EP1913013A2 - Truncated oxidized thymosin beta4 and derivatives thereof - Google Patents

Truncated oxidized thymosin beta4 and derivatives thereof

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Publication number
EP1913013A2
EP1913013A2 EP06788075A EP06788075A EP1913013A2 EP 1913013 A2 EP1913013 A2 EP 1913013A2 EP 06788075 A EP06788075 A EP 06788075A EP 06788075 A EP06788075 A EP 06788075A EP 1913013 A2 EP1913013 A2 EP 1913013A2
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EP
European Patent Office
Prior art keywords
oxidized
thymosin
disease
truncated
peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP06788075A
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German (de)
French (fr)
Inventor
Elizabeth Taylor
Robert Goodenow
John Young
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Grannus Biosciences
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Grannus Biosciences
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Filing date
Publication date
Application filed by Grannus Biosciences filed Critical Grannus Biosciences
Publication of EP1913013A2 publication Critical patent/EP1913013A2/en
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/57581—Thymosin; Related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a synthetic peptide portion of oxidized thymosin ⁇ 4 or variants thereof. More particularly, the invention relates to a peptide containing 14 N-terminal amino acids of oxidized thymosin ⁇ 4 or variants thereof. Such peptides have oxidized thymosin ⁇ 4 activity which are useful for treating various diseases and conditions, for example, diseases and conditions associated with inflammation and those for which steroid therapy is useful.
  • Steroids are effectively used in the treatment of inflammatory diseases, such as asthma, eczema, dermatitis, psoriasis, allergic reactions, and rheumatic diseases such as rheumatoid arthritis.
  • inflammatory diseases such as asthma, eczema, dermatitis, psoriasis, allergic reactions, and rheumatic diseases such as rheumatoid arthritis.
  • Steroids both inhaled and oral forms, are also used for the clinical management of cystic fibrosis.
  • steroids have serious side effects and are therefore only used intermittently or in cases where non-steroidal anti-inflammatory drugs are not effective.
  • Peripheral blood mononuclear cells such as monocytes and lymphocytes are important immune effector cells that play a fundamental role in cellular immunity.
  • monocytes In addition to their antigen-presenting and phagocytic activities at the sites of inflammation, monocytes are also involved in the synthesis and release of a variety of pro-inflammatory enzymes and polypeptide cytokines which modulate neutrophil responses. The production of these proinflammatory components can be suppressed by glucocorticoids and this has been suggested as the basis for their anti-inflammatory action.
  • Cytokines, cheniokines and mitogens are a diverse group of soluble proteins and peptides with immunomodulatory properties. These agents regulate the activities of individual cells and tissues either under normal or pathological conditions. These immunomodulatory agents are produced by multiple immune cell types and have pleiotropic effects on multiple target cells thus resulting in multiple biological actions and outcomes. Constitutive cytokine and chemokine production is usually low or absent and its production is promoted by certain biochemical stimuli. Given that the functions of these humoral immunomodulators are pleiotropic they also tend to be redundant.
  • these immunomodulatory agents In host defense responses to infection, inflammation, autoimmune reactions, wounds, trauma and metabolic injury, these immunomodulatory agents not only stimulate immune cells to proliferate and differentiate but also regulate the progression and termination of immunological reactions.
  • Pro-inflammatory agents cause inflammation to progress, notable among these are TNF- ⁇ and IL- 12 and IL-18 the later of which often act in concert. When these early and mid acting agents are produced they act to destroy normal and diseased tissues. They also enhance the synthesis of other pro-inflammatory cytokines by various immunological cells, and can induce systemic inflammatory reactions such as fever and shock.
  • Another class of immunoregulatory agents serves to counteract inflammation by inhibiting the production of proinflammatory agents.
  • Chettibi et al. (1993, 1994) have investigated the steroid induced stimulatory effect on neutrophil migration using an automated cell tracking assay enabling study of the behavior of cells migrating on protein-coated glass coverslip.
  • STMS Steroid-treated monocyte supernatant
  • Huff T. et al (Eur J Biochem. (1995) 230(2):650-7) and Heintz D. et al. (Eur J Biochem. (1994) 223(2):345-50) describe studies involving beta-thymosins and how they interact with G-actin in a biomolecular complex and inhibit the polymerization to F-actin under high salt conditions.
  • the oxidized form of thymosin ⁇ 4 is disclosed as inhibiting actin polymerization, however, only at a 20-fold higher concentration than thymosin ⁇ 4. Neither document however implicates any medical role for oxidized thymosin ⁇ 4. In fact the papers appear to teach away from a positive role for oxidized thymosin ⁇ 4.
  • Huff et al. (Intl J. of Biochem & Cell Biol. (2001) 33:205-220) reported that oxidation of thymosin ⁇ 4 increases the dissociation constant of the actin thymosin ⁇ 4 complex 20 fold and suggested that the oxidation of thymosin ⁇ 4 to its sulfoxide could regulate the binding to G and F-actin in vivo. This teaches that oxidation of the methionine at residue six decreases the actin binding activity of thymosin ⁇ 4. Moreover, Huff et al.
  • U.S. Patent No. 5,578,570 discloses a method of treating septic shock by administering thymosin ⁇ 4. There is no disclosure however of oxidized thymosin ⁇ 4 or suggestion that this may have a role in treating septic shock.
  • WO 03/0060405, WO 02/36143, WO 02/091969, WO 03/020215 identify thymosin ⁇ 4 peptides useful for various diseases or conditions that contain an amino acid sequence LKKTET, which is not a N-terminal amino acid sequence.
  • US Pat. No. 6,602,519 discusses the physiological activity of oxidized thymosin ⁇ 4 in comparison to thymosin ⁇ 4. It would be advantageous to the art to provide a molecule that can achieve better results than oxidized thymosin ⁇ 4 for various indications such as those requiring small molecular weight compounds suitable for inhalation, parenteral or injection therapies.
  • the invention provides an isolated synthetic peptide where the peptide comprises at least seven consecutive amino acids that correspond to the 14 N-terminal amino acids of oxidized thymosin ⁇ 4 or variants thereof, wherein at least one amino acid of the peptide is an oxidized methionine and wherein the peptide has oxidized thymosin ⁇ 4 activity.
  • the invention also includes compositions comprising the peptide in combination with a pharmaceutically-acceptable carrier, as well as methods of treating a condition or disease using the truncated peptide.
  • FIG. 1 illustrates human umbilical vein endothelial cells (HUVEC) cell migration results for oxidized thymosin ⁇ 4 and various truncated peptides GBLOl -GBL05 at a 2 nm level.
  • GBLOl and GBL02 are ac-SDKPDMAEIEKFDK (SEQ ID NO:1).
  • GBL 04 and GBL 05 are described below and GBL 05 is MLLPATMSDKPDMAEIEKFDK (SEQ ID NO:2), where M is an oxidized sulfur in the methionine.
  • Fibroblast growth factor (FGF) is the control.
  • Figure 2 illustrates HUVEC cell migration results for oxidized thymosin ⁇ 4 and various truncated peptides at a 20 nm level.
  • Figure 3 illustrates HUVEC cell migration results based on percent wound closure for oxidized thymosin illustrates HUVEC cell migration results for oxidized thymosin ⁇ 4 and various truncated peptides at a 2 nm level.
  • Figure 4 illustrates HUVEC cell migration results based on percent wound closure for oxidized thymosin illustrates HUVEC cell migration results for oxidized thymosin
  • Figure 7 shows two bar graphs illustrating the ability of oxidized thymosin ⁇ 4 to inhibit the ability of pro-inflammatory cytokines and mitogens to induce TNF- ⁇ in peripheral blood mononuclear cells (PBMCs) at levels equivalent to the corticosteroid dexamethasone.
  • Figure 7A shows GBLlOl inhibition on TNF- ⁇ production by PBMCs in the presence of IL- 12/18.
  • Figure 7B shows GBLlOl inhibition on TNF- ⁇ production by PBMCs in the presence of LPS.
  • Figure 8 is a bar graph illustrating that GBLlOl induces apoptosis in human peripheral blood neutrophils in a dose dependant fashion.
  • Figure 9 shows a bar graph illustrating that the presence of an oxidized methionine residue in the truncated oxidized thymosin ⁇ 4 (T ⁇ 4so) peptide GBLl 06 is required for biological activity in the wound scratch assay.
  • truncated oxidized thymosin ⁇ 4" compounds or variants thereof include compounds that are less than the full length of oxidized thymosin ⁇ 4, or variants thereof, but still retain the activity of oxidized thymosin ⁇ 4
  • the oxidized methionine in the N-terminal portion of the protein is responsible for at least a part of the overall activity of oxidized thymosin ⁇ 4.
  • substitution of methionine with nor-leucine which contains a methyl group in place of the sulfur group of Met, destroys the anti- inflammatory and wound healing properties of the truncated 14 mer.
  • the truncated oxidized thymosin ⁇ 4 of the invention is a peptide which is 14 amino acids of the N- terminal portion of oxidized thymosin ⁇ 4 or variants thereof that retain oxidized thymosin ⁇ 4 activity.
  • peptides are of sufficient length, for example, seven amino acids in length, and have secondary structure in solution such as alpha-helical configurations.
  • peptides of such sufficient length include acetylated or unacetylated peptides such as SDKPDMA (SEQ ID NO:3), DKPDMAE (SEQ ID NO:4), KPDMAEI (SEQ ID NO:5), PDMAEIE (SEQ ID NO:6), DMAEIEK (SEQ ID NO:7), MAEIEKF (SEQ ID NO:8), or variants thereof that retain oxidized thymosin ⁇ 4 activity.
  • Native thymosin ⁇ 4 is acetylated and thus truncated oxidized thymosin ⁇ 4 are preferably acetylated.
  • the truncated oxidized thymosin ⁇ 4 peptide may further comprise a carrier, such as a prodrug known to one of skill in the art, or a protein, peptide, or amino acid moiety.
  • a carrier such as a prodrug known to one of skill in the art
  • the amino acid or protein moiety that is attached to the truncated oxidized thymosin ⁇ 4 contains at least one oxidized methionine, and preferably one or two oxidized methionines.
