Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/64—Cyclic peptides containing only normal peptide links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/02—Nasal agents, e.g. decongestants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/08—Bronchodilators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/04—Antipruritics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/06—Antipsoriatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/10—Anti-acne agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/12—Antihypertensives
• • 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 synthetic cyclic peptides.
• the present invention also relates to uses of these peptides, in particular for treating diseases and/or conditions associated with inhibition of T-cell activation or function, allergic airways disease, microbial infections and cancer.
• CP Core Peptide
• CP acts as an immunomodulatory agent and inhibits IL-2 production in T-cells following antigen recognition (Manolios et. at., 1997).
• CP has been shown to reduce T-cell mediated inflammation in animal models of adjuvant induced arthritis, allergic encephalomyelitis and delayed type contact hypersensitivity.
• CP is 9 amino acids in length (GLRILLLKV), the sequence of which is derived from the T-cell antigen receptor (TCR- ⁇ ) transmembrane region.
• linear peptides are poorly absorbed, enzyme and pH sensitive, easily biodegradable and have low solubility. Accordingly, there is a need to improve the delivery and stability of peptides to overcome the problems associated with linear molecules.
• a cyclic peptide comprising alternating D- and L-form amino acids, and wherein the peptide possesses immunomodulatory activity.
• the immunomodulatory activity may comprise the ability to inhibit T cell activation, proliferation or stimulation.
• the cyclic peptide may comprise between 6 and 14 amino acids.
• the cyclic peptide may comprise between 8 and 12 amino acids.
• the cyclic peptide comprises 10 amino acids, in which 5 amino acids are L-form amino acids and 5 amino acids are D-form amino acids.
• the cyclic peptide may have a net positive charge. The net positive charge may be at least +2.
• the peptide may comprise two positively charged amino acid residues separated by 1 to 5 hydrophobic residues. At least one of the positively charged residues may be a lysine residue.
• the cyclic peptide comprises a lysine residue and a second positively charged residue separated by a spacer of five amino acid residues, wherein the second positively charged residue is an arginine or lysine residue.
• These amino acids may be D- or L-form amino acids.
• the five amino acid spacer may comprise no net charge or a net positive charge.
• a cyclic peptide of the invention may comprise a structure as depicted in any one
• a cyclic peptide selected from the group consisting of, LDRLDLLDLLDKVDG, LDRDLDLDLDLDLDKDVDG, LRLLLLLKVG, LDKLDLLDLLDKVDG, LRDLLDLLDLKDVGD, L D LLDRL D LL D KVDG, R D LL D LLDLLDKVDG, LDLLDLLDRLDKVDG, LDRLDKLDLVDLLDG, LDRLDLLDLLDRVDG, LDKLDKLDKLDKVDG, LDRLDL ⁇ DKLDKVDG, LDRLDKLDLLDKVDG, LDRLDKLDKLDKVDG, LDKLDKKDKLDKVDG, LDKLDKKDKLDKVDG, LDRLDKKDKLDKVDG, LDKLDKKDKVDG, LDRLDKKDKKDKVDG, LDRLDKKDKKDKVDG, LDRLDKKDKKDKVDG, LDRLDKVDG, LD
• a cyclic peptide of the first, second, third or fourth aspect may be conjugated to one or more chemical moieties. Conjugation may be via linkage at one or more carboxyl, hydroxyl and/or amide groups.
• the chemical moiety may be a polypeptide, peptide, lipid, sugar or other chemical compound.
• the chemical moiety may be a carrier or a therapeutic agent.
• a pharmaceutical composition comprising a peptide of any one of the first, second, third or fourth aspects.
• the pharmaceutical composition may include one or more pharmaceutically acceptable carriers, adjuvants or diluents.
• an antibody that selectively binds to a cyclic peptide of any one of the first, second, third or fourth aspects.
• the present invention provides a method for treating or preventing a disease state in a subject, said method comprising administering to the subject an effective amount of a peptide of any one of the first, second, third or fourth aspects or a pharmaceutical composition of the fourth aspect.
• the disease is an auto-immune disease.
• the disease may be neural, endocrinal, skeletal, dermal, gastrointestinal, cardiac, respiratory, vascular, of the immune system or a transplantation disease.
• the disease may be a cancer.
• the disease may be an airway disease such as asthma, chronic eosinophilic pneumonia, COPD, COPD associated with chronic bronchitis, pulmonary emphysema, dyspnea associated or not associated with COPD, COPD characterized by irreversible, progressive airway obstruction, adult respiratory distress syndrome (ARDS), exacerbation of airway hyper-reactivity consequent to other drug therapy, airway disease associated with pulmonary hypertension, rhinitis, bronchial oedema, bronchial asthma, pulmonary oedema, anaphylaxis or angioedema.
• airway disease such as asthma, chronic eosinophilic pneumonia, COPD, COPD associated with chronic bronchitis, pulmonary emphysema, dyspnea associated or not associated with COPD, COPD characterized by irreversible, progressive airway obstruction, adult respiratory distress syndrome (ARDS), exacerbation of airway hyper-reactivity consequent to other drug therapy, airway disease associated
• the present invention provides a method for treating or preventing a disease state in a subject in which the inhibition of T-cell activation, proliferation or function is desirable, said method comprising administering to the subject an effective amount of a peptide of any one of the first, second, third or fourth aspects or a pharmaceutical composition of the fourth aspect.
• the inhibition of T-cell activation, proliferation or function may involve the inhibition of IL-2 production in T-cells.
• the present invention provides a method for treating or preventing a microbial infection in a subject, said method comprising administering to the subject a therapeutically effective amount of a peptide of any one of the first, second, third or fourth aspects or a pharmaceutical composition of the fifth aspect.
• the infection may be a Staphylococcus infection.
