WO2004099768A1 - Method for detecting development of a physiological disorder in a subject - Google Patents

Abstract

Development of a physiological disorder in a subject is detected by measuring a test concentration of an LKKTET peptide in at least a portion of the subject or in a sample of the subject, and determining if the test concentration is outside a normal concentration or concentration range of the LKKTET peptide for the subject.

Description

METHOD FOR DETECTING DEVELOPMENT OF A PHYSIOLOGICAL DISORDER IN A SUBJECT

BACKGROUND OF THE INVENTION

CROSS-REFERENCE TO RELATED APPLICATION [001] This application claims benefit of U.S. Provisional application No. 60/467,626, filed May 5, 2003.

Field of the Invention [002] The present invention relates to the field of diagnosing physiological disorders.

Description of the Background Art

[003] Healthcare has progressed significantly from the days when a physician had to rely on visual or other sensual observations of a patient, or the patient's description of a health problem, in order to attempt to diagnose a malady. There now exist many diagnostic tools, including mechanical, ultrasonic, electrical, chemical and optical tools, to assist physicians in diagnosing physiological disorders. Nevertheless, there remains a need in the art for new methods of detecting physiological disorders. SUMMARY OF THE INVENTION

[004] In accordance with the present invention, a method for detecting development of a physiological disorder in a subject includes a step of measuring a test concentration of an LKKTET peptide in at least a portion of the subject or in a sample of the subject. The method includes a step of determining if the test concentration is outside a normal concentration or concentration range of the LKKTET peptide for the subject, so as to detect development of a physiological disorder in the subject. DETAILED DESCRIPTION OF THE INVENTION [005] The present invention measures actin-sequestering peptides such as thymosin B4 (TB4) and other actin-sequestering peptides or peptide fragments containing amino acid sequence LKKTET or conservative variants thereof. Sometimes these are referred to herein as LKKTET peptides. Included are N- or C-terminal variants such as KLKKTET and LKKTETQ. [006] The present invention relates generally to the detection and/or diagnosis of inflammatory, degenerative, immunological and other disorders of the skin, eye and surrounding tissue, other organs, and body fluids that occur due to a variety of factors (including but not limited to aging, disease or trauma), by detecting the levels of an LKKTET peptide in the tissue, organ, gland, or body fluid, as well as the levels of an LKKTET peptide circulating in the blood. [007] Millions of people suffer from chronic skin disorders and eye disorders, which may be due, in part or in whole, to the lack of production of an LKKTET peptide such as TB4. Many such disorders may result from the natural aging process, disease, or trauma. These disorders occur in both men and women. As people age, chronic wounds and the inability to heal either rapidly or completely becomes more commonplace. This may account for increased incidence of disorders such as "dry eye" for example, or chronic dermal wounds such as pressure sores. People with underlying disease states, such as diabetes, or who are on medications such as steroids, experience difficulties with chronic wounds and the inability to heal properly or rapidly. [008] In many cases, dry eye results from disorders of the various glands which work together to produce normal tears. Many different things may cause dry eye syndrome. The normal aging of tear glands, as well as specific diseases and disorders, may cause changes in the amount or condition of tears produced. For example, Sjδgren's syndrome is an immune system disorder characterized by inflammation and dryness of the mouth, eyes, and other mucous membranes, and damage to the lacrimal glands, and this damage affects tear production. Hormonal changes can also affect secretions from the tear glands. The incidence of dry eye increases in post-menopausal women and post-menopausal women have a reduced amount of TB4 circulating in the blood.

[009] Recently it has been found that as people age, the levels of TB4 in human tears diminishes, thus this may allow for detection or confirmation of ocular indications that may be treatable by any number of means, including the administration of an LKKTET peptide such as TB4.

