; wherein the domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, an analog of the alpha domain of metallothionein, and an analog of the beta domain of metallothionein; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of (i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) one or more repetitions of the primary sequence of the alpha domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, (iii) one or more primary sequences of the beta domain of metallothionein are fused to the N-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (iv) one or more primary sequences of the beta domain of metallothionein are fused to the C-terminal end of the alpha domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (v) one or more additional amino acid residues fused to the N-terminal end of the alpha domain of metallothionein, (vi) one or more additional amino acid residues fused to the C-terminal end of the alpha domain of metallothionein, and (vii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v) and (vi); and wherein the primary amino acid sequence of the analog of the beta domain of metallothionein differs from the primary amino acid sequence of the beta domain of metallothionein in a way selected from the group consisting of

(i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids,

(ii) one or more repetitions of the primary sequence of the beta domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, (iii) one or more primary sequences of the alpha domain of metallothionein are fused to the N-terminal end of the beta domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (iv) one or more primary sequences of the alpha domain of metallothionein are fused to the C-terminal end of the beta domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (v) one or more additional amino acid residues fused to the N-terminal end of the beta domain of metallothionein, (vi) one or more additional amino acid residues fused to the C-teπninal end of the beta domain of metallothionein, and (vii) a combination of two or more of the differences in (i), (ii), (iii), (iv),

(v) and (vi).

2. The method according to claim 1, wherein step (a) is accomplished using an automated solid-phase synthesizer.

3. The method according to claim 1 , wherein said domain is the alpha domain of metallothionein or the beta domain of metallothionein.

4. The method according to claim 2, wherein said domain is the alpha domain of metallothionein or the beta domain of metallothionein.

5. The method according to claim 1, wherein the alpha amino groups are protected with an Fmoc protecting group, the carboxylate groups are protected with a t-Butyl ester protecting group, the hydroxyl groups are protected with a t-butyl ether protecting group, and the amine groups are protected with a t-Boc protecting group.

6. The method according to claim 1, wherein the cleaving step (b) is accomplished using a solution comprising about 75 parts by weight phenol, about 28 parts by weight ethanedithiol, about 53 parts by weight thioanisole, about 50 parts by weight water, and about 142 parts by weight trifluoroacetic acid; wherein the purifying step (c) is accomplished by gel filtration chromatography using a gel prepared from beads comprising dextran that has been cross linked with epichlorohydrin under alkaline conditions wherein the dry beads have a diameter in a range from about 20 micrometers to about 150 micrometers, and wherein the gel is prepared and eluted with an aqueous solution comprising 0.1 % trifluoroacetic acid.

7. The method according to claim 1, wherein the removing step (e) is accomplished with a solution comprising silver(I) nitrate in acetic acid.

8. The method according to claim 1, wherein said domain is the alpha domain of metallothionein or the beta domain of metallothionein; and wherein the alpha amino groups are protected with an Fmoc protecting group, the carboxylate groups are protected with a t-Butyl ester protecting group, the hydroxyl groups are protected with a t-butyl ether protecting group, and the amine groups are protected with a t-Boc protecting group; wherein the synthesizing step (a) is accomplished using a solid-phase synthesizer wherein the cleaving step (b) is accomplished using a solution comprising about 75 parts by weight phenol, about 28 parts by weight ethanedithiol, about 53 parts by weight thioanisole, about 50 parts by weight water, and about 142 parts by weight trifluoroacetic acid; wherein the purifying step (c) is accomplished by gel filtration chromatography using a gel prepared from beads comprising dextran that has been cross linked with epichlorohydrin under alkaline conditions wherein the dry beads have a diameter in a range from about 20 micrometers to about 150 micrometers, and wherein the gel is prepared and eluted with an aqueous solution comprising 0.1 % trifluoroacetic acid; and wherein the removing step (e) is accomplished with a solution comprising silver(I) nitrate in acetic acid.

9. A composition comprising a domain, wherein said domain comprises no more than 5 percent impurities arising from the synthesis and isolation of the domain, and wherein said domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, and analogs thereof; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of

(v) and (vi); and wherein the primary amino acid sequence of the analog of the beta domain of metallothionein differs from the primary amino acid sequence of the beta domain of metallothionein in a way selected from the group consisting of (i) substitution of one or more non-cysteine residues with different amino acids wherein the amino acids are selected from the group consisting of naturally occurring or non-naturally occurring amino acids, (ii) one or more repetitions of the primary sequence of the beta domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, (iii) one or more primary sequences of the alpha domain of metallothionein are fused to the N-terminal end of the beta domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (iv) one or more primary sequences of the alpha domain of metallothionein are fused to the C-terminal end of the beta domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids,

(v) one or more additional amino acid residues fused to the N-terminal end of the beta domain of metallothionein,

(vi) one or more additional amino acid residues fused to the C-terminal end of the beta domain of metallothionein, and (vii) a combination of two or more of the differences in (i), (ii), (iii), (iv),

(v) and (vi).

10. A composition according to claim 9, wherein said domain comprises no more than 2 percent impurities arising from the synthesis and isolation of the domain.

11. A composition comprising a domain, wherein said domain comprises at least 95 % of the total protein in said composition, and wherein said domain is selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, and analogs thereof; wherein the primary amino acid sequence of the analog of the alpha domain of metallothionein differs from the primary amino acid sequence of the alpha domain of metallothionein in a way selected from the group consisting of

(ii) one or more repetitions of the primary sequence of the beta domain of metallothionein wherein the one or more repetitions optionally are separated by a spacer sequence of one or more amino acids, (iii) one or more primary sequences of the alpha domain of metallothionein are fused to the N-terminal end of the beta domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (iv) one or more primary sequences of the alpha domain of metallothionein are fused to the C-terminal end of the beta domain of metallothionein, wherein the primary sequences of the alpha and beta domains of metallothionein optionally are separated by a spacer sequence of one or more amino acids, (v) one or more additional amino acid residues fused to the N-terminal end of the beta domain of metallothionein,

(vi) one or more additional amino acid residues fused to the C-terminal end of the beta domain of metallothionein, and (vii) a combination of two or more of the differences in (i), (ii), (iii), (iv), (v) and (vi).