• the Staphylococcus infection may be a Staphylococcus aureus infection.
• the microbial infection is associated with increased IL-13.
• the microbial infection may be a parasitic infection.
• the parasitic infection may be an intracellular parasite.
• the intracellular parasite may be bacterium or a protozoan.
• the bacterium may be selected from the group comprising Chlamydia, Rickettsia, Coxiella or
• Mycobacterium may be Mycobacterium leprae or Mycobacterium tuberculosis.
• the Protozoan may be selected from the group comprising Plasmodium sp,
• Leishmania sp, Toxoplasma or Trypanosoma Leishmania sp, Toxoplasma or Trypanosoma.
• the present invention provides the use of a peptide of any one of the first, second, third or fourth aspects for the manufacture of a medicament for the treatment or prevention of a disease state in a subject
• the present invention provides the use of a peptide of any one of the first, second, third or fourth aspects for the manufacture of a medicament for the treatment or prevention of a microbial infection in a subject.
• the present invention provides a method for treating or preventing a disease state in a subject in which the control of blood glucose levels is desirable, said method comprising administering to the subject an effective amount of a peptide of any one of claims first, second, third or fourth aspects or a pharmaceutical composition of the fifth aspect.
• the disease state may be diabetes.
• FIG. 1 Cyclized structure of peptide C1. Amino acids are numbered from 1 to 10, according to the order of residues provided in the linear single letter amino acid code. D subscript represents a D-amino acid.
• Figure 2. Cyclized structure of peptide C2. Amino acids are numbered from 1 to 10, according to the order of residues provided in the linear single letter amino acid code. D subscript represents a D-amino acid.
• Figure 6 Viability and proliferation assay. % viability and proliferation of 2B4 cells in theo presence of C1 at 50 ⁇ M, relative to a control of 0.5% DMSO.
• FIG. 7 Antigen presentation assay. %IL-2 produced, relative to a DMSO control (0.5%) under various T-cell stimulants in the presence of C1 at 50 ⁇ M.
• FIG. 1 Comparative binding of peptide analogues (50 ⁇ M) to DMPC (A) and DMPG (B) liposomes immobilised onto L1 sensor chip.
• FIG. 12 Transmission Electron micrograph of C1 (100 ⁇ M in Water/HCI) showing rectangular structures.
• FIG. 19 Effect of cyclic C1 peptide administration on bronchoalveolar lavage fluid (BALF) inflammatory cell infiltrates in allergic mice: total white blood cells.
• Data represents the mean + SEM for a minimum of 4 mice per group compared to historical na ⁇ ve, PBS/OVA and OVA/OVA mice.
• Student's unpaired t-test *** P ⁇ 0.001, ** p ⁇ 0.005, * p ⁇ 0.05 compared to OVA/OVA.
• Figure 20 Effect of peptide administration on bronchoalveolar lavage fluid (BALF) inflammatory cell infiltrates in allergic mice: differential white blood cell counts. Data represents the mean + SEM for a minimum of 4 mice per group compared to historical naive, PBS/OVA and
• Figure 21 The effect of peptide administration on bronchoalveolar lavage fluid (BALF) inflammatory cell infiltrates in allergic mice: total white blood cells. Data represents the mean +SEM for a minimum of 7 mice per group. Student's unpaired t-test ** * P ⁇ 0.001, **p ⁇ 0.005, * p ⁇ 0.05 compared to OVA/OVA.
• BALF bronchoalveolar lavage fluid
• Figure 22 The effect of peptide administration on bronchoalveolar lavage fluid (BALF)inflammatory cell infiltrates in allergic mice: differential white blood cell counts. Data represents the mean + SEM for a minimum of 6 mice per group. Student's unpaired t-test *** P ⁇ 0.001 , * * p ⁇ 0.005,* p ⁇ 0.05 compared to OVA/OVA.
• BALF bronchoalveolar lavage fluid
• Figure 23 The effect of peptide administration on spleen and peribronchial lymph node OVA-specific Th2 cytokine production. Data represents the mean + SEM for a minimum of 6 mice per group. Student's unpaired t-test ***P ⁇ 0.001, * * p ⁇ 0.005, * p ⁇ 0.05 compared to OVA/OVA.
• Figure 24 The effect of peptide administration on airways hyperreactivity. Airway resistance (RL) is presented as a percentage of the baseline reactivity to saline in the absence of cholinergic stimuli and represent the mean + SEM for a minimum of 5 mice per group. 2Way ANOVA ** *P ⁇ 0.001 , * *p ⁇ 0.005, * p ⁇ 0.05 compared to OVA/OVA.
• Figure 25 The effect of peptide administration on airways hyperreactivity. Dynamic compliance (CDyn) is presented as a percentage of the baseline reactivity to saline in the absence of cholinergic stimuli and represent the mean + SEM for a minimum of 5 mice per group.2Way ANOVA ***P ⁇ 0.001, ** p ⁇ 0.005, * p ⁇ 0.05 compared to OVA/OVA.
• FIG. 27 Blood glucose levels of NOD mice over time after the administration of water.
• peptide means a polymer made up of amino acids linked together by peptide bonds.
• analogue as used herein with reference to a peptide means a peptide which is a derivative of a cyclic peptide of the invention which derivative comprises addition, deletion, substitution of one or more amino acids, such that the peptide retains substantially the same function as the cyclic peptide of the invention.
• treatment refers to any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever.
• the term "effective amount” includes within its meaning a non-toxic but sufficient amount of an agent or compound to provide the desired therapeutic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact “effective amount” . However, for any given case, an appropriate
• the terms "subject” and “patient” are used interchangeably and include humans and individuals of any species of social, economic or research importance including but not limited to members of the genus ovine, bovine, equine, porcine, feline, canine, primates, rodents.