[0010] It has been found that in patients with Alzheimer's Disease the levels of TB4 are abnormally low. Likewise in post-menopausal women the levels of TB4 circulating in the blood is abnormally low. [0011] Thymosin B4 was initially identified as a protein that is up-regulated during endothelial cell migration and differentiation in vitro. Thymosin B4 is a 43 amino acid, 4.9 kDa ubiquitous polypeptide identified in a variety of tissues. Several roles have been ascribed to this protein including a role in a endothelial cell differentiation and migration, T cell differentiation, actin sequestration and vascularization. [0012] Thymosin beta 4 is a member of the beta-thymosin family of highly conserved polar 5-kDa polypeptides found in various tissues and cell types. Originally purified from thymus and regarded as a thymic hormone, thymosin B4 was then found to be involved in multiple biological processes. As the main G-actin sequestering peptide, it plays an important role in regulation of actin assembly during cell proliferation, migration, and differentiation. Numerous studies implicate thymosin B4 in regulation of cancerogenesis, inflammation, angiogenesis, and wound healing. It was found that thymosin B4 expression regulated tumorigenicity and metastatic activity in malignant cell lines through actin-based cytoskeletal organization. Thymosin B4 was found to be elevated in tube forming endothelial cells; it increases their attachment, spreading and migration thus promoting angiogenesis. Thymosin B4 was also found in ulcer extracts and wound fluids at high concentrations and was suggested to function as an antibacterial factor.

The stimulating role of thymosin B4 in wound healing was demonstrated in several studies with animal models. When added topically or administered intraperitoneally, thymosin B4 enhanced dermal wound healing in a rat full thickness model. The ability to accelerate dermal wound healing has also been observed in db/db diabetic mice, steroid-immunosuppressed mice and in aged mice. Thymosin B4 has also been shown to accelerate healing of the corneal epithelium after burn injuries and to down regulate a number of corneal cytokines and chemokines reducing the inflammatory response.

[0013] As noted above, the present invention is a method for detecting development of a physiological disorder in a subject. The invention is particularly applicable to mammalian subjects such as human patients. [0014] The invention includes a step of measuring a test concentration of an LKKTET peptide in at least a portion of the subject or in a sample of the subject. A determination is made if the test concentration is outside a normal concentration or concentration range of the LKKTET peptide for the subject, so as to detect or identify development of a physiological disorder in the subject. [0015] During the determining step, the test concentration may be compared with a normal concentration or concentration range of the LKKTET peptide for the subject. The normal concentration or concentration range may be easily be determined by any person skilled in the art utilizing well known techniques. [0016] The ability of detect LKKTET peptides such as TB4 using any number of detection methods has been available for many years, although no correlation has been reported between levels of TB4 and a physiological disorder or disease state or other medical abnormality. Nor has there been any reported information indicating TB4 levels as they related to the aging of the skin or other body organs and tissues. [0017] An assay for LKKTET peptides such as TB4 (or its analogues, isoforms, or derivatives) which correlates to a physiological disorder, or the aging process, or other abnormality, has great diagnostic value and offers the possibility of earlier or better treatment or possibly even prevention of manifestation of disease or disease prevention. [0018] A simple one-step procedure for the determination of thymosin beta 4 in whole blood, various blood corpuscles, plasma and serum by reversed-phase high-performance liquid chromatography (HPLC) has been previously described in the art. The blood cells were isolated by a Ficoll density gradient technique. The isolated cells were lysed and deproteinized with perchloric acid in the presence of an internal standard. The supernatant solution was neutralized by potassium hydroxide solution and thymosin beta 4 was measured by HPLC without further manipulations. The following amounts (fg per cell) were determined: polymorphonuclear leukocytes (409), mononuclear leukocytes (267) and platelets (22.3). Erythrocytes contained no thymosin beta 4. The average concentration of thymosin beta 4 in an extract of whole blood was 16.3 micrograms/ml. The concentration in plasma was less than 1% of that value. However, if serum was prepared and not immediately removed from the clotted blood, the concentration of the peptide increased from 0.04 to 2.1 micrograms/ml within 24 h. [OO 19] Thymosin beta 4 has been determined by a simple and fast one-step procedure in different tissue samples. The tissues (1 to 40 mg) were disintegrated and deproteinized by homogenization in perchloric acid. After neutralization by potassium hydroxide the supernatant solution was used for determining thymosin beta 4 by reverse-phase HPLC without further manipulations. Not only does this procedure avoid artificial proteolysis as effectively as extraction of tissues by guanidinium chloride or boiling buffer, but it offers two further advantages. First, no additional steps— as for example desalting—are necessary prior to HPLC and thus the risk of losing thymosin beta 4 is eliminated. Using this procedure thymosin beta 4 is recovered quantitatively. The method is linear over the range 0.04 to 1.13 nmol and thymosin beta 4 is well separated from other thymosin beta 4-like peptides known to be present in mammals; i.e., thymosin beta Ala4, thymosin beta 9, thymosin beta 10, and thymosin beta