  • Preferred embodiments include ac-MSDKPDMAEIEKFDK (SEQ ID NO:9) (GBL03) or ac-LLP ATMSDKPDMAEIEKFDK (SEQ ID NO: 10) (GBL04), wherein the letter M in the preceding truncated peptides is methionine sulphoxide.
  • the truncated portion does not contain the amino acid sequence LKKTET (SEQ ID NO:1 1) or a conservative variant thereof.
  • the truncated peptide of the invention preferably does not contain a G-actin binding region and, without being bound by theory, it is believed that this portion does not play as important a role as was thought in thymosin ⁇ 4 activity, but rather the oxidized methionine is important for such activity.
  • variants of oxidized thymosin ⁇ 4 are conservative amino acid variants.
  • 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.
  • the present invention provides a replacement to steroid therapy.
  • the present invention provides use of a truncated oxidized thymosin ⁇ 4, preferably a 14 N-terminal amino acid peptide thereof, or physiologically active variant in therapy.
  • oxidized thymosin ⁇ 4 is a form of thymosin ⁇ 4 in which a methionine residue, 6 amino acids from the N-terminus, (Met6), is oxidized such that the residue is converted to methionine sulphoxide.
  • the methionine residue (Met6) may be further oxidized to the methionine sulphone and this as such is also encompassed by the present invention.
  • Other modifications of the methionine residue may also be envisaged, such as complexing the sulfur with metals, which may result in an active form of thymosin ⁇ 4 similar to the oxidized form described herein.
  • the truncated oxidized thymosin ⁇ 4 may be obtained for example by reacting a native truncated thymosin ⁇ 4 under oxidizing conditions, for example by treating with hydrogen peroxide, to form oxidized thymosin ⁇ 4, which is then truncated.
  • native thymosin ⁇ 4 may first be obtained and thereafter oxidized to the oxidized form.
  • truncated oxidized thymosin ⁇ 4 is made synthetically by procedures known in the art.
  • the present invention provides the use of purified truncated oxidized thymosin ⁇ 4 variants and compositions thereof.
  • Preferred compositions contain truncated oxidized thymosin ⁇ 4 which comprise at least 30%, 60%, or 80%, and more preferably 90%, 95%, 98%, or 99% of truncated oxidized thymosin ⁇ 4 or variants thereof.
  • isolated and purified truncated, oxidized thymosin ⁇ 4 is preferred.
  • Thymosin ⁇ 4 in an oxidized or non-oxidized form may be obtained from any suitable source, for example from steroid treated monocytes.
  • the thymosin ⁇ 4 may be derived from any suitable species, but is typically of mammalian origin, such as bovine, equine, murine or human origin. It is to be noted that bovine, equine, murine, rat and human thymosin ⁇ 4 are all identical in sequence.
  • bovine thymosin ⁇ 4 may provide a suitable source of thymosin ⁇ 4 for subsequent oxidation and administration to other species, such as humans.
  • Physiologically active truncated oxidized thymosin ⁇ 4 peptides or variants thereof display similar physiological properties as displayed by oxidized thymosin ⁇ 4. It is preferred that such variants would include the oxidized methionine, but such variants may be truncated, deleted or mutated forms thereof.
  • Group VIB elements e.g., Selenium, Tellurium, Polonium.
  • IX oxidized methionine and other oxidized methionine analogues (e.g., Group VIB analogues, methionine sulfoximine).
  • X methionine and other sulfur-containing amino acids (e.g., et al S and R cysteine) including their oxidized analogues.
  • peptides that contain less than the full length amino acid sequence of oxidized thymosin ⁇ 4 retains the activity thereof. Indeed this may assist in the development of peptide mimetics which display the same physiological function as the oxidized thymosin ⁇ 4.
  • the truncated oxidized thymosin ⁇ 4 may have a blocked N-terminal.
  • a synthetic truncated oxidized thymosin ⁇ 4 comprising the peptide sequence of thymosin ⁇ 4 in oxidized form or physiologically active variant thereof.
  • the synthetic oxidized thymosin ⁇ 4 may be modified and/or amino acid substituted as described above, as long as the physiological activity remains.
  • selenomethionine could be introduced in place of methionine and oxidized in the same manner.
  • the invention further provides the use of a truncated oxidized thymosin ⁇ 4 compounds as described herein in the preparation of a medicament for the treatment of a chronic or acute inflammatory condition or to treat inflammation.
  • the disclosed truncated oxidized thymosin ⁇ 4 compounds have potent anti-inflammatory and immunosuppressive properties, and thus are effective to treat a wide range of diseases with these etiologies.
  • Conditions for which truncated oxidized thymosin ⁇ 4 is an effective treatment include inflammatory arthropathies such as rheumatoid arthritis, psoriatic arthritis, crystal arthritis, reactive arthritis, ankylosing spondylitis, infectious arthritis, juvenile chronic arthritis; connective tissue diseases, such as systemic lupus erythematosis, Sjogren's syndrome, polymyalgia rheumatica, cranial arteritis; vasculitic syndromes, such as Wegener's granulomatosis, polyarteritis nodosa, Churg-Strauss syndrome; respiratory diseases, such as asthma, chronic obstructive pulmonary disease (COPD) 5 fibrosing alveolitis, hypersensitivity pneumonitis, sarcoidosis, allergic aspergillosis, cryptogenic pulmonary eosinophilia, bronchiolitis obliterans organising pneumonia; dermatological diseases, such as inflammatory arthropathie
  • inventive compounds are also useful in treating other diseases and conditions such as neutrophil related diseases such as cystic fibrosis, restenosis, ischemia or stroke, skin degeneration and/or blisters or sores such as those associated with skin aging and/or Epidermolysis Bullosa, eye degeneration or coronary damage.
  • neutrophil related diseases such as cystic fibrosis, restenosis, ischemia or stroke
  • skin degeneration and/or blisters or sores such as those associated with skin aging and/or Epidermolysis Bullosa, eye degeneration or coronary damage.
  • the ability of a pharmacologic agent to inhibit neutrophil function and/or inhibit TNF ⁇ production is relevant to the medical treatment of a wide number of autoimmune and inflammatory processes involving the skin, joints, vasculature, smooth muscle, bone, respiratory tract and muscle for which these functions play a central role in the etiology of the disease.
  • the truncated oxidized thymosin ⁇ 4 may prevent or modulate active polymerization and influences the production of laminin-5.
  • the compounds are also useful for treating venous stasis ulcers, septic shock, and alopecia.
  • compounds of the invention may be used for wound healing, such as dermal and ocular wound healing and treating diseases involving tissue and organ repair, such as cardiac-related repair.
  • wound healing such as dermal and ocular wound healing and treating diseases involving tissue and organ repair, such as cardiac-related repair.
  • truncated oxidized thymosin ⁇ 4 reduces tissue damage, ventricular scarring and dilation and improves cardiac function after a myocardial infarction.
  • Truncated oxidized thymosin ⁇ 4 prevents or reduces damage to the heart, although not bound by theory, by preventing apoptosis resulting from a cardiac infarction and may protect cardiac cells from dying by causing them to withstand hypoxia caused by a heart attack.
  • the treatment may include repair and/or regeneration of tissue.
  • Figures 1-4 show full-length oxidized thymosin ⁇ 4 and various truncated peptides are positive in the wound scratch assay, thus suggesting that these compounds are useful for wound healing.
  • the peptides disclosed produce a positive result in a neutrophil burst assay, which will serve as an indication that the truncated oxidized thymosin ⁇ 4 is helpful in managing chronic inflammation and associated tissue damage caused by neutrophils, for example, in cystic fibrosis, or COPD patients.
  • truncated TB4So peptide GBLlOl 14mer induces apoptosis in human peripheral blood neutrophils thus negatively impacting their function and role in potential inflammatory responses.
  • peptide GBLlOl was effective to inhibit f-MLP-induced respiratory burst from neutrophils.
  • Treating" a subject with truncated oxidized thymosin ⁇ 4 for conditions described above includes promoting the healing and/or preventing persistence of the condition or disease.
  • truncated oxidized thymosin ⁇ 4 compounds may be administered in conjunction with other drugs, e.g., cytokines such as interferon which may induce an inflammatory response as a side effect.
  • cytokines such as interferon which may induce an inflammatory response as a side effect.
  • oxidized thymosin ⁇ 4 may serve to minimize or reduce physiological or disease states which are characterized in part by inappropriate inflammation.
  • truncated oxidized thymosin ⁇ 4 compounds mentioned above do not only extend to human conditions.
  • truncated oxidized thymosin ⁇ 4 may be used in the treatment of animals such as cats, dogs, horses, cows, sheep, pigs and goats with similar conditions to those mentioned above.
  • the truncated oxidized thymosin ⁇ 4 compounds are in a purified form as described above.
  • the invention further provides a pharmaceutical composition comprising oxidized thymosin ⁇ 4 as described herein.
  • the invention further provides use of a nucleotide molecule having a sequence capable of encoding thymosin ⁇ 4 as described herein for subsequently preparing truncated oxidized thymosin ⁇ 4.
  • the invention provides the use of a vector or vectors comprising the nucleotide molecule in the preparation of truncated oxidized thymosin ⁇ 4 and deleted and mutated forms thereof as described herein.
  • the present invention provides the use of a vector or vectors comprising the nucleotide molecule in the preparation of a medicament comprising truncated oxidized thymosin ⁇ 4 and deleted and mutated forms thereof for the treatment of inflammation such as that which accompanies an inflammatory condition or the other diseases or conditions described herein.
  • the invention provides spliced variants.
  • Such splice variants may provide peptides of varying lengths, such as oxidized forms of those described with respect to thymosin ⁇ 4 in Girardi, et al, Immunology 109, 1-7 (2002), which is incorporated herein by reference.
  • the truncated oxidized thymosin ⁇ 4 can be used for treatment of patients where non steroidal anti-inflammatory drugs are currently used as an alternative to steroids because of the risks of side-effects.