12. A composition according to claim 11, wherein said domain comprises at least 98 % of the total protein in said composition.

13. A composition according to claim 9, wherein said domain is metal containing.

14. A composition according to claim 13, wherein said domain contains one or more 5 metals selected from the group consisting of main group metals, transition metals, lanthanides, and actinides.

15. A composition according to claim 13, wherein said domain contains one or more metals selected from the group consisting of zinc, copper, gold, cadmium, iron,

10 cobalt, calcium, selenium, manganese, nickel, silver, arsenic, molybdenum, tungsten, aluminum, barium, strontium, bismuth, hafnium, technicium, lanthanum, and combinations thereof.

16. A composition according to claim 15 wherein said metal is zinc. 15

17. A method of treating a subject having a pathological condition responsive to treatment with a domain selected from the group consisting of the alpha domain of metallothionein, the beta domain of metallothionein, and analogs thereof, comprising administering to the subject an amount of a composition according to claim 9,

20 wherein said amount is effective to treat said condition.

18. The method according to claim 17, wherein said condition is selected from the group consisting of disorders of the central nervous system, inflammatory diseases, disorders of the endocrine system, pathological apoptosis, injury from radiation,

25 injury from chemotherapy, carcinogenesis, immune diseases, infectious diseases, skeletal diseases, circulatory disorders, visual disorders, skin diseases, gastrointestinal disorders, immunologic phenomena, metal deficiencies, and metal- overload diseases.

30 19. The method according to claim 17 wherein said domain is metal- free.

20. The method according to claim 17, wherein said condition is selected from the group consisting of Alzheimer's disease, neurodegenerative disease, epilepsy, bulimia, obesity, prostate cancer, prostate hypertrophy, a disorder of the thyroid, a disorder of 35 the parathyroid, a disorder of the ovaries, a disorder of the adrenal gland, cancer,

AIDS, HIV infection, hepatitis C, measles, osteoarthritis, cardiomyopathy, and cardiomyopathy.

21. The method according to claim 17 wherein said domain contains one or more metals.

22. The method according to claim 21 wherein said domain contains a metal selected 5 from the group consisting of zinc, copper, gold, cadmium, iron, cobalt, calcium, selenium, manganese, nickel, silver, arsenic, molybdenum, tungsten, aluminum, barium, strontium, bismuth, hafnium, technicium, lanthanum, and combinations thereof.

10 23. The method according to claim 22, wherein said metal is zinc.

24. The method according to claim 23, wherein said condition is selected from the group consisting of Parkinson's disease, addiction, severe mental illness, anorexia nervosa, neuropathy, colitis, Crohn's disease, asthma, cancer, systemic lupus erythematosus,

15 altered immune function, papilloma viral carcinogenesis, Semliki Forest virus, complication of kidney dialysis, retinitis pigmentosa, acrodermatitis enteropathica, baldness, parakeratosis, diarrhea, and the common cold.

25. The method according to claim 24, wherein said addiction is addiction to alcohol, 20 and further comprising administering disulfiram to said subject within 120 hours of the time of administering the composition.

26. The method according to claim 22, wherein said condition rheumatoid arthritis, and said metal is gold.

25

27. The method according to claim 22, wherein said metal is copper and said condition is selected from the group consisting of peripheral vascular disease, and Wilson's disease.

30 28. The method according to claim 22, wherein said metal is iron and said condition is hemochromatosis,

29. The method according to claim 22, wherein said metal is selenium and said condition is white muscle disease and said subject is a lamb.

35

30. The method according to claim 17 wherein said domain is bound to an antibody to a cellular antigen.

31. The method according to claim 17 wherein said domain is adrninistered as part of a pharmaceutical composition.

5 32. The method according to claim 31 wherein said pharmaceutical composition is adapted for intravenous administration to human beings.

33. The method according to claim 31 wherein said domain is formulated as a suppository.

10

34. A pharmaceutical composition comprising a composition according to claim 9 further comprising one or more ingredients selected from the group consisting of a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, a wetting agent, a buffering agent, an emulsifying agent, and a binding agent.

15

35. The composition according to claim 34 wherein the domain is immobilized on a biologically-inert support selected from the group consisting of a non-polymeric and a polymeric support

20 36. The composition according to claim 35 wherein the support comprises a material selected from the group consisting of a polymeric support in a form selected from a bead, a fiber, and a sheet; paper; and cotton thread.

37. The composition according to claim 36 wherein the polymeric support comprises a 25 functionalized polystyrene.

38. A kit comprising, in one or more containers, a pharmaceutical composition according to claim 34.

30 39. The method according to claim 17 further comprising administering to the subject a cofactor selected from the group consisting of glutathione, ATP and GTP within 120 hours of the time of administering the composition.

40. A composition according to claim 9, further comprising a probe characteristic. 35

41. A composition according to claim 40, wherein the probe characteristic is selected from the group consisting of incoφoration of one or more radioactive metals into the domain; isotopic substitution of one or more atoms of the domain; fusion to the domain of one or more compounds selected from the group consisting of a fluorophore, an NMR contrast reagent, and a Foerster radiation-less energy transfer reagent; and arsenoazo modification of one or more amino acids of the domain.