ArglO. Second, the acid-insoluble pellet of the same extract can be used to determine the DNA content of the sample. Thus it is possible to relate thymosin beta 4 to DNA, which then allows comparing cells of different tissues and cell lines to one another. This procedure is also applicable to other small peptides soluble in perchloric acid.

[0020] A method was described in the art for the isolation of peptides from thymus tissue. Frozen, powdered tissue was suspended in boiling buffer to inactivate endogenous proteinases, the suspension was homogenized, and the peptides were isolated by a two-step procedure including gel filtration and purification by HPLC. The recoveries from thymus tissue were, in micrograms per gram of whole tissue, 60-80 for prothymosin alpha, 50-80 for thymosin beta 4, and 20-30 for thymosin beta 10. The procedure also yielded smaller quantities of a fourth peptide, designated para thymosin alpha. The quantities of these peptides in vertebrate tissues can be evaluated by applying radioimmunoassays for prothymosin alpha and thymosin beta 4 to the boiled tissue extract.

[0021] In order to indicate development of a physiological disorder, the test concentration of LKKTET peptide can be, for example, about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or even 99% or more outside a normal concentration or concentration range of LKKTET peptide for the subject.

[0022] The present invention is based on the discovery that anti-inflammatory peptides and actin-sequestering LKKTET peptides such as TB4, TB4 sulfoxide, TB4 splice variants, and a number of other TB4-like peptides (or analogues, isoforms, or derivatives) may be useful for the diagnosis of certain physiological states, disorders, diseases, or other abnormal condition of the skin, eye, other tissues or organs, or body fluids which should normally contain certain levels of TB4 (or its analogues, isoforms, or derivatives). Since these peptides have the capacity to promote repair and healing by having the ability to induce terminal deoxynucleotidyl transferase (a non-template directed DNA polymerase), to decrease the levels of one or more inflammatory cytokines, regulate actin, or to act as a chemotactic and angiogenic factor for endothelial cells and thus heal and reverse degenerative changes in the eye and other organs, their levels, absence, or over abundance serve as a way to detect or treat certain diseases or disorders. [0023] The invention involves a method of determining a physiological disorder comprising measuring an LKKTET peptide such as TB4 concentration (or analogues, isoforms, derivatives such as TB4 sulfoxide and spliced variants of TB4) in a patient or patient sample.

[0024] The invention involves evaluating the presence of an LKKTET peptide such as TB4 (or analogues, isoforms, derivatives) using monoclonal or polyclonal antibody-based radioimmunoassay, enzyme-linked immunoassays or any other suitable method, such as capillary electrophoresis or mass spectrometry analysis, that can determine such levels for the purpose of determining the likelihood of diseases or disorders which are a result of abnormal levels of an LKKTET peptide such as TB4 (or analogues, isoforms, derivatives). [0025] The invention involves a measurement of abnormal levels of an LKKTET peptide such as TB4 (or analogues, isoforms, derivatives) indicating an underlying disorder, for example in human tears as humans age, or at the site of wounds, or circulating in the blood or other body fluids such as cerebral spinal fluid, tears, seminal fluids, milk, saliva and lymphatic fluids, which may be treated by administering LKKTET peptides such as TB4 (or analogues, isoforms, derivatives), or by biopsy of tissues in disease states, such as the diabetic cornea which has abnormally low levels of TB4, or in cancer, such as melanoma, prostate cancer, breast cancer, thyroid cancer and fϊbro cancers which have elevated levels of TB4, TB10, TB15 or other LKKTET peptide(s).