  • truncated oxidized thymosin ⁇ 4 or of synthetic or expressed (e.g., recombinantly) thymosin ⁇ 4 which is subsequently oxidized will be safe and reliable, since it will generally not be foreign to the body to which it is being administered.
  • the truncated oxidized thymosin ⁇ 4 may also be made synthetically or may be made recombinantly. Synthetic methods for preparing peptides are known in the art and include building a peptide step- wise in a column, e.g., a solid resin support to build a full-length peptide, such as solid phase peptide synthesis (SPPS).
  • SPPS solid phase peptide synthesis
  • the amount of oxidized thymosin ⁇ 4 required to be effective in a treatment will, of course, vary and is ultimately at the discretion of the medical or veterinary practitioner.
  • the factors to be considered include the condition being treated, the route of administration, and nature of the formulation, the recipients body weight, surface area, age and general condition, and the particular compound to be administered.
  • a suitable effective dose may lie in the range of about 0.001 to about 120 mg/kg bodyweight, e.g., 0.01 to about 120 mg/kg body weight, preferably in the range of about 0.01 to 50 mg/kg, for example 0.05 to 20 mg/lcg.
  • the total daily dose may be given as a single dose, multiple doses, e.g., two to six times per day or by intravenous infusion for selected duration.
  • multiple doses e.g., two to six times per day or by intravenous infusion for selected duration.
  • the dose range may be about 8 to 9000 mg per day, and a typical dose could be about 50 mg per day. If discrete multiple doses are indicated treatment might typically be 15 mg of truncated oxidized thymosin ⁇ 4 given up to 4 times per day.
  • Formulations of the present invention for medical use, comprise truncated oxidized thymosin ⁇ 4, or a salt thereof together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients.
  • the carrier(s) should be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.
  • the present invention therefore, further provides a pharmaceutical formulation comprising truncated oxidized thymosin ⁇ 4 or a pharmaceutically acceptable salt or physiologically functional derivative thereof together with a pharmaceutically acceptable carrier therefor.
  • Formulations according to the present invention include those suitable for oral, nasal, topical, vaginal, rectal or parenteral (including subcutaneous, intraarthrodial (i.e., within joints) intramuscular and intravenous) administration including biolistic e.g., Powderject R administration.
  • Preferred formulations are those suitable for oral, topical or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active compound into association with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier or a finely divided solid carrier or both and then, if necessary, shaping the product into desired formulations.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units as capsules, cachets, tablets, lozenges, comprising the active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • Each formulation generally contains a predetermined amount of the active compound; as a powder or granules; or a solution or suspension in an aqueous or non-aqueous liquid such as a syrup, an elixir, an emulsion or draught and the like.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed, tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form such as a powder or granules, optionally mixed with a binder, (e.g., povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycollate, cross-linked povidone, cross- linked sodium carboxymethyl cellulose), surface active or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile.
  • a syrup may be made adding the active compound to a concentrated, aqueous solution of a sugar, for example sucrose, to which may also be added any accessory ingredients.
  • a sugar for example sucrose
  • Such accessory ingredient(s) may include flavorings, an agent to retard crystallization of the sugar or an agent to increase the solubility of any other ingredients, such as a polyhydric alcohol for example glycerol or sorbitol.
  • Formulations for rectal administration may be presented as a suppository with a conventional carrier such as cocoa butter.
  • Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active compound which is preferably isotonic with the blood of the recipient.
  • Such formulations suitably comprise a solution of a pharmaceutically and pharmacologically acceptable salt of truncated oxidized thymosin ⁇ 4, that is isotonic with the blood of the recipient.
  • Useful formulations also comprise concentrated solutions or solids containing truncated oxidized thymosin ⁇ 4, which upon dilution with an appropriate solvent give a solution for parental administration as above.
  • the truncated oxidized thymosin ⁇ 4 or physiologically active variant thereof disclosed herein may be administered to the lungs of a subject by any suitable means, but are preferably administered by generating an aerosol comprised of respirable particles, the respirable particles comprised of the active compound, which particles the subject inhales (i.e., by inhalation administration).
  • the respirable particles may be liquid or solid.
  • An example of respirable particles is found, e.g., in U.S. Patent Nos. 5,740,794, and 6,051,256.
  • Particles comprised of truncated oxidized thymosin ⁇ 4 for practising the present invention should include particles of respirable size: that is, particles of a size sufficiently small to pass through the mouth and larynx upon inhalation and into the bronchi and alveoli of the lungs. In general, particles ranging from about 0.5 to 10 microns in size (more particularly, less than about 5 microns in size) are respirable. Particles of non-respirable size which are included in the aerosol tend to deposit in the throat and be swallowed, and the quantity of non-respirable particles in the aerosol is preferably minimized. For nasal administration, a particle size in the range of 10-500 ⁇ m is preferred to ensure retention in the nasal cavity.
  • Liquid pharmaceutical compositions or truncated oxidized thymosin ⁇ 4 for producing an aerosol can be prepared by combining the truncated oxidized thymosin ⁇ 4 with a suitable vehicle, such as sterile pyrogen free water.
  • Solid particulate compositions containing respirable dry particles of micronized truncated oxidized thymosin ⁇ 4 may be prepared by grinding dry truncated oxidized thymosin ⁇ 4 with a mortar and pestle, and then passing the micronized composition through a 400 mesh screen to break up or separate out large agglomerates.
  • a solid particulate composition comprised of the truncated oxidized thymosin ⁇ 4 may optionally contain a dispersant which serves to facilitate the formation of an aerosol.
  • a suitable dispersant is lactose, which may be blended with the oxidized thymosin ⁇ 4 in any suitable ratio (e.g., a 1 to 1 ratio by weight).
  • Aerosols of liquid particles comprising the truncated oxidized thymosin ⁇ 4 may be produced by any suitable means, such as with a nebulizer. See, e.g., U.S. Patent No. 4,501,729.
  • Nebulizers are commercially available devices which transform solutions or suspensions of the oxidized thymosin ⁇ 4 into a therapeutic aerosol mist either by means of acceleration of a compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation.
  • compositions for use in nebulizers consist of the truncated oxidized thymosin ⁇ 4 in a liquid carrier, the truncated oxidized thymosin ⁇ 4 comprising up to 40% w/w of the compositions, but preferably less than 20% w/w.
  • the carrier is typically water or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride.
  • Optional additives include preservatives if the composition is not prepared sterile, for example, methyl hydroxybenzoate, antioxidants, flavouring agents, volatile oils, buffering agents and surfactants.
  • Aerosols of solid particles comprising the truncated oxidized thymosin ⁇ 4 may likewise be produced with a solid particulate medicament aerosol generator.
  • Aerosol generators for administering solid particulate medicaments to a subject produce particles which are respirable, as explained above, and generate a volume of aerosol containing a predetermined metered dose of a medicament at a rate suitable for human administration. Examples of such aerosol generators include metered dose inhalers and insufflators.
  • the formulations are preferably applied as a topical ointment, gel, cream, foam or time-release suspension containing the active ingredient in an amount of, for example, 0.075 to 20% w/w, preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w.
  • the active ingredients may be employed with either a paraffinic or a water-miscible ointment base.
  • the active ingredients may be formulated in a cream with an oil-in-water cream base.
  • the aqueous phase of the cream may include, for example, at least 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane- 1,3-diol, mannitol, sorbitol, glyercol and polyethylene glycol and mixtures thereof.
  • the topical formulations may desirably include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulphoxide and related analogues.
  • the oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While the phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat.
  • the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax
  • the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
  • Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyle alcohol, glycerol mono- stearate and sodium lauryl sulphate.
  • suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is very low.
  • the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers.
  • Straight or branched chain, mono-or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
  • the formulations of this invention may further include one or more accessory ingredient(s) selected from diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including antioxidants) and the like.
  • truncated oxidized thymosin ⁇ 4 or variants thereof may be used as a coating or may be impregnated in prosthetic devices such as stents.
  • a cell monolayer (HUVEC, HeLa) was grown.
  • the cell monolayer was scratched with a sterile pipette tip and cell debris was washed away. Thereafter, the scratched cell monolayer was treated ⁇ vith full-length oxidized thymosin ⁇ 4 or one of the peptides listed in Figures 1 or 2, a control, or was left untreated.
  • the gap closure of the scratched epithelium was measured over time.
  • Cell migration was measured in Figure 1 , which shows the oxidized thymosin ⁇ 4 or the truncated peptides at a 2 nm level, and Figure 2, which shows treatment at a 20 nm level.
  • Figures 3 and 4 measure percent wound closure instead of cell migration as in Figures 1 and 2, using the same peptides.
  • Figures 1-4 Other truncated oxidized thymosin ⁇ 4 peptides are expected to also have such effect.
  • Figures 1 and 3 show the effect of the various peptides in comparison to thymosin ⁇ 4 and an FGF control, as well as a negative control, as a 2 nm level and Figures 2 and 4 shows roughly the same effect for oxidized thymosin ⁇ 4 and four of the five truncated oxidized thymosin ⁇ 4 peptides.
  • Figure 1 and 3 show that a greater effect may be achieved on a per molar basis at lower dosages for four of the five truncated peptides tested in comparison to oxidized thymosin ⁇ 4.
  • Oxidized thymosin ⁇ 4 and the 14-mer GBLOl were tested in a carrageenan assay.
  • Carrageenan induces an inflammatory response and tests for mouse paw edema.
  • Groups of mice were injected subcutaneously in one hind paw with 300 ⁇ g carrageenan mixed with PBS, DEX, and oxidized thymosin ⁇ 4 or the 14-mer GBLOl .
  • Foot pad swelling was measured using a spring-dial caliper and expressed as the difference in swelling between the carrageenan injected paw and the uninjected contralateral paw.
  • the animals were injected intraperitoneally with the same dose of test material and measurements were made at 6, 12, and 24 hours. The results are shown in Figure 5. The results show that the oxidized thymosin ⁇ 4 and the truncated 14-mer have approximately the same effect.
  • Dihydrorhodamine 123 is a dye that oxidizes to form the brightly fluorescent compound rhodamine 123.