• the patient will be a mammal and more preferably the patient will be a human.
• Amino acids can take two mirror forms, the L-isomer or the D-isomer.
• Naturally occurring amino acids in mammalian polypeptides and proteins contain L-isomer amino acids only.
• Peptide (CP) derived from the transmembrane region of the T-cell antigen receptor (TCR- ⁇ ) consists of 9 L-isomer amino acids and has the ability to inhibit T-cell antigen specific activation in vitro and in vivo.
• Gerber et al. (2005) compared the activity of the two stereoisomers of CP, that is, CP consisting of L-amino acids only and CP consisting of D-amino acids only. Gerber et al. showed that both the L- and D- forms of CP possessed similar inhibitory activity of T-ceil proliferation in vitro. Furthermore both the L- and D- form of CP were equally active in alleviating adjuvant arthritis in vivo.
• the cyclic peptide of formula I possesses not only desirable biological activity, including the inhibition of T-cell activation and antimicrobial activity, but also possesses desirable stability is characteristics, increasing the utility of the compound.
• the present invention provides isolated cyclic peptides (C1-C4) having the cyclized structures as depicted in Figures 1 to 4 respectively, represented below by the formulas I to Il respectively.
• the present invention also provides cyclized structures represented below by the 0 formulas v to xxvii respectively:
• peptides of the invention comprise between 6 and 14 amino acids, between 7 and 13 amino acids, between 8 and 12 amino acids, between 9 and 11 amino acids, or more typically 10 amino acids.
• the present invention also contemplates peptide conjugates wherein peptides of the invention are linked to one or more additional chemical moieties. Conjugation may be via linkage at one or more carboxyl, hydroxyl and/or amide groups.
• the chemical moiety may be a polypeptide, peptide, lipid, sugar or other chemical compound.
• the chemical moiety may be a carrier agent, facilitating or enhancing the in vivo delivery of the peptide and/or targeting and delivering the peptide to the region, tissue or cell in which activity of the peptide is desired.
• Suitable carrier molecules include lipids, such as short or long chain fatty acids.
• the chemical moiety may be a therapeutic agent such as a pharmaceutical compound.
• Cyclic D-, L- ⁇ - peptides are proteolytically stable, easy to synthesise, and can be derived from a vast potentially membrane-active sequence space.
• the unique abiotic structure of the cyclic peptides and their rapid bactericidal action may also limit temporal acquirement by drug resistant bacteria.
• the low molecular weight D, L- ⁇ -peptides disclosed herein therefore offer an attractive complement to the current arsenal of naturally derived antibiotics, and hold considerable potential in combating a variety of existing and emerging infectious diseases.
• the cyclic peptide of formula I is an active biological compound with potent immunosuppression. Biophysical studies indicate that C1 does not damage the bilayer of model membranes like other cyclic and antimicrobial peptides.
• the cyclic peptide C1 is stable over a large pH range (1-11) and when incubated with trypsin. The ability of C1 to inhibit immune reactions provides a new and exciting alternative to existing therapies.
• Peptides of the present invention may be synthesised first as linear molecules by standard methods of liquid or solid phase chemistry well known to those of ordinary skill in the art. For example such molecules may be synthesised following the solid phase chemistry procedures of Steward and Young (Steward, J. M. & Young, J. D., Solid Phase Peptide Synthesis. (2nd Edn.) Pierce Chemical Co., Illinois, USA (1984). Linear peptides can then be cyclized using techniques and procedures also well known to those of skill in the art. In particular embodiments a linear peptide of the invention may be cyclized in accordance with the following method.
• a solution of a peptide of the invention in dichloromethane (DCM ; ⁇ 1 mM) may be stirred with (benzotriazol-i-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP; 4 eq.) and N,N-diisopropylethylamine (DIEA; 10 eq.) overnight.
• Cyclized peptide may then be dried on a rotary evaporator prior to deprotection with a mixture of 95% trifluoroacetic acid (TFA): 2.5%water: and 2.5% triisopropylsilane (TIPSI).
• TIPSI triisopropylsilane Crude peptide may be characterized by mass spectrometry.
• cyclization may be performed as described above but the protected cyclized peptide may be first precipitated in water and the precipitate washed with 0.5M sodium hydrogen carbonate (NaHCOs), ether and acetonitrile respectively. Protecting groups may then be removed in TFA/scavenger mixture. The peptide may be characterized by mass spectrometry.
• cyclization may be performed under anhydrous conditions, 1.5equiv of pentafluorophenyl diphenylphosphinate (FDPP; 39mg, 0.1 mmol) may be added to a solution of linear protected C1 (100mg, O.OOTmmol) in acetonitrile (14ml), followed by addition of 3equiv of DIEA (0.2mmol).
• FDPP pentafluorophenyl diphenylphosphinate
• DIEA 3equiv of DIEA
• Purification of a cyclic peptide of the invention may be performed by HPLC.
• HPLC typically the hydrophobic nature of the cyclised peptides of the invention have made purification of the peptides difficult using conventional HPLC techniques.
• the inventors have used an At-Column Dilution (ACD) technique to purify the peptides using HPLC. This technique was developed for injecting relatively large volumes of strong sample diluents. ACD technique also prevents bulk precipitation in the sample loop or in the column itself.
• Figures 16 and 17 are schematic diagrams of conventional and ACD HPLC techniques.
• a peptide of the invention may be dissolved in approximately 0.5ml of acetonitrile, 200 ⁇ l of 10% TFA. DMSO may then be added until a clear solution is obtained. The sample may be centrifuged prior to collecting the supernatant for loading on to a reverse phase column, such as a c18 column. The sample may be loaded using standard ACD techniques.
• a peptide of the invention may be loaded on to the column at a flow rate of, for example, 0.1ml/min. Simultaneously the mobile phase may be pumped through the column using another pump at a flow rate of, for example, 5ml/min.
• Peptides of the present invention find application in the treatment or prevention of a variety of diseases and conditions such as diseases in which inhibition of T-cell function is desirable, particularly auto-immune diseases and cancers.
• diseases and disorders to which methods and compositions of the present invention are applicable include, but are not limited to, neural diseases such as multiple sclerosis and Guillain Barre Syndrome, endocrine diseases such as diabetes, Hashimotos disease and pernicious anaemia, skeletal diseases such as rheumatoid arthritis, ankylosing spondylitis, reactive arthritis and systemic lupus erythematosus, immune diseases such as transplant rejection syndrome, urticaria and drug allergy, dermal diseases such as pemphigus, eczema, contact dermatitis and psoriasis, gastrointestinal tract diseases such as ulcerative colitis and Crohn's disease, respiratory diseases such as asthma and pneumonitis, transplantation disease, cardiac diseases, vascular diseases and cancer.
• neural diseases such as multiple sclerosis and Guillain Barre Syndrome
• endocrine diseases such as diabetes, Hashimotos disease and pernicious anaemia
• skeletal diseases such as rheumatoid arthritis, ankylos
• lnterleukin-5 is associated with allergic diseases including allergic rhinitis and asthma which are characterised by increased eosinophils in the circulation, in airway tissue and in sputum
• IL-13 lnterleukin 13
• Parasitic infection often leads to granulomas the formation of which is at least partly controlled by IL-13. Granulomas result in organ damage and thus profound or sometimes fatal disease rather than resolution of the infection.
• IL-13 is thought to antagonize Th1 responses required to resolve intracellular infections.
• immune dysregulation which is characterised by recruitment of large numbers of Th2 cells IL-13 inhibits the destruction of intracellular pathogens by the hosts immune system.
• IL-13 also induces features of allergic lung disease, including airway hyperresponsiveness, goblet cell metaplasia and mucus hypersecretion which contribute to airway obstruction.
• peptides of the present invention reduce IL-5 and IL-13 levels (See for example Figure 23) the peptides of the present invention find application in the treatment of diseases characterised by increased IL-5 or IL-13 or both and where the increase in IL-5 and/or IL-13 contributes to the pathology of the disease.
• Peptides of the present invention also find application in the inhibition of T-cell function for the treatment or prevention of a variety of diseases and conditions such as non-autoimmune diseases in which the T-cell may be involved by virtue of providing CD4 help.
• diseases and disorders to which methods and compositions of the present invention are applicable include, but are not limited to airway diseases, dermal diseases, osteoporosis, atherosclerosis, multiple sclerosis, and ischaemic heart disease.
• the airway disease may be an airway disease such as asthma, chronic eosinophilic pneumonia, COPD (chronic obstructive pulmonary disease), COPD associated with bronchitis, pulmonary emphysema, dyspnea associated or not associated with COPD, COPD characterized by irreversible, progressive airway obstruction, adult respiratory distress syndrome (ARDS), exacerbation of airway hyper-reactivity consequent to other drug therapy, airway disease associated with pulmonary hypertension, rhinitis, bronchial oedema, bronchial asthma, pulmonary oedema, anaphylaxis or angioedema.
• COPD chronic eosinophilic pneumonia
• COPD chronic obstructive pulmonary disease
• COPD associated with bronchitis
• pulmonary emphysema pulmonary emphysema
• dyspnea associated or not associated with COPD COPD characterized by irreversible, progressive airway obstruction,
• Examples of dermal diseases include vitiligo, psoriasis, eczema, Herpes simplex, erythema nodosum, acne vulgaris, erythema, erythema multiforme, neurodermatitis, drug rash, urticaria, contact dermatitis, dermatomycosis and herpes zoster.
• Methods and compositions of the present invention also find application in the treatment of a variety of infectious diseases and microbial infections. Such infections may result from, or be associated with, either Gram-negative or Gram-positive bacteria.
• peptides of the invention may be administered alone or in conjunction with one or more additional agents.
• a peptide of the invention may be administered together with one or more agents for inhibiting T-cell activation and/or function, such as cyclosporin.
• a peptide of the invention may be administered in conjunction with one or more antibiotics or antimicrobial agents. Suitable agents which may be used in combination with the peptides of the present invention will be known to those of ordinary skill in the art.
• each component of the combination may be administered at the same time, or sequentially in any order, or at different times, so as to provide the desired effect.
• the components may be formulated together in a single dosage unit as a combination product.
• Suitable agents which may be used in combination with the compositions of the present invention will be known to those of ordinary skill in the art.
• Cyclic peptides of the invention, or antibodies thereof, may be administered in the form of pharmaceutical compositions either therapeutically or preventively.
• compositions are administered to a patient already suffering from a disease, in an amount sufficient to cure or at least partially arrest the disease and its complications.
• the composition should provide a quantity of the active agent sufficient to effectively treat the patient.
• the effective dose level for any particular patient will depend upon a variety of factors including: the disorder being treated and the severity of the disorder; activity of the agent employed; the composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of sequestration of the agent; the duration of the treatment; drugs used in combination or coincidental with the treatment, together with other related factors well known in medicine.
• an effective dosage is expected to be in the range of about 0.0001 mg to about 1000mg per kg body weight per 24 hours; typically, about 0.001 mg to about 750mg per kg body weight per 24 hours; about 0.01 mg to about 500mg per kg body weight per 24 hours; about 0.1 mg to about 500mg per kg body weight per 24 hours; about 0.1 mg to about 250mg per kg body weight per 24 hours; about 1.Omg to about 250mg per kg body weight per 24 hours.
• an effective dose range is expected to be in the range about 1.Omg to about 200mg per kg body weight per 24 hours; about 1.0mg to about 100mg per kg body weight per 24 hours; about 1.Omg to about 50mg per kg body weight per 24 hours; about 1.Omg to about 25mg per kg body weight per 24 hours; about ⁇ .Omg to about 50mg per kg body weight per 24 hours; about ⁇ .Omg to about 20mg per kg body weight per 24 hours; about ⁇ .Omg to about 15mg per kg body weight per 24 hours, about ⁇ .Omg to about 10 mg per kg body weight per 24 hours or about 1.Omg to about 6 mg per kg body weight per 24 hours
• an effective dosage may be up to about 500mg/m 2 .
• an effective dosage is expected to be in the range of about 25 to about 500mg/m 2 , preferably about 25 to about 350mg/m 2 , more preferably about 25 to about 300mg/m 2 , still more preferably about 25 to about 250mg/m 2 , even more preferably about 50 to about 250mg/m 2 , and still even more preferably about 75 to about 150mg/m 2 .
• the treatment would be for the duration of the disease state.
• compositions may be prepared according to methods which are known to those of ordinary skill in the art and accordingly may include a pharmaceutically acceptable carrier, diluent and/or adjuvant.
• compositions can be administered by standard routes.
• the compositions may be administered by the parenteral (e.g., intravenous, intraarticular, intraspinal, subcutaneous or intramuscular), oral or topical route.
• parenteral e.g., intravenous, intraarticular, intraspinal, subcutaneous or intramuscular
• the carriers, diluents and adjuvants must be "acceptable” in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof.
• Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glyco
• compositions of the invention may be in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (such as capsules, tablets, caplets, elixirs, for example), in the form of an ointment, cream or lotion suitable for topical administration, in a form suitable for delivery as an eye drop, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, in a form suitable for parenteral administration, that is, subcutaneous, intramuscular or intravenous injection.
• a formulation suitable for oral ingestion such as capsules, tablets, caplets, elixirs, for example
• an ointment cream or lotion suitable for topical administration
• an eye drop in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation
• parenteral administration that is, subcutaneous, intramuscular or intravenous injection.
• non-toxic parenterally acceptable diluents or carriers can include, Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1,2 propylene glycol.
• suitable forms for oral administration include tablets, lozenges, pills, capsules, aerosols, elixirs, powders, granules, solutions, suspensions and emulsions.
• suitable carriers, diluents, excipients and adjuvants for oral use include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine and lecithin.
• these oral formulations may contain suitable flavouring and colourings agents.
• the capsules When used in capsule form the capsules may be coated with compounds such as glyceryl monostearate or glyceryl distearate which delay disintegration.
• Adjuvants typically include emollients, emulsifiers, thickening agents, preservatives, bactericides and buffering agents.
• Solid forms for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and/or time delay agents.
• Suitable binders include gum acacia, gelatine, com starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol.
• Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine.
• Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar.
• Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate.
• Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring.
• Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten.
• Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite.
• Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc.
• Suitable time delay agents include glyceryl monostearate or glyceryl distearate.
• Liquid forms for oral administration may contain, in addition to the above agents, a liquid carrier.
• suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, arachis oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides or mixtures thereof.
• Suspensions for oral administration may further comprise dispersing agents and/or suspending agents.
• Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, poly-vinyl-pyrrolidone, sodium alginate or acetyl alcohol.
• Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate, polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate and the like.
• the emulsions for oral administration may further comprise one or more emulsifying agents.
• Suitable emulsifying agents include dispersing agents as exemplified above or natural gums such as guar gum, gum acacia or gum tragacanth.
• Methods for preparing parenterally administrable compositions are apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa., hereby incorporated by reference herein.
• the topical formulations according to the present invention comprise an active ingredient together with one or more acceptable carriers, and optionally any other therapeutic ingredients.
• Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site of where treatment is required, such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.
• Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions. These may be prepared by dissolving the active ingredient in an aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and optionally including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container and sterilised. Sterilisation may be achieved by: autoclaving or maintaining at 9O 0 C-IOO 0 C for half an hour, or by filtration, followed by transfer to a container by an aseptic technique.
• bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%).
• Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.
• Lotions according to the present invention include those suitable for application to the skin or eye.
• An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those described above in relation to the preparation of drops.
• Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisturiser such as glycerol, or oil such as castor oil or arachis oil.
• Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely-divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with a greasy or non-greasy basis.
• the basis may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol such as propylene glycol or macrogols.
• the composition may incorporate any suitable surfactant such as an anionic, cationic or non-ionic surfactant such as sorbitan esters or polyoxyethylene derivatives thereof.
• Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.
• the compositions may also be administered in the form of liposomes. Liposomes are generally derived from phospholipids or other lipid substances, and are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used.
• compositions in liposome form may contain stabilisers, preservatives, excipients and the like.
• the preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art, and in relation to this specific reference is made to: Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic
• the present invention also provides antibodies that selectively bind to the cyclic peptides of the present invention.
• Suitable antibodies include, but are not limited to polyclonal, monoclonal, chimeric, humanised, single chain, Fab fragments, and an Fab expression library.
• Antibodies of the present invention may act as agonists or antagonists of the cyclic peptides of the present invention.
• a monoclonal antibody typically containing Fab portions, may be prepared using the hybridoma technology described in Antibodies-A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, N.Y. (1988).
• any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include the hybridoma technique originally developed by Kohler et ah, Nature, 256:495-497 (1975), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today, 4:72 (1983)), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R. Liss, Inc., (1985)).
• Immortal, antibody-producing cell lines can be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al., "Hybridoma Techniques” (1980); Hammerling et al., “Monoclonal Antibodies and T-cell Hybridomas” (1981); Kennett etal., “Monoclonal Antibodies” (1980).
• a means of producing a hybridoma from which the monoclonal antibody is produced a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunised with a recognition factor-binding portion thereof, or recognition factor, or an origin-specific DNA-binding portion thereof.
• Hybridomas producing a monoclonal antibody useful in practicing this invention are identified by their ability to immunoreact with the present recognition factor and their ability to inhibit specified transcriptional activity in target cells.
• a monoclonal antibody useful in practicing the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity.
• the culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium.
• the antibody-containing medium is then collected.
• the antibody molecules can then be further isolated by well-known techniques.
• cyclic peptides of the invention there are various procedures known in the art which may be used for the production of polyclonal antibodies to cyclic peptides of the invention.
• various host animals can be immunized by injection with the cyclic peptide, including but not limited to rabbits, mice, rats, sheep, goats, etc.
• the cyclic peptides can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).
• BSA bovine serum albumin
• KLH keyhole limpet hemocyanin
• various adjuvants may be used to increase the immunological response, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminium hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
• Freund's complete and incomplete
• mineral gels such as aluminium hydroxide
• surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol
• BCG Bacille Calmette-Guerin
• Corynebacterium parvum bacille Calmette-Guerin
• Assays for immunospecific binding of antibodies may include, but are not limited to, radioimmunoassays, ELISAs (enzyme-linked immunosorbent assay), sandwich immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays, Western blots, precipitation reactions, agglutination assays, complement fixation assays, immunofluorescence assays, protein A assays, and Immunoelectrophoresis assays, and the like (see, for example, Ausubel etal., eds, 1994, Current Protocols in Molecular Biology, Vol.
• Antibody binding may be detected by virtue of a detectable label on the primary antibody.
• the antibody may be detected by virtue of its binding with a secondary antibody or reagent which is appropriately labelled.
• a variety of methods are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
• Antibodies of the present invention can be used in diagnostic methods and kits that are well known to those of ordinary skill in the art.
• An antibody raised against a cyclic peptide of the invention has binding affinity for the cyclic peptide.
• the antibody has binding affinity or avidity greater than about 10 5 M '1 , more preferably greater than about 10 6 IVI- 1 , more preferably still greater than about 10 7 M' 1 and most preferably greater than about 10 8 M- 1 .
• the antibody may be first separated by Protein A affinity chromatography and then treated with solvent/detergent to inactivate any lipid enveloped viruses. Further purification, typically by anion and cation exchange chromatography may be used to remove residual proteins, solvents/detergents and nucleic acids. The purified antibody may be further purified and formulated into 0.9% saline using gel filtration columns. The formulated bulk preparation may then be sterilised and viral filtered and dispensed.
• the present invention will now be described with reference to specific examples, which should not be construed as in any way limiting the scope of the invention.
• DMPG N-octyl- ⁇ -D-glucopyranoside
• Sigma St.Louis, MO, USA
• Pioneer Chips L1 were purchased from Biacore (Uppsala, Sweden).
• Peptides were synthesised by standard solid phase peptide synthesis methods, using side-chain protected Fmoc-conjugated amino acids in the manual mode.
• Fmoc-protected amino acids were purchased from Auspep or Novabiochem.
• the linear sequences were constructed using tritylchloride polystyrene resin (Novabiochem) loaded with the first protected amino acid.
• the addition of subsequent amino acids was achieved by first deprotecting the Fmoc group followed by coupling of the next amino acid.
• Example 1 Methods of cyclization and purification
• Method A (below) is a standard method while Methods B and C (below) have been developed to test for greater yield of product.
• Method B cyclization was carried out as per method A but a purification step was introduced before removing protecting groups.
• Method C uses a different cyclizing reagent as reported by Skropeta et al. Method A. Cyclization of C1 using PyBOP o The linear sequence of protected C1 was purchased from Auspep.
• C1 was dissolved in approximately 0.5ml of acetonitrile, 200 ⁇ l of 10% TFA and then DMSO was added until a clear solution was obtained. The sample was centrifuged prior to collecting the supernatant for loading on to a C18 reverse phase column. Sample was loaded using ACD techniques. C1 was loaded on to the column at 0.1ml/min flow rate. Simultaneously mobile phase was pumped through the column using another pump at a flow rate of 5ml/min. Presence of C1 in fraction collected at a retention time of 31.8-32.2min was confirmed by mass spectrometry (Figure 18).
• the cyclic peptides were tested in an antigen presentation assay to examine their effects on T cell stimulation.
• An established assay (Manolios et al., 1997) was used to assess T cell activation by measuring IL-2 produced in response to antigen. Briefly, 2B4.11 hybridoma (5 ⁇ 10 cells) and LK35.2 (5x10 ) were incubated with pigeon cytochrome C (50 ⁇ M) in 96-well microtiter plates for 24 h in the presence and absence of peptides (50, 25, and 10 ⁇ M respectively). An aliquot of each supernatant (100 ⁇ l) was removed and serial twofold dilutions prepared using medium lacking IL-2.
• C1 and C2 inhibited IL-2 production appreciably in comparison to a solvent only (DMSO) control.
• C1 inhibited IL-2 production to only 7.73% ⁇ 4.97SD of the solvent only control value, which decreased to 59.69% ⁇ 23.54SD of the solvent only control value upon lowering the C1 concentration to 25 ⁇ M.
• C2 was the second most active peptide with IL-2 production dropping to 24.51% + 8.81SD of the control when tested at 50 ⁇ M, and 58.74% ⁇ 7.63SD IL-2 produced when tested at 25 ⁇ M.
• C3 and C4 showed only minimal activity in this assay at 50 ⁇ M, resulting in 91.69% ⁇ 25.86SD and 74.54% ⁇ 11.22SD respectively.
• CP normally inhibits IL-2 production to 37.08% ⁇ 10.49SD at equivalent doses (50 ⁇ M).
• Uncyclized C1 (C1-L) also showed only minimal activity at 50 ⁇ M, Table 1. Pe tide list.
• a cyclic peptide may have an Arginine in position 2 and a Lysine in position 8. Active cyclic peptides may also include an indeterminate number of hydrophobic amino acids between R and K. Furthermore, active cyclic polypeptides with ano indeterminate number of charged amino acids between R and K may retain an angle between K and R of 36-140 degrees.
• 2B4.11 T-cells (1 x 10 5 ) weres cultured in microtiter wells in the presence of the peptide conjugates at 50 ⁇ M. After 20 hours,
• T-cells were activated using 2C11-145 and H57-
• the H57-597 antibody is directed against the TCR ⁇ chain, and whens administered at sub-optimal doses in conjunction with accessory cells (LK35.2), is capable of activating T cells.
• T-cell hybridoma 2B4.11 cells (1 x 10 5 ) were cultured in 96-well, round bottom plates in the presence of LK cells (5 x 10 4 ), 0.5 ⁇ g/mL of either soluble 145-2C11 (lab purified) to crosslink the CD3 ⁇ chains of the TCR, or H57-597 (Biolab) to crosslink the TCR ⁇ chain.
• C1 was added to give a final reaction volume of 250 ⁇ l_ at 50 ⁇ M and 0.5% DMSO.
• T cells were also activated using the 2C11-145 antibody to crosslink the CD3 chains of the TCR, thereby bypassing the antigen recognition sub-units of the TCR ⁇ / ⁇ .
• CD3 crosslinking in the presence of C1 resulted in a %IL-2 production of 50.00% +/- 15.79SD (see Figure 7).
• T-cells were also activated using Staphylococcal enterotoxin A (SEA).
• SEA Staphylococcal enterotoxin A
• SEA is capable of activating T-cells through the TCR.
• the MHC molecule of the macrophage (LK35.2) is crosslinked to the TCR ⁇ subunit resulting in T-cell activation.
• the T- cell hybridoma, 2B4.11 (5 x 10 4 CeIIs) were incubated with an equal amount of the macrophage, LK35.2 (5 x 10 4 cells), at 100ng/mL of SEA, 50 ⁇ M of C1, and 0.5% DMSO to a total volume of 250 ⁇ L. Stimulation was allowed to proceed for 24 hours at 37°C / 5% CO2 in 96-well in round bottom microtiter plates. Supernatants were then collected and analysed for IL-2 production. C1 inhibited IL-2 production to 71.42% +/- 14.27SD using the SEA model of T-cell activation (see Figure 7). The results for other peptides are shown in Table 4.
• T-cells were activated using PMA and ionomycin stimulation.
• the method employed to activate T cells utilized the protein kinase C activating PMA in combination with the calcium ionophore, ionomycin.
• PMA/ionomycin effectively activate T cells downstream from the TCR.
• Phorbol 12-myristate 13-acetate (PMA) (40ng/mL) and ionomycin (5 ⁇ g/mL) were added to 2B4 cells (5 x 10 4 ) and incubated with each CP-conjugate at 50 ⁇ M in 0.5% DMSO in 96-well, round bottom microtiter plates for 24 h at 37°C with 5%CO2 in a total volume of 250 ⁇ L.
• each IL-2 column represents individual experiments performed in triplicate. The values show the average amount of IL-2 produced (%) relative to DMSO control (100%). That is, the smaller the values the more inhibition (effectiveness).
• C1 in vivo was compared to commercially available cyclosporin.
• C1 given subcutaneously had a comparable effect to cyclosporin in reducing inflammation in the adjuvant induced arthritis model. This was more significantly favourable when compared with CP or placebo treated rats.
• the difference in swelling observed between C1 treated rats and those treated with the placebo was statistically significant (P ⁇ 0.0001), whereas the difference between C1 and cyclosporine treated rats was not statistically significant.
• Samples containing 25 ⁇ mol of lipid were prepared by dissolving the dry lipids in 500 ⁇ L of methanol/chloroform (1/1 v/v), followed by addition of the appropriate amount of peptide in 500 ⁇ L of trifluoroethanol. Subsequently, the solvents were removed by drying in a rotary evaporator followed by overnight incubation under high vacuum. The peptide/lipid mixed films obtained were hydrated with 150 ⁇ L of distilled water, and the resulting dispersions of multilamellar vesicles were subjected to 10 freeze-thaw cycles to ensure that the samples were homogeneous.
• NMR spectra of the peptide-lipid dispersions were recorded at 34 0 C on a Varian (Palo Alto, CA) Inova 300 wide-bore NMR spectrometer, using a broadband probe with a 5 mm sample coil.
• Proton-decoupled 31 P experiments were carried out at 121.46 MHz (4 ⁇ s 90° pulse, 1.5 s recycle time. Approximately 10,000 scans were acquired using a sweep width of 25 kHz and 1024 complex data points. Prior to Fourier transformation an exponential multiplication was applied, resulting in a 100 Hz line broadening.
• 2 H NMR measurements were performed at 46.06 MHz, using a quadrupolar echo sequence (Davis et a/., 1976) (8 ⁇ s 90° pulse, 30 ⁇ s pulse separation), a recycle time of 0.5 s, and a spectral width of 100 kHz, with 4000 complex data points in the time domain. Approximately 60,000 scans were accumulated. The free induction decays were left-shifted to begin at the top of the echo and multiplied with an exponential window function equivalent to a line broadening of 50 Hz. All spectra were scaled to the same height. 3 1 P and 2 H NMR measurements were performed to investigate whether the C1 peptide is capable of perturbing model membrane structure.
• the conformation of the lipid acyl chains were then assessed by 2 H NMR measurements on membranes that contain lipids with perdeuterated acyl chains.
• the 2 H NMR spectrum of a dispersion of DMPC-cfe4 /DMPG in the absence and presence of peptide is presented in Figure 6.
• the spectrum consists mostly of overlapping doublet resonances that resulted from the different CD2 segments of the acyl chain, while the central high-intensity doublet corresponds to the terminal CD3 moiety.
• the above solid state NMR studies of C1 at 10% concentration in DMPC/DMPG membranes show that the peptide appeared to order the lipid suggesting a transmembrane orientation. Based on these observations, although C1 appears to enter membranes and adopt a transmembrane orientation, it does not appear likely that it is forming lethal pores.
• SPR is a useful method in the study of peptide binding and possible insertion into membranes.
• Successful applications include studies of antimicrobial peptides and their mode of action such as melittin, its analogues and magainin and to understand the mode of action of membrane-active peptides using anionic and zwitterionic lipids.
• the technique of SPR offers the ability to study structure/function relationships while overcoming the need to use labelled peptides as well as cumbersome separation procedures (see Salamon et al., 1999). It allows real time monitoring of peptide binding to, and dissociation from, these model membranes.
• Model membranes composed of zwitterionic and anionic phospholipids were prepared as previously described (Bender et al., 2004). Briefly, DMPC and DMPG were dissolved in dry chloroform and chloroform/ methanol (2:1) respectively to give 10 mg/ml solutions. These were evaporated under reduced pressure and the resulting lipid films dried overnight in vacuo. Lipids were hydrated by resuspending in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer for 60 min at 34 0 C to give 0.5mM concentration in respect of phospholipids. The solution was sonicated in an ultrasonic bath for 20 min.
• HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
• HEPES HBS-N, Biacore, Upssala, Sweden
• the chip surface was cleaned with 40 mM octyl glycoside (30 ⁇ l, 10 ⁇ l/min) followed liposomes (SUV) (100 ⁇ l, 5 ⁇ l/min).
• 1OmM NaOH 40 ⁇ l, 10 ⁇ l/min
• the dissociation stage was 1200 sec. Regeneration of the sensor chip was achieved with 4OmM octyl glycoside (30 ⁇ l, 10 ⁇ l/min).
• the analogues C3 and C4 showed no binding at all to either of the model membranes and the uncyclized C1 (C1-L) only very limited binding. Sensorgrams of C2 showed that there was some binding to the anionic liposome (3000 RU) but only very limited binding to the zwitterionic (data not shown). With the exception of C2, C1 demonstrated in excess of 10-fold binding to all the other analogues examined.
• the inventors have recently reported that there is a strong correlation between the membrane binding affinity and functional activity of CP and its linear analogues in inhibiting IL-2 production (Bender et al., 2004). Here it is shown that this correlation also holds true for C1.
• C1 has a higher binding affinity to model membranes than that of CP and also exhibits a stronger biological activity.
• Transmission Electron Microscopy was used to identify if C1 formed nanotubes.
• a suspension of C1 in water, 90%acetonitrile: 10% water, methanol, water/DMSO and water/HCI were prepared. These suspensions were made at a range of concentrations (6 ⁇ M-2mM). None of the suspensions, except for water/HCI indicated any tube like structures shown in Figure 12. Further analysis is currently underway to assess if these tube-like structures are nanotubes.
• C1 in water/HCI sample was prepared as follows: C1 (1.5mg) was suspended in 100 ⁇ l of water and 50 ⁇ l of 1OmM HCI to make an approximately 9mM stock solution. The stock solution was then diluted to a final concentration of 100 ⁇ M using water.
• the synthetic C1 peptide and its analogues were tested using Standard disk susceptibility testing by National Committee for Calibration Laboratory Standards (NCCLS) against, S. aureus, E. coli and S. pneumoniae. From all the cyclic peptides tested only C1 showed antimicrobial activity against S .aureus and the MIC was determined to be 100 ⁇ g/ml.
• NCCLS National Committee for Calibration Laboratory Standards
• mice The effect of administering an immunosuppressive peptide on the development of allergic airways disease in a mouse model was determined using female Balb/c WT mice at 7 weeks old housed in IVC cages.
• a 400 ⁇ g intraperitoneal dose of cyclic C1 peptide results in death of mice 50
• 100 or 200 ⁇ g doses of cyclic C1 were administered to groups of mice either subcutaneously or intraperitoneally according to the design set out below.
• mice Following the initial cyclic C1 peptide intraperitoneal injection mice were continuously monitored. Mice in group 3 were lying on the bottom of the cage within 10-15 minutes and their breathing rate had slowed. These animals were not moving around or grooming as mice usually do. The eyes of a few of these mice also appeared darker. In group 3, one mouse's tail began to appear blue and this animal was euthanased immediately and the remainder of the group were also euthanased (total of 4 mice) as by this stage most had begun to exhibit signs of distress (approximately 1 hour and 15 minutes after the initial injection). Mice in groups 1 & 2 appeared slightly distressed (scruffy and hunched) for the first 60 minutes however showed no further signs of distress were observed such as those observed with group 3. By 1 hour and 15 minutes after injection the mice in groups 1 and 2 mice appeared to be improving
• mice in groups 1 & 2 appeared slightly scruffy and hunched following the injection but fully recovered by 1 hour. Mice in groups 4-6 did not show any adverse effects.
• mice did not tolerate 200 ⁇ g cyclic C1 delivered by intraperitoneal injection.
• a 200 ⁇ g dose of cyclic C1 delivered by subcutaneous