[0026] The invention involves a measurement of abnormal levels of an LKKTET peptide such as TB4 (or analogues, isoforms, derivatives) circulating in human blood or present on or within the skin associated with the propensity of the skin to wrinkle, or result in chronic inflammation, or may be treated or ameliorated by administering LKKTET peptides such as TB4 (or analogues, isoforms, derivatives). [0027] In one aspect of the method, the diagnostic test may be performed in vivo, systematically or locally.

[0028] In another aspect of the method, the diagnostic test may be performed in vitro or by any other suitable means available for identifying the presence of molecules, peptides and other chemicals in the skin, eye, other body organs, body fluids (such as cerebral spinal fluid, tears, seminal fluids, milk, saliva and lymphatic fluid) and blood.

[0029] In preferred embodiments, after development of the physiological disorder has been detected, an LKKTET peptide is administered to the subject so as to treat or ameliorate the physiological disorder. [0030] In order to detect a physiological disorder, the test concentration of LKKTET peptide may be determined to be above or below the normal concentration or concentration range. For physiological disorders such as Alzheimer's Disease and disorders present in post-menopausal women, test concentrations of LKKTET peptide are below a normal concentration or concentration range for the subject. One example of a physiological disorder present in some post-menopausal women is dry eye syndrome.

[0031] In preferred embodiments, the LKKTET peptide comprises amino acid sequence KLKKTET or LKKTETQ, Thymosin B4 (TB4), an N-terminal variant of TB4, a C-terminal variant of TB4, an isoform of TB4, a splice-variant of TB4, oxidized TB4, TB4 sulfoxide, lymphoid TB4, pegylated TB4 or any other actin sequestering or bundling proteins having actin binding domains, or peptide fragments comprising or consisting essentially of the amino acid sequence LKKTET or conservative variants thereof. International Application Serial No. PCT/US99/17282, incorporated herein by reference, discloses isoforms of TB4 which may be useful in accordance with the present invention as well as amino acid sequence LKKTET and conservative variants thereof, which may be utilized with the present invention. International Application Serial No. PCT/GB99/00833 (WO 99/49883), incorporated herein by reference, discloses oxidized Thymosin B4 which may be utilized in accordance with the present invention. Although the present invention is described primarily herein with respect to TB4 and TB4 isoforms, it is to be understood that this description is intended to be equally applicable to amino acid sequence LKKTET, LKKTETQ, peptides and fragments comprising or consisting essentially of LKKTET or LKKTETQ, conservative variants thereof, as well as oxidized Thymosin B4.

[0032] For treatment, the LKKTET peptide such as TB4, or its analogues, isoforms or derivatives, may be administered in any suitable effective amount. For example, TB4 may be administered in dosages within the range of about 0.1-50 micrograms of TB4, more preferably in amounts within the range of about 1-25 micrograms. [0033] A composition in accordance with the present invention can be administered daily, every other day, etc., with a single administration or multiple administrations per day of administration, such as applications 2, 3, 4 or more times per day of administration. [0034] TB4 isoforms have been identified and have about 70%, or about 75%, or about 80% or more homology to the known amino acid sequence of TB4. Such isoforms include, for example, TB4^ala, TB9, TBIO, TBl l, TB12, TB13, TB14 and TB15. Similar to TB4, the TBIO and TB 15 isoforms, as well as the TB4 splice-variants, have been shown to sequester actin. TB4, TBIO and TB15, as well as these other isoforms share an amino acid sequence, LKKTET, that appears to be involved in mediating actin sequestration or binding. Although not wishing to be bound to any particular theory, the activity of TB4 isoforms may be due, in part, to the ability to regulate the polymerization of actin. Beta-thymosins appear to depolymerize F-actin by sequestering free G-actin. TB4's ability to modulate actin polymerization may therefore be due to all, or in part, its ability to bind to or sequester actin via the LKKTET sequence. Thus, as with TB4, other proteins which bind or sequester actin, or modulate actin polymerization, including TB4 isoforms having the amino acid sequence LKKTET, are likely to be effective, alone or in a combination with TB4, as set forth herein. [0035] Thus, it is specifically contemplated that known TB4 isoforms, such as TB4^ala, TB9, TBIO, TB1 1, TB12, TB13, TB14 and TB15, as well as TB4 isoforms and TB4 splice-variants not yet identified, will be useful in the methods of the invention. As such TB4 isoforms are useful in the methods of the invention, including the methods practiced in a subject. The invention therefore utilizes pharmaceutical compositions comprising TB4, as well as TB4 isoforms TB4^ala, TB9, TBIO, TB11, TB12, TB13, TB14 and TB15, and a pharmaceutically acceptable carrier.

[0036] In addition, other proteins having actin sequestering or binding capability, or that can mobilize actin or modulate actin polymerization, as demonstrated in an appropriate sequestering, binding, mobilization or polymerization assay, or identified by the presence of an amino acid sequence that mediates actin binding, such as LKKTET, for example, can similarly be employed in the methods of the invention. Such proteins include gelsolin, vitamin D binding protein (DBP), profilin, cofϊlin, adsevertin, propomyosin, fincilin, depactin, Dnasel, villin, fragmin, severin, capping protein, B-actinin and acumentin, for example. As such methods include those practiced in a subject, the invention further provides pharmaceutical compositions comprising gelsolin, vitamin D binding protein (DBP), profilin, cofϊlin, depactin, Dnasel, villin, fragmin, severin, capping protein, B-actinin and acumentin as set forth herein. Thus, the invention includes utilization of a polypeptide comprising the amino acid sequence LKKTET (which may be within its primary amino acid sequence) and conservative variants thereof. [0037] As used herein, the term "conservative variant" or grammatical variations thereof denotes the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the replacement of a hydrophobic residue such as isoleucine, valine, leucine or methionine for another, the replacement of a polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like. [0038] TB4 has been localized to a number of tissue and cell types and thus, agents which stimulate the production of TB4 can be added to or comprise a composition to effect TB4 production from a tissue and/or a cell. Such agents include members of the family of growth factors, such as insulin-like growth factor (IGF-1), platelet derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor beta (TGF-B), basic fibroblast growth factor (bFGF), thymosin l (T l), prothymosin a (PTα) and vascular endothelial growth factor (VEGF). More preferably, the agent is transforming growth factor beta (TGF-B) or other members of the TGF-B superfamily. [0039] Additionally, agents that assist or stimulate healing may be utilized along with TB4 or a TB4 isoform. Such agents include angiogenic agents, growth factors, agents that direct differentiation of cells. For example, and not by way of limitation, TB4 or a TB4 isoform alone or in combination can be utilized in combination with any one or more of the following agents: VEGF, KGF, FGF, PDGF, TGFB, IGF-1, IGF-2, IL-1, prothymosin and thymosin αl 1 in an effective amount. [0040] The actual dosage, formulation or composition that heals or prevents inflammation, damage and degeneration may depend on many factors, including the size and health of a subject. However, persons of ordinary skill in the art can use teachings describing the methods and techniques for determining clinical dosages as disclosed in PCT/US99/17282, supra, and the references cited therein, to determine the appropriate dosage to use. [0041] Suitable formulations for administration may include TB4 or a TB4 isoform at a concentration within the range of about 0.001 - 10% by weight, within the range of about 0.01 - 0.1% by weight, or even about 0.05% by weight.

[0042] The invention includes use of antibodies which interact with TB4 peptide or functional fragments thereof. Antibodies which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided. Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known to those skilled in the art as disclosed in PCT/US99/ 17282, supra. The term antibody as used in this invention is meant to include monoclonal and polyclonal antibodies.

[0043] In yet another embodiment, the invention utilizes an effective amount of an agent which modulates TB4 gene expression. The term "modulate" refers to inhibition or suppression of TB4 expression when TB4 is over expressed, and induction of expression when TB4 is under expressed. The term "effective amount" means that amount of TB4 agent which is effective in modulating TB4 gene expression resulting in effective treatment. An agent which modulates TB4 or TB4 isoform gene expression may be a polynucleotide for example. The polynucleotide may be an antisense, a triplex agent, or a ribozyme. For example, an antisense directed to the structural gene region or to the promoter region of TB4 may be utilized.

[0044] In another embodiment, the invention utilizes compounds that modulate TB4 activity. Compounds that affect TB4 activity (e.g., antagonists and agonists) include peptides, peptidomimetics, polypeptides, chemical compounds, minerals such as zincs, and biological agents.

[0045] Although the invention has been described herein in detail, the above description is not intended to be limiting.

Claims

1. A method for detecting development of a physiological disorder in a subject, comprising steps of measuring a test concentration of an LKKTET peptide in at least a portion of said subject or in a sample of said subject, and determining if said test concentration is outside a normal concentration or concentration range of said LKKTET peptide for said subject, so as to detect development of physiological disorder in said subject.

2. The method of claim 1 wherein, during the determining step, said test concentration is compared with a normal concentration or concentration range of said LKKTET peptide for said subject.

3. The method of claim 1 wherein said test concentration is measured using a monoclonal or polyclonal antibody-based radioimmunoassay, enzyme-linked immunoassay, capillary electrophoresis or mass spectrometry analysis.

4. The method of claim 1 wherein said portion or sample comprises a wound site, body fluid, tear sample, blood, cerebral spinal fluid, seminal fluid, saliva, lymphatic fluid, or skin.

5. The method of claim 4 wherein said LKKTET peptide comprises amino acid sequence KLKKTET or LKKTETQ, Thymosin β4 (TB4), an N-terminal variant of TB4, a C- terminal variant of TB4, an isoform of TB4, a splice-variant of TB4, oxidized TB4, TB4 sulfoxide, lymphoid TB4 or pegylated TB4.

6. The method of claim 1 wherein, after development of said physiological disorder has been detected, further including a step of administering said LKKTET peptide to said subject so as to treat or ameliorate said physiological disorder.

7. The method of claim 6 wherein said LKKTET peptide comprises amino acid sequence KLKKTET or LKKTETQ, Thymosin B4 (TB4), an N-terminal variant of TB4, a C- terminal variant of TB4, an isoform of TB4, a splice-variant of TB4, oxidized TB4, TB4 sulfoxide, lymphoid TB4 or pegylated TB4.

8. The method of claim 1 wherein the measuring step is performed in vivo in said subject.

9. The method of claim 8 wherein said LKKTET peptide comprises amino acid sequence KLKKTET or LKKTETQ, Thymosin B4 (TB4), an N-terminal variant of TB4, a C- terminal variant of TB4, an isoform of TB4, a splice-variant of TB4, oxidized TB4, TB4 sulfoxide, lymphoid TB4 or pegylated TB4.

10. The method of claim 9 wherein said measuring step is performed systemically in said subject.

11. The method of claim 9 wherein said measuring step is performed locally in said subject.

12. The method of claim 1 wherein the measuring step is performed in vitro with said sample.

13. The method of claim 12 wherein said LKKTET peptide comprises amino acid sequence KLKKTET or LKKTETQ, Thymosin B4 (TB4), an N-terminal variant of TB4, a C- terminal variant of TB4, an isoform of TB4, a splice-variant of TB4, oxidized TB4, TB4 sulfoxide, lymphoid TB4 or pegylated TB4.

14. The method of claim 13 wherein said sample comprises organ tissue, skin tissue, eye tissue, body fluid, cerebral spinal fluid, tear fluid, seminal fluid, milk, saliva, lymphatic fluid or blood.

15. The method of claim 14 wherein said sample comprises blood.

16. The method of claim 1 wherein said test concentration is determined to be below said normal concentration or concentration range.

17. The method of claim 16 wherein said LKKTET peptide comprises amino acid sequence KLKKTET or LKKTETQ, Thymosin B4 (TB4), an N-terminal variant of TB4, a C- terminal variant of TB4, an isoform of TB4, a splice-variant of TB4, oxidized TB4, TB4 sulfoxide, lymphoid TB4 or pegylated TB4.

18. The method of claim 1 wherein said physiological disorder is a disease in which said LKKTET peptide is overexpressed or underexpressed in body fluids or tissues of said subject.

19. The method of claim 1 wherein said physiological disorder is Alzheimer's Disease, dry eye syndrome, diabetes or cancer.

20. The method of claim 1 wherein said physiological disorder is present in post- menopausal women.