  • Neutrophils can oxidize DHR following stimulation by irritants or oxidative stress, chemoattractants, etc., subsequent to the metabolic burst generated through the hexose monophosphate (HMP) shunt.
  • HMP hexose monophosphate
  • a series of metabolic events are triggered in the cell, including increased oxygen consumption and increased superoxide, hydroxyl radical, and hydrogen peroxide formation.
  • DHR 123 is oxidized to R 123, which is measured on the FACS.
  • the results with respect to oxidized thymosin ⁇ 4 are shown in Figure 6.
  • Example 7 A similar burst assays is discussed in Example 7. Data from this additional example further supports the position that the peptides of the disclosed invention are effective to inhibit respiratory burst.
  • Example 4 TNF- ⁇ Production in PBMCs Inhibited bv GBLlOl
  • PBMCs peripheral blood mononuclear cells
  • LPS lipopolysaccharide
  • IL-12 IL-12
  • IL- 18 IL-12
  • PBMCs were cultured overnight in ImI culture (24- well tissue culture plate) at 2 x 10 6 cells/ml in RPMI 10%FCS, and treated with various agonists (i.e., GBLlOl, dexamethasone or TNF- ⁇ ).
  • FIG. 7A shows the amounts of TNF- ⁇ produced in the presence of lOOul of IL- 12/18 (3ng/ml and lOOng/ml final added, respectively).
  • Figure 7B shows the amount of TNF- ⁇ produced in the presence of lOOul LPS (250ng/ml final concentration) was added.
  • Cells were cultured for 24 hours at 37 0 C 5%CO 2 in humidified atmosphere. 150ul of supernatant was removed at 24 hours. TNF- ⁇ concentration determined by ELISA (Biosource).
  • neutrophils were prepared fresh from peripheral blood. Cells were washed, counted and adjusted to 1 million cells/ml.
  • Figure 8 shows that increasing concentrations, the truncated TB4So peptide GBLlOl 5 is shown to induce programmed cell death (apoptosis) in human peripheral blood neutrophils. This is an important anti-inflammatory response that modulates the activity and function of a key inflammatory cell type.
  • HUVEC cells cultured in 24-well tissue culture plate A scratch wound was made at time 0 with yellow Gilson tip. Dislodged cells were removed by washing the plates twice. Measurements of cell migration were made at 1Ox magnification, using micrometer. Migration values shown in Figure 9 are the average migration distance into the scratch wound (Time 0 - time 2.5hr/6hr), measured in 5 marked locations along the scratch, in triplicate wells. Conclusions: TB4so and GBLlOl have induced HUVEC cell migration to a similar level as FGF (positive control). This effect is statistically significant versus negative control, at all concentrations tested. GBLl 06 has not produced a statistically significant effect over negative control, at any concentration tested.
  • Example 7 GBLlOl Inhibits fMLP-Induced Respiratory Burst from Isolated Human Neutrophils
  • Neutrophils were prepared fresh from peripheral blood. Cells were washed, counted and brought to a concentration of 1 million cells/ml. One milliliter of these cells were incubated with various concentrations of GBLOl for 3 hours at 37 0 C. Cells were then incubated in the presence of DHR (30 ⁇ M final) for 10 minutes at 37 0 C. The cells were then incubated in the presence of 10 '6 M fMLP to induce a respiratory burst, for 10 minutes at 37 0 C. The reaction was stopped by washing cells in ice cold PBS with 0.02% EDTA. The cells were isolated and resuspended in 300 ⁇ l of PBS/EDTA and analyzed by FACS.
  • MFI Median Fluorecence Intensity

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Abstract

The present invention relates to the use of truncated oxidized thymosin β4 in therapy, such as in the treatment of diseases or conditions associated with inflammation.

Description

TRUNCATED OXIDIZED THYMOSIN β4 AND DERIVATIVES THEREOF
Field of the Invention
[0001] The present invention relates to a synthetic peptide portion of oxidized thymosin β4 or variants thereof. More particularly, the invention relates to a peptide containing 14 N-terminal amino acids of oxidized thymosin β4 or variants thereof. Such peptides have oxidized thymosin β4 activity which are useful for treating various diseases and conditions, for example, diseases and conditions associated with inflammation and those for which steroid therapy is useful.
Background of the Invention
[0002] Steroids are effectively used in the treatment of inflammatory diseases, such as asthma, eczema, dermatitis, psoriasis, allergic reactions, and rheumatic diseases such as rheumatoid arthritis. Steroids, both inhaled and oral forms, are also used for the clinical management of cystic fibrosis. However, steroids have serious side effects and are therefore only used intermittently or in cases where non-steroidal anti-inflammatory drugs are not effective.
[0003] Peripheral blood mononuclear cells, such as monocytes and lymphocytes are important immune effector cells that play a fundamental role in cellular immunity. In addition to their antigen-presenting and phagocytic activities at the sites of inflammation, monocytes are also involved in the synthesis and release of a variety of pro-inflammatory enzymes and polypeptide cytokines which modulate neutrophil responses. The production of these proinflammatory components can be suppressed by glucocorticoids and this has been suggested as the basis for their anti-inflammatory action.
[0004] Cytokines, cheniokines and mitogens are a diverse group of soluble proteins and peptides with immunomodulatory properties. These agents regulate the activities of individual cells and tissues either under normal or pathological conditions. These immunomodulatory agents are produced by multiple immune cell types and have pleiotropic effects on multiple target cells thus resulting in multiple biological actions and outcomes. Constitutive cytokine and chemokine production is usually low or absent and its production is promoted by certain biochemical stimuli. Given that the functions of these humoral immunomodulators are pleiotropic they also tend to be redundant. [0005] In host defense responses to infection, inflammation, autoimmune reactions, wounds, trauma and metabolic injury, these immunomodulatory agents not only stimulate immune cells to proliferate and differentiate but also regulate the progression and termination of immunological reactions. Pro-inflammatory agents cause inflammation to progress, notable among these are TNF-α and IL- 12 and IL-18 the later of which often act in concert. When these early and mid acting agents are produced they act to destroy normal and diseased tissues. They also enhance the synthesis of other pro-inflammatory cytokines by various immunological cells, and can induce systemic inflammatory reactions such as fever and shock. Another class of immunoregulatory agents serves to counteract inflammation by inhibiting the production of proinflammatory agents. These mediators act mainly by inhibiting the production of proinflammatory agents or by neutralizing the biological effects of pro-inflammatory mediators. Homeostatic mechanisms that balance the effects of pro and anti-inflammatory agents are thought to be responsible for the resolution of inflammation (Ref Hanada, T. et al. Cytokine Growth Factor Rev. 2002 Aug-Oct;13(4-5):413-21).
[0006] The effect of steroid-induced factors on neutrophil migration is primarily of interest in elucidating anti-inflammatory mechanisms as λvell as the development of therapeutic treatments for immune mediated disease and conditions. Corticosteroids can function by down regulating the synthesis of many pro-inflammatory mediators (Lew et al 1988; Almawi et al 1991; Standford et al. 1992) as well as up-regulation of anti-inflammatory mediators.
[0007] The neutrophil migration stimulating activity of steroid induced factors suggests that dispersive locomotion tends to prevent cells collecting at a focus and this may be important in terminating inflammatory responses.
[0008] Stevenson (1973, 1974, 1978) demonstrated that human monocytes when incubated in the presence of anti-inflammatory corticosteroids released a protease sensitive factor that enhanced the migration of neutrophils from a cell pellet contained in a short capillary tube.
[0009] Later studies demonstrated that the phenomenon of stimulated neutrophil migration was also observed with leucocytes from patients receiving steroid therapy.
[0010] Recently, Chettibi et al. (1993, 1994) have investigated the steroid induced stimulatory effect on neutrophil migration using an automated cell tracking assay enabling study of the behavior of cells migrating on protein-coated glass coverslip.
[0011] These studies determined:
1. Steroid-treated monocyte supernatant (STMS) causes a dramatic increase in the speed of locomotion of human neutrophils and a significant decrease in their adhesion to protein-coated glass. In contrast, control monocyte supernatants have a smaller effect on the speed of locomotion, but cause a large increase in adhesiveness.
2. The supernatant activity was produced equally well in the presence or absence of serum after 24 h culture at 370C with 10"6 M dexamethasone.
3. The effect of the steroid-treated monocyte supernatant on the speed of locomotion of human peripheral blood neutrophils was not altered by rabbit polyclonal antisera against lipocortins 1-6.
4. Rabbit anti-inter leukin- 8 antibody which blocked the effect of IL-8 on the speed of locomotion of neutrophils did not antagonize the locomotion stimulating action of steroid- treated monocyte supernatant.
5. The extracellular release of this factor(s) by human mononuclear leucocytes suggests that it may be an in vivo mediator of the anti-inflammatory effect of glucocorticoids.
[0012] However, there is no disclosure of what the active agent(s) in STMS might be.
[0013] According to Huff T. et al. (Eur J Biochem. (1995) 230(2):650-7), it is well documented that the β thymosins play an important role in organization of the cytoskeleton sequestering G-actin. Very little is known about the possible interactions of thymosin β 4 with proteins other than the actins. The numerous reports that thymosin β 4 may be involved in other physiological events such as angiogenesis, wound healing, inflammation, apoptosis and carcinogenesis raises questions about whether this is due to actin sequestration or other unproven cytokine or other activities of thymosin β 4. Therefore, proof that removal of the actin sequestration ability of the molecule through truncation leaves other biological functions intact, suggests that the molecule may have other functions.
[0014] Huff T. et al (Eur J Biochem. (1995) 230(2):650-7) and Heintz D. et al. (Eur J Biochem. (1994) 223(2):345-50) describe studies involving beta-thymosins and how they interact with G-actin in a biomolecular complex and inhibit the polymerization to F-actin under high salt conditions. The oxidized form of thymosin β4 is disclosed as inhibiting actin polymerization, however, only at a 20-fold higher concentration than thymosin β4. Neither document however implicates any medical role for oxidized thymosin β4. In fact the papers appear to teach away from a positive role for oxidized thymosin β4.
[0015] Huff et al. (Intl J. of Biochem & Cell Biol. (2001) 33:205-220) reported that oxidation of thymosin β 4 increases the dissociation constant of the actin thymosin β 4 complex 20 fold and suggested that the oxidation of thymosin β 4 to its sulfoxide could regulate the binding to G and F-actin in vivo. This teaches that oxidation of the methionine at residue six decreases the actin binding activity of thymosin β 4. Moreover, Huff et al. and others report that truncation of the first six to 12 amino acid residues of thymosin β 4 increase the dissociation constant to a similar extent as the oxidation of thymosin β 4. Truncation of the first 23 or last 26 amino acids of the molecule completely abolishes the interaction with G actin. Huff concluded that: "amino acid residues and possibly the secondary structure of the polypeptide thymosin β 4 chain ahead of position 7 are indispensable for inhibition of actin polymerization while amino acid residues between position 13 and 23 are necessary of binding to actin." Certainly, the X- ray analysis suggests that 17LKKTETQEK23 is involved in actin binding and claimed for antiinflammatory and wound healing effects by Goldstein et al.
[0016] These references suggest that an N terminal 14 mer with alpha helical properties ascribed to this portion of the molecule would have no ability to sequester actin and as a consequence could not play a role in the physiological functions requiring actin sequestration. As such, the immunologic activity of an N terminal 14 mer therefore would consequently have to be related to some other mechanism of action for immunological activity.
[0017] U.S. Patent No. 5,578,570 (Goldstein et al.) discloses a method of treating septic shock by administering thymosin β4. There is no disclosure however of oxidized thymosin β4 or suggestion that this may have a role in treating septic shock.
[0018] WO 03/0060405, WO 02/36143, WO 02/091969, WO 03/020215 identify thymosin β4 peptides useful for various diseases or conditions that contain an amino acid sequence LKKTET, which is not a N-terminal amino acid sequence.
[0019] US Pat. No. 6,602,519 discusses the physiological activity of oxidized thymosin β4 in comparison to thymosin β4. It would be advantageous to the art to provide a molecule that can achieve better results than oxidized thymosin β4 for various indications such as those requiring small molecular weight compounds suitable for inhalation, parenteral or injection therapies.
Summary of the Invention
[0020] Thus, the invention provides an isolated synthetic peptide where the peptide comprises at least seven consecutive amino acids that correspond to the 14 N-terminal amino acids of oxidized thymosin β4 or variants thereof, wherein at least one amino acid of the peptide is an oxidized methionine and wherein the peptide has oxidized thymosin β4 activity. The invention also includes compositions comprising the peptide in combination with a pharmaceutically-acceptable carrier, as well as methods of treating a condition or disease using the truncated peptide.
Brief Description of the Figures
[0021] Figure 1 illustrates human umbilical vein endothelial cells (HUVEC) cell migration results for oxidized thymosin β4 and various truncated peptides GBLOl -GBL05 at a 2 nm level. GBLOl and GBL02 are ac-SDKPDMAEIEKFDK (SEQ ID NO:1). GBL 04 and GBL 05 are described below and GBL 05 is MLLPATMSDKPDMAEIEKFDK (SEQ ID NO:2), where M is an oxidized sulfur in the methionine. Fibroblast growth factor (FGF) is the control.
[0022] Figure 2 illustrates HUVEC cell migration results for oxidized thymosin β4 and various truncated peptides at a 20 nm level.
[0023] Figure 3 illustrates HUVEC cell migration results based on percent wound closure for oxidized thymosin illustrates HUVEC cell migration results for oxidized thymosin β4 and various truncated peptides at a 2 nm level.
[0024] Figure 4 illustrates HUVEC cell migration results based on percent wound closure for oxidized thymosin illustrates HUVEC cell migration results for oxidized thymosin |34 and various truncated peptides at a 20 nm level.
[0025] Figure 5 shows the results of treatment of oxidized thymosin β4 and a truncated peptide in a carrageenan-induced mouse paw edema test. * p<0.05, n=7/group.
[0026] Figure 6 shows oxidized thymosin β4 inhibits fMLP-induced respiratory burst in a dose-dependent response. N=2; * p<0.05: 50% inhibition observed at 0.02μM exposure.
[0027] Figure 7 shows two bar graphs illustrating the ability of oxidized thymosin β4 to inhibit the ability of pro-inflammatory cytokines and mitogens to induce TNF-α in peripheral blood mononuclear cells (PBMCs) at levels equivalent to the corticosteroid dexamethasone. Figure 7A shows GBLlOl inhibition on TNF-α production by PBMCs in the presence of IL- 12/18. Figure 7B shows GBLlOl inhibition on TNF-α production by PBMCs in the presence of LPS.
[0028] Figure 8 is a bar graph illustrating that GBLlOl induces apoptosis in human peripheral blood neutrophils in a dose dependant fashion.
[0029] Figure 9 shows a bar graph illustrating that the presence of an oxidized methionine residue in the truncated oxidized thymosin β4 (Tβ4so) peptide GBLl 06 is required for biological activity in the wound scratch assay. [0030] Figure 10 shows a bar graph illustrating that the truncated oxidized thymosin β4 peptide GBLlOl is effective to block fMLP-induce respiratory burst in isolated neutrophils. N=2, p<0.05.
Detailed Description of the Invention
[0031] As used herein "truncated oxidized thymosin β4" compounds or variants thereof include compounds that are less than the full length of oxidized thymosin β4, or variants thereof, but still retain the activity of oxidized thymosin β4 Although not bound by theory, it is believed that the oxidized methionine in the N-terminal portion of the protein is responsible for at least a part of the overall activity of oxidized thymosin β4. As discussed here, substitution of methionine with nor-leucine (which contains a methyl group in place of the sulfur group of Met), destroys the anti- inflammatory and wound healing properties of the truncated 14 mer. However, analogs having other oxidized metals in place of the sulfur in methionine are also contemplated and have requisite oxidized thymosin β4 activity. In a preferred embodiment, the truncated oxidized thymosin β4 of the invention is a peptide which is 14 amino acids of the N- terminal portion of oxidized thymosin β4 or variants thereof that retain oxidized thymosin β4 activity. Preferably, peptides are of sufficient length, for example, seven amino acids in length, and have secondary structure in solution such as alpha-helical configurations. Examples of peptides of such sufficient length include acetylated or unacetylated peptides such as SDKPDMA (SEQ ID NO:3), DKPDMAE (SEQ ID NO:4), KPDMAEI (SEQ ID NO:5), PDMAEIE (SEQ ID NO:6), DMAEIEK (SEQ ID NO:7), MAEIEKF (SEQ ID NO:8), or variants thereof that retain oxidized thymosin β4 activity. Native thymosin β4 is acetylated and thus truncated oxidized thymosin β4 are preferably acetylated. In addition, the truncated oxidized thymosin β4 peptide may further comprise a carrier, such as a prodrug known to one of skill in the art, or a protein, peptide, or amino acid moiety. In a preferred embodiment, the amino acid or protein moiety that is attached to the truncated oxidized thymosin β4 contains at least one oxidized methionine, and preferably one or two oxidized methionines. Preferred embodiments include ac-MSDKPDMAEIEKFDK (SEQ ID NO:9) (GBL03) or ac-LLP ATMSDKPDMAEIEKFDK (SEQ ID NO: 10) (GBL04), wherein the letter M in the preceding truncated peptides is methionine sulphoxide. In another embodiment, the truncated portion does not contain the amino acid sequence LKKTET (SEQ ID NO:1 1) or a conservative variant thereof. The truncated peptide of the invention preferably does not contain a G-actin binding region and, without being bound by theory, it is believed that this portion does not play as important a role as was thought in thymosin β4 activity, but rather the oxidized methionine is important for such activity. Preferably, variants of oxidized thymosin β4 are conservative amino acid variants. 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.
[0032] In one aspect, the present invention provides a replacement to steroid therapy.
[0033] In another aspect, the present invention provides use of a truncated oxidized thymosin β4, preferably a 14 N-terminal amino acid peptide thereof, or physiologically active variant in therapy.
[0034] Typically oxidized thymosin β4 is a form of thymosin β4 in which a methionine residue, 6 amino acids from the N-terminus, (Met6), is oxidized such that the residue is converted to methionine sulphoxide. Moreover, the methionine residue (Met6) may be further oxidized to the methionine sulphone and this as such is also encompassed by the present invention. Other modifications of the methionine residue may also be envisaged, such as complexing the sulfur with metals, which may result in an active form of thymosin β4 similar to the oxidized form described herein.
[0035] It is understood that the truncated oxidized thymosin β4 may be obtained for example by reacting a native truncated thymosin β4 under oxidizing conditions, for example by treating with hydrogen peroxide, to form oxidized thymosin β4, which is then truncated. Thus, native thymosin β4 may first be obtained and thereafter oxidized to the oxidized form. In a more preferred embodiment, however, truncated oxidized thymosin β4 is made synthetically by procedures known in the art.
[0036] The present invention provides the use of purified truncated oxidized thymosin β4 variants and compositions thereof. Preferred compositions contain truncated oxidized thymosin β4 which comprise at least 30%, 60%, or 80%, and more preferably 90%, 95%, 98%, or 99% of truncated oxidized thymosin β4 or variants thereof. In another embodiment isolated and purified truncated, oxidized thymosin β4 is preferred.
[0037] Thymosin β4 in an oxidized or non-oxidized form may be obtained from any suitable source, for example from steroid treated monocytes. Moreover, the thymosin β4 may be derived from any suitable species, but is typically of mammalian origin, such as bovine, equine, murine or human origin. It is to be noted that bovine, equine, murine, rat and human thymosin β4 are all identical in sequence. Thus, for example, bovine thymosin β4 may provide a suitable source of thymosin β4 for subsequent oxidation and administration to other species, such as humans.
[0038] Physiologically active truncated oxidized thymosin β4 peptides or variants thereof display similar physiological properties as displayed by oxidized thymosin β4. It is preferred that such variants would include the oxidized methionine, but such variants may be truncated, deleted or mutated forms thereof.
[0039] It will be understood that for the particular truncated oxidized thymosin β4 embraced herein, variations (natural or otherwise) can exist. These variations may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. All such derivatives are included within the scope of this invention provided that the derivatives are physiologically active (i.e., display oxidized thymosin β4 activity as defined herein). For example, for the purpose of the present invention conservative replacements may be made between amino acids, within the following groups:
(I) alanine, serine and threonine; (II) glutamic acid and aspartic acid;
(III) arginine and lysine;
(IV) asparagine and glutamine; (V) isoleucine, leucine and valine;
(VI) phenylalanine, tyrosine and tryptophan. (VII) methionine and other methionine analogues (VIII) methionine and other methionine analogues where the sulfur is replaced by
Group VIB elements (e.g., Selenium, Tellurium, Polonium). (IX) oxidized methionine and other oxidized methionine analogues (e.g., Group VIB analogues, methionine sulfoximine). (X) methionine and other sulfur-containing amino acids (e.g., et al S and R cysteine) including their oxidized analogues.
[0040] Early molecular modeling studies suggest that the methionine residue (met-6) is at the top of one of three helices in the peptide.
[0041] Not all peptides that contain less than the full length amino acid sequence of oxidized thymosin β4 retains the activity thereof. Indeed this may assist in the development of peptide mimetics which display the same physiological function as the oxidized thymosin β4. [0042] Moreover, it may be possible to increase the half life of truncated oxidized thymosin β4 or physiologically active variants thereof by use of appropriate chemical modification (e.g., acetylation) or use of D amino acids.
[0043] The truncated oxidized thymosin β4 may have a blocked N-terminal.
[0044] According to the present invention there is also provided a synthetic truncated oxidized thymosin β4 comprising the peptide sequence of thymosin β4 in oxidized form or physiologically active variant thereof.
[0045] The synthetic oxidized thymosin β4 may be modified and/or amino acid substituted as described above, as long as the physiological activity remains. For example selenomethionine could be introduced in place of methionine and oxidized in the same manner.
[0046] The invention further provides the use of a truncated oxidized thymosin β4 compounds as described herein in the preparation of a medicament for the treatment of a chronic or acute inflammatory condition or to treat inflammation. The disclosed truncated oxidized thymosin β4 compounds have potent anti-inflammatory and immunosuppressive properties, and thus are effective to treat a wide range of diseases with these etiologies.
[0047] Conditions for which truncated oxidized thymosin β4 is an effective treatment include inflammatory arthropathies such as rheumatoid arthritis, psoriatic arthritis, crystal arthritis, reactive arthritis, ankylosing spondylitis, infectious arthritis, juvenile chronic arthritis; connective tissue diseases, such as systemic lupus erythematosis, Sjogren's syndrome, polymyalgia rheumatica, cranial arteritis; vasculitic syndromes, such as Wegener's granulomatosis, polyarteritis nodosa, Churg-Strauss syndrome; respiratory diseases, such as asthma, chronic obstructive pulmonary disease (COPD)5 fibrosing alveolitis, hypersensitivity pneumonitis, sarcoidosis, allergic aspergillosis, cryptogenic pulmonary eosinophilia, bronchiolitis obliterans organising pneumonia; dermatological diseases, such as inflammatory dermatosis including psoriasis, eczema, dermatitis, urticaria; gastro-intestinal diseases, such as ulcerative colitis, Crohn's disease, lupoid hepatitis; haematological disease, such as haemolytic anaemia, idiopathic thrombocytopenic purpura, multiple myeloma, lymphoma/leukaemia; transplantation/prosthetics, such as graft rejection, graft versus host disease, tissue reaction to implanted prostheses; infections, such as tuberculosis, malaria Pneumocystis carinii pneumonia, leprosy; chronic dermal wounds, such as decubitous ulcers, diabetic ulcers, and pressure sores; skin disorders, such as epidermolysis bullosa (EB); ophthalmic diseases and wounds; hair loss; stent surgery; organ/tissue regeneration; adult respiratory distress syndrome, allergic contact dermatitis, allergic eosophagitis, ANCA vasculitis, arthritis, atopic dermatitis, atopic keratoconjunctivitis, balanitis (plasma cell/xerotica obliterans), Bechet's disease, bulanoposthitis, bullous disorders, burns, cellulites, cholestytis, chondrioretinitis, chondrodermatitis nodularis helices, chronic pelvic pain syndrome, cutis laxa, bioterrorism agent dermatoses, foliculitis, gout, granuloma annulare, granuloma faciale, halogenoderma, Hersoch- Schonlein disease, idiopathic pulmonary fibrosis (alveolitis), immune complex disease, inflammatory bowel disease, irritant contact dermatitis, ischemic reperfusion injury, Kawasaki disease, keloids, keratosis follicularis (Darier- White syndrome), lichen planus, multiple sclerosis, myocardial infarction, pemphagus, photoallergenic dermatitis, phototoxic dermatitis, phototraunia, polyglandular autoimmune syndrome, radiation dermatitis, radiodermatitis (acute and chronic), relapsing polychondritis, scleritis, seborrheic dermatitis, spondyloarthropathy, Sweet's disease, and vasculitis.
[0048] The inventive compounds are also useful in treating other diseases and conditions such as neutrophil related diseases such as cystic fibrosis, restenosis, ischemia or stroke, skin degeneration and/or blisters or sores such as those associated with skin aging and/or Epidermolysis Bullosa, eye degeneration or coronary damage. The ability of a pharmacologic agent to inhibit neutrophil function and/or inhibit TNFα production is relevant to the medical treatment of a wide number of autoimmune and inflammatory processes involving the skin, joints, vasculature, smooth muscle, bone, respiratory tract and muscle for which these functions play a central role in the etiology of the disease. Previously, Young et al. showed that the oxidized form of thymosin β4, inhibited neutrophil function and TNF production to predict its potential use equivalent to that of a steroidal anti-inflammatory agent. Similarly, the finding that the N terminal 14 amino acid sequence of thymosin β 4 sulfoxide- while being novel for its unexplained and documented function based on the documented actin sequestration of the parental molecule- demonstrates several of the key features of the full length thymosin β 4 sulfoxide, also predicts that the 14 mer will possess the anti-inflammatory or wound healing properties relevant to the full length molecule as previously disclosed.
[0049] The truncated oxidized thymosin β4 may prevent or modulate active polymerization and influences the production of laminin-5. The compounds are also useful for treating venous stasis ulcers, septic shock, and alopecia. In addition compounds of the invention may be used for wound healing, such as dermal and ocular wound healing and treating diseases involving tissue and organ repair, such as cardiac-related repair. For example, truncated oxidized thymosin β4 reduces tissue damage, ventricular scarring and dilation and improves cardiac function after a myocardial infarction. Truncated oxidized thymosin β4 prevents or reduces damage to the heart, although not bound by theory, by preventing apoptosis resulting from a cardiac infarction and may protect cardiac cells from dying by causing them to withstand hypoxia caused by a heart attack.
[0050] With respect to wound healing, for example, the treatment may include repair and/or regeneration of tissue. Figures 1-4 show full-length oxidized thymosin β4 and various truncated peptides are positive in the wound scratch assay, thus suggesting that these compounds are useful for wound healing.
[0051] As discussed below, the peptides disclosed produce a positive result in a neutrophil burst assay, which will serve as an indication that the truncated oxidized thymosin β4 is helpful in managing chronic inflammation and associated tissue damage caused by neutrophils, for example, in cystic fibrosis, or COPD patients. As shown in Example 5 and Figure 8, truncated TB4So peptide GBLlOl ( 14mer) induces apoptosis in human peripheral blood neutrophils thus negatively impacting their function and role in potential inflammatory responses. As shown in Example 7, peptide GBLlOl was effective to inhibit f-MLP-induced respiratory burst from neutrophils.
[0052] "Treating" a subject with truncated oxidized thymosin β4 for conditions described above includes promoting the healing and/or preventing persistence of the condition or disease.
[0053] Moreover, truncated oxidized thymosin β4 compounds may be administered in conjunction with other drugs, e.g., cytokines such as interferon which may induce an inflammatory response as a side effect. Thus, in one aspect oxidized thymosin β4 may serve to minimize or reduce physiological or disease states which are characterized in part by inappropriate inflammation.
[0054] Reduced forms of truncated oxidized thymosin β4 as well as full-length oxidized thymosin β4 will also be effective in these diseases, although perhaps not to the same extent as the truncated oxidized thymosin β4 compound.
[0055] Additionally, it should be appreciated that the uses of truncated oxidized thymosin β4 compounds mentioned above do not only extend to human conditions. Thus, truncated oxidized thymosin β4 may be used in the treatment of animals such as cats, dogs, horses, cows, sheep, pigs and goats with similar conditions to those mentioned above.
[0056] Typically the truncated oxidized thymosin β4 compounds are in a purified form as described above.
[0057] The invention further provides a pharmaceutical composition comprising oxidized thymosin β4 as described herein. [0058] The invention further provides use of a nucleotide molecule having a sequence capable of encoding thymosin β4 as described herein for subsequently preparing truncated oxidized thymosin β4.
[0059] In a particular embodiment the invention provides the use of a vector or vectors comprising the nucleotide molecule in the preparation of truncated oxidized thymosin β4 and deleted and mutated forms thereof as described herein.
[0060] Alternatively the present invention provides the use of a vector or vectors comprising the nucleotide molecule in the preparation of a medicament comprising truncated oxidized thymosin β4 and deleted and mutated forms thereof for the treatment of inflammation such as that which accompanies an inflammatory condition or the other diseases or conditions described herein.
[0061] In another aspect, the invention provides spliced variants. Such splice variants may provide peptides of varying lengths, such as oxidized forms of those described with respect to thymosin β4 in Girardi, et al, Immunology 109, 1-7 (2002), which is incorporated herein by reference.
[0062] The use of truncated oxidized thymosin β4 as described herein in place of steroid treatment will alleviate the side effects which are normally associated with the use of steroids.
[0063] The truncated oxidized thymosin β4 can be used for treatment of patients where non steroidal anti-inflammatory drugs are currently used as an alternative to steroids because of the risks of side-effects.
[0064] Use of highly purified truncated oxidized thymosin β4 or of synthetic or expressed (e.g., recombinantly) thymosin β4 which is subsequently oxidized will be safe and reliable, since it will generally not be foreign to the body to which it is being administered. The truncated oxidized thymosin β4 may also be made synthetically or may be made recombinantly. Synthetic methods for preparing peptides are known in the art and include building a peptide step- wise in a column, e.g., a solid resin support to build a full-length peptide, such as solid phase peptide synthesis (SPPS).
[0065] The amount of oxidized thymosin β4 required to be effective in a treatment will, of course, vary and is ultimately at the discretion of the medical or veterinary practitioner. The factors to be considered include the condition being treated, the route of administration, and nature of the formulation, the recipients body weight, surface area, age and general condition, and the particular compound to be administered. A suitable effective dose may lie in the range of about 0.001 to about 120 mg/kg bodyweight, e.g., 0.01 to about 120 mg/kg body weight, preferably in the range of about 0.01 to 50 mg/kg, for example 0.05 to 20 mg/lcg. The total daily dose may be given as a single dose, multiple doses, e.g., two to six times per day or by intravenous infusion for selected duration. For example, for a 75 kg mammal (e.g., a human) the dose range may be about 8 to 9000 mg per day, and a typical dose could be about 50 mg per day. If discrete multiple doses are indicated treatment might typically be 15 mg of truncated oxidized thymosin β4 given up to 4 times per day.
[0066] Whilst it is possible for the active compound to be administered alone, it is preferable to present the active compound in a pharmaceutical formulation. Formulations of the present invention, for medical use, comprise truncated oxidized thymosin β4, or a salt thereof together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients. The carrier(s) should be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.
[0067] The present invention, therefore, further provides a pharmaceutical formulation comprising truncated oxidized thymosin β4 or a pharmaceutically acceptable salt or physiologically functional derivative thereof together with a pharmaceutically acceptable carrier therefor.
[0068] There is also provided a method for the preparation of a pharmaceutical formulation comprising bringing into association truncated oxidized thymosin β4 or a pharmaceutically acceptable salt or physiologically functional derivative thereof, and a pharmaceutically acceptable carrier therefor.
[0069] Formulations according to the present invention include those suitable for oral, nasal, topical, vaginal, rectal or parenteral (including subcutaneous, intraarthrodial (i.e., within joints) intramuscular and intravenous) administration including biolistic e.g., PowderjectR administration. Preferred formulations are those suitable for oral, topical or parenteral administration.
[0070] The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active compound into association with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier or a finely divided solid carrier or both and then, if necessary, shaping the product into desired formulations.
[0071] Formulations of the present invention suitable for oral administration may be presented as discrete units as capsules, cachets, tablets, lozenges, comprising the active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier. Each formulation generally contains a predetermined amount of the active compound; as a powder or granules; or a solution or suspension in an aqueous or non-aqueous liquid such as a syrup, an elixir, an emulsion or draught and the like.
[0072] A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed, tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form such as a powder or granules, optionally mixed with a binder, (e.g., povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycollate, cross-linked povidone, cross- linked sodium carboxymethyl cellulose), surface active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide the desired release profile.
[0073] A syrup may be made adding the active compound to a concentrated, aqueous solution of a sugar, for example sucrose, to which may also be added any accessory ingredients. Such accessory ingredient(s) may include flavorings, an agent to retard crystallization of the sugar or an agent to increase the solubility of any other ingredients, such as a polyhydric alcohol for example glycerol or sorbitol.
[0074] Formulations for rectal administration may be presented as a suppository with a conventional carrier such as cocoa butter.
[0075] Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active compound which is preferably isotonic with the blood of the recipient. Such formulations suitably comprise a solution of a pharmaceutically and pharmacologically acceptable salt of truncated oxidized thymosin β4, that is isotonic with the blood of the recipient.
[0076] Useful formulations also comprise concentrated solutions or solids containing truncated oxidized thymosin β4, which upon dilution with an appropriate solvent give a solution for parental administration as above. [0077] The truncated oxidized thymosin β4 or physiologically active variant thereof disclosed herein may be administered to the lungs of a subject by any suitable means, but are preferably administered by generating an aerosol comprised of respirable particles, the respirable particles comprised of the active compound, which particles the subject inhales (i.e., by inhalation administration). The respirable particles may be liquid or solid. An example of respirable particles is found, e.g., in U.S. Patent Nos. 5,740,794, and 6,051,256.
[0078] Particles comprised of truncated oxidized thymosin β4 for practising the present invention should include particles of respirable size: that is, particles of a size sufficiently small to pass through the mouth and larynx upon inhalation and into the bronchi and alveoli of the lungs. In general, particles ranging from about 0.5 to 10 microns in size (more particularly, less than about 5 microns in size) are respirable. Particles of non-respirable size which are included in the aerosol tend to deposit in the throat and be swallowed, and the quantity of non-respirable particles in the aerosol is preferably minimized. For nasal administration, a particle size in the range of 10-500 μm is preferred to ensure retention in the nasal cavity.
[0079] Liquid pharmaceutical compositions or truncated oxidized thymosin β4 for producing an aerosol can be prepared by combining the truncated oxidized thymosin β4 with a suitable vehicle, such as sterile pyrogen free water. Solid particulate compositions containing respirable dry particles of micronized truncated oxidized thymosin β4 may be prepared by grinding dry truncated oxidized thymosin β4 with a mortar and pestle, and then passing the micronized composition through a 400 mesh screen to break up or separate out large agglomerates. A solid particulate composition comprised of the truncated oxidized thymosin β4 may optionally contain a dispersant which serves to facilitate the formation of an aerosol. A suitable dispersant is lactose, which may be blended with the oxidized thymosin β4 in any suitable ratio (e.g., a 1 to 1 ratio by weight).
[0080] Aerosols of liquid particles comprising the truncated oxidized thymosin β4 may be produced by any suitable means, such as with a nebulizer. See, e.g., U.S. Patent No. 4,501,729. Nebulizers are commercially available devices which transform solutions or suspensions of the oxidized thymosin β4 into a therapeutic aerosol mist either by means of acceleration of a compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation. Suitable compositions for use in nebulizers consist of the truncated oxidized thymosin β4 in a liquid carrier, the truncated oxidized thymosin β4 comprising up to 40% w/w of the compositions, but preferably less than 20% w/w. the carrier is typically water or a dilute aqueous alcoholic solution, preferably made isotonic with body fluids by the addition of, for example, sodium chloride. Optional additives include preservatives if the composition is not prepared sterile, for example, methyl hydroxybenzoate, antioxidants, flavouring agents, volatile oils, buffering agents and surfactants.
[0081] Aerosols of solid particles comprising the truncated oxidized thymosin β4 may likewise be produced with a solid particulate medicament aerosol generator. Aerosol generators for administering solid particulate medicaments to a subject produce particles which are respirable, as explained above, and generate a volume of aerosol containing a predetermined metered dose of a medicament at a rate suitable for human administration. Examples of such aerosol generators include metered dose inhalers and insufflators.
[0082] For inflammation of external tissues, e.g., skin, the formulations are preferably applied as a topical ointment, gel, cream, foam or time-release suspension containing the active ingredient in an amount of, for example, 0.075 to 20% w/w, preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w. Mien formulated in an ointment, the active ingredients may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with an oil-in-water cream base.
[0083] If desired, the aqueous phase of the cream may include, for example, at least 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane- 1,3-diol, mannitol, sorbitol, glyercol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulphoxide and related analogues.
[0084] The oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner. While the phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabilizer(s) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
[0085] Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyle alcohol, glycerol mono- stearate and sodium lauryl sulphate. [0086] The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is very low. Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono-or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
[0087] In addition to the aforementioned ingredients, the formulations of this invention may further include one or more accessory ingredient(s) selected from diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including antioxidants) and the like.
[0088] In another aspect of the invention, truncated oxidized thymosin β4 or variants thereof may be used as a coating or may be impregnated in prosthetic devices such as stents.
[0089] The following examples are not intended to limit the scope of the invention.
Example 1
Human Umbilical Vein Endothelial Cells (HUVEC) Cell Migration Wound
Healing Scratch Assay
[0090] A cell monolayer (HUVEC, HeLa) was grown. The cell monolayer was scratched with a sterile pipette tip and cell debris was washed away. Thereafter, the scratched cell monolayer was treated λvith full-length oxidized thymosin β4 or one of the peptides listed in Figures 1 or 2, a control, or was left untreated. The gap closure of the scratched epithelium was measured over time. Cell migration was measured in Figure 1 , which shows the oxidized thymosin β4 or the truncated peptides at a 2 nm level, and Figure 2, which shows treatment at a 20 nm level. Figures 3 and 4 measure percent wound closure instead of cell migration as in Figures 1 and 2, using the same peptides.
[0091] The truncated oxidized thymosin β4 peptides, in comparison to full-length oxidized thymosin β4, have a greater effect at a lower dosage in a molar-to-molar comparison. Please see
Figures 1-4. Other truncated oxidized thymosin β4 peptides are expected to also have such effect. Figures 1 and 3 show the effect of the various peptides in comparison to thymosin β4 and an FGF control, as well as a negative control, as a 2 nm level and Figures 2 and 4 shows roughly the same effect for oxidized thymosin β4 and four of the five truncated oxidized thymosin β4 peptides. Figure 1 and 3 show that a greater effect may be achieved on a per molar basis at lower dosages for four of the five truncated peptides tested in comparison to oxidized thymosin β4.
Example 2 Truncated Peptide Inhibits Inflammation In Vivo
[0092] Oxidized thymosin β4 and the 14-mer GBLOl were tested in a carrageenan assay. Carrageenan induces an inflammatory response and tests for mouse paw edema. Groups of mice were injected subcutaneously in one hind paw with 300 μg carrageenan mixed with PBS, DEX, and oxidized thymosin β4 or the 14-mer GBLOl . Foot pad swelling was measured using a spring-dial caliper and expressed as the difference in swelling between the carrageenan injected paw and the uninjected contralateral paw. The animals were injected intraperitoneally with the same dose of test material and measurements were made at 6, 12, and 24 hours. The results are shown in Figure 5. The results show that the oxidized thymosin β4 and the truncated 14-mer have approximately the same effect.
Example 3 Burst Assay
[0093] Dihydrorhodamine 123 (DHR) is a dye that oxidizes to form the brightly fluorescent compound rhodamine 123. Neutrophils can oxidize DHR following stimulation by irritants or oxidative stress, chemoattractants, etc., subsequent to the metabolic burst generated through the hexose monophosphate (HMP) shunt. A series of metabolic events are triggered in the cell, including increased oxygen consumption and increased superoxide, hydroxyl radical, and hydrogen peroxide formation. When the later events occur, DHR 123 is oxidized to R 123, which is measured on the FACS. The results with respect to oxidized thymosin β4 are shown in Figure 6. A similar burst assays is discussed in Example 7. Data from this additional example further supports the position that the peptides of the disclosed invention are effective to inhibit respiratory burst. Example 4 TNF-α Production in PBMCs Inhibited bv GBLlOl
[0094] To study the effect of GBLlOl on TNF-a production from peripheral blood mononuclear cells (PBMCs) in the presence of pro-inflammatory cytokines and mitogens, PBMCs were isolated and cultured in the presence of lipopolysaccharide (LPS) or IL-12 or IL- 18. Specifically, PBMCs were cultured overnight in ImI culture (24- well tissue culture plate) at 2 x 106 cells/ml in RPMI 10%FCS, and treated with various agonists (i.e., GBLlOl, dexamethasone or TNF-α).
[0095] At twenty four (24) hours, addition of agonists was repeated. Cells were aspirated from the plate, scraping with a blue Gilson tip, and 100 ml x 3 added to a 96-well flat-bottomed tissue culture plate. Figure 7A shows the amounts of TNF-α produced in the presence of lOOul of IL- 12/18 (3ng/ml and lOOng/ml final added, respectively). Figure 7B shows the amount of TNF-α produced in the presence of lOOul LPS (250ng/ml final concentration) was added. Cells were cultured for 24 hours at 370C 5%CO2 in humidified atmosphere. 150ul of supernatant was removed at 24 hours. TNF-α concentration determined by ELISA (Biosource).
[0096] As can be seen in Figure 7 A, 10 ng/ml of GBLlOl was approximately as effective as dexamethasone in inhibiting TNF-α production from PBMCs treated with the mitogen LPS. Similarly, 10 ng/ml of GBLlOl was at least as effective as dexamethasone in TNF-α production from PBMCs treated with the pro-inflammatory cytokines IL-12 and IL- 18. These results clearly demonstrate that GBLlOl possesses anti-inflammatory characteristics.
Example 5
GBLlOl Induces Apoptosis in Human Peripheral Blood Neutrophils in a Dose Dependent Fashion
[0097] To study the apoptotic properties of the truncated thymosin β4 oxidized peptide, isolated neutrophils were prepared fresh from peripheral blood. Cells were washed, counted and adjusted to 1 million cells/ml.
[0098] 1 ml of these cells was incubated with various concentrations of TB4so (lug, 100 ng/ml, 10 ng/ml, 1 ng/ml) or TNF-α ( 100ng/ml) as a positive control for the induction of apoptosis for 4 hours at 370C. Cells were aspirated into FACS tubes, centrifuged at 1500 ipm for 5 minutes. The supernatant was aspirated. lOOul of CM added then 5 microlitres of AnnexinV-FITC and propidium iodide (PI). Cells then analyzed by FACS. Data is presented as percentage increase in AnnexinV binding to peripheral blood neutrophils, relative to untreated cells.
[0099] Figure 8 shows that increasing concentrations, the truncated TB4So peptide GBLlOl5 is shown to induce programmed cell death (apoptosis) in human peripheral blood neutrophils. This is an important anti-inflammatory response that modulates the activity and function of a key inflammatory cell type.
Example 6
Presence of Oxidized Met in GBL 106 is Required for Biological Activity in the
Wound Scratch Assay
[0100] HUVEC cells cultured in 24-well tissue culture plate. A scratch wound was made at time 0 with yellow Gilson tip. Dislodged cells were removed by washing the plates twice. Measurements of cell migration were made at 1Ox magnification, using micrometer. Migration values shown in Figure 9 are the average migration distance into the scratch wound (Time 0 - time 2.5hr/6hr), measured in 5 marked locations along the scratch, in triplicate wells. Conclusions: TB4so and GBLlOl have induced HUVEC cell migration to a similar level as FGF (positive control). This effect is statistically significant versus negative control, at all concentrations tested. GBLl 06 has not produced a statistically significant effect over negative control, at any concentration tested.
[0101] In Figure 9 above the presence of oxidized methionine at position 6 in the truncated TB4So peptide is required for biological activity. Peptides GBL 106 and GBLlOl are identical except for the substitution of norleucine at position 6 in GBLl 06. The data clearly show that in order to achieve directed cell migration of human endothelial cells, a measure of wound healing, position 6 of the peptide must be oxidized.
Example 7 GBLlOl Inhibits fMLP-Induced Respiratory Burst from Isolated Human Neutrophils
[0102] Neutrophils were prepared fresh from peripheral blood. Cells were washed, counted and brought to a concentration of 1 million cells/ml. One milliliter of these cells were incubated with various concentrations of GBLOl for 3 hours at 370C. Cells were then incubated in the presence of DHR (30 μM final) for 10 minutes at 370C. The cells were then incubated in the presence of 10'6M fMLP to induce a respiratory burst, for 10 minutes at 370C. The reaction was stopped by washing cells in ice cold PBS with 0.02% EDTA. The cells were isolated and resuspended in 300 μl of PBS/EDTA and analyzed by FACS. As shown in Figure 10, increasing concentrations of GBLlOl inhibited the respiratory burst response from human neutrophils as correlated and measured by DHR uptake of the incubated cells, which is indicated by Median Fluorecence Intensity (MFI). This data demonstrates that the truncated oxidized peptides are effective to inhibit respiratory burst.

Claims

Claims
1. An isolated synthetic peptide comprising at least seven consecutive amino acids that correspond to the 14 N-teraiinal amino acids of oxidized thymosin β4, or variants thereof, wherein at least one amino acid of the peptide is an oxidized methionine, and wherein the peptide has oxidized thymosin β4 activity.
2. The peptide of claim 1 , selected from the group consisting of ac-SDKPDMAEIEKFDK (SEQ ID NO:1), ac-MSDKPDMAEIEKFDK (SEQ ID NO:9), and ac-LLP ATMSDKPDMAEIEICFDK (SEQ ID NO: 10), wherein ac represents an acetyl group on the peptide and M represents a methionine containing an oxidized sulfur.
3. The peptide of claim 1, selected from the group consisting of acetylated or non- acetylated SDKPDMA (SEQ ID NO:3), DKPDMAE (SEQ ID NO:4), KPDMAEI (SEQ ID NO:5), PDMAEIE (SEQ ID NO:6), DMAEIEK (SEQ ID NO:7), and MAEIEIvF (SEQ ID NO:8); wherein M represents a methionine containing an oxidized sulfur.
4. A composition comprising the peptide in any of claims 1-3 and a pharmaceutically acceptable carrier.
5. The composition of claim 4, wherein the composition is adapted for topical, enteral, or parenteral administration
6. The composition of claim 4, wherein the composition is adapted for systemic, cutaneous, ocular, enema, intranasal, oral, gastric, duodenal, rectal, intravenous, intra-arterial, intramuscular, intracardiac, subcutaneous, intraosseous, subcutaneous, transdermal, transmucosal, inhalational, intraperitoneal, epidural, intrathecal, intravitreal, or intracranial administration.
7. A method of treating a subject with inflammation, comprising, identifying the subject with the condition or disease; and administering to the subject a pharmaceutical composition of claims 4 or 5, whereby the inflammation is reduced.
8. The method of claim 7, wherein the inflammation causes or exacerbates a condition or disease.
9. The method of claim 8, wherein the condition or disease is a skin disorder.
10. The method of claim 9, wherein the skin disorder is selected from the group consisting of psoriasis, contact or atopic dermatitis, chronic dermal wounds, decubitous ulcers, venous stasis ulcers, diabetic ulcers, pressure sores, skin disorders, epidermolysis bullosa (eb), pemphagus, photoallergenic dermatitis, phototoxic dermatitis, phototrauma, radiation dermatitis, radiodermatitis (acute and chronic), blisters, sores, skin aging, tissue reaction to implanted prostheses, and conditions associated with transplantation or prosthetics.
1 1. The method of claim 8, wherein the condition or disease is an autoimmune disease.
12. The method of claim 1 1, wherein the autoimmune disease is selected from the group consisting of organ/tissue regeneration, adult respiratory distress syndrome, allergic contact dermatitis, allergic eosophagitis, arthritis, asthma, Crohn's disease, inflammatory bowel disease, inflammatory dermatosis, irritant contact dermatitis, ischemic reperfusion injury, Kawasaki disease, keloids, keratosis follicularis (Darier- White syndrome), lichen planus, multiple sclerosis, pemphagus, rheumatoid arthritis, scleritis, seborrheic dermatitis, systemic lupus erytliromatosis, graft rejection, graft versus host disease, and lupoid hepatitis.
13. The method of claim 8, wherein the condition or disease is an inflammatory respiratory disorder.
14. The method of claim 13, wherein the inflammatory respiratory disorder is selected from the group consisting of asthma, COPD, emphysema and cystic fibrosis.
15. The method of claim 8, where in condition or disease is a dermal wound.
16. The method of claim 8, wherein the condition or disease is associated with an ischemic event.
17. The method of claim 16, wherein the ischemic event is a cardiovascular ischemia event.
18. The method of claim 17, wherein the cardiovascular ischemic event is a myocardial infarction or restenosis.
19. The method of claim 16, wherein the ischemic event is a stroke.
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