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EP1913013A2 - Thymosine beta4 oxydee tronquee et derives de celle-ci - Google Patents

Thymosine beta4 oxydee tronquee et derives de celle-ci

Info

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
Authority
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
Other languages
German (de)
English (en)
Inventor
Elizabeth Taylor
Robert Goodenow
John Young
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Grannus Biosciences
Original Assignee
Grannus Biosciences
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Grannus Biosciences filed Critical Grannus Biosciences
Publication of EP1913013A2 publication Critical patent/EP1913013A2/fr
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

L'invention concerne l'utilisation de la thymosine ß4 oxydée tronquée dans le traitement, tel que celui de maladies ou d'états associés aux inflammations.
EP06788075A 2005-07-19 2006-07-19 Thymosine beta4 oxydee tronquee et derives de celle-ci Ceased EP1913013A2 (fr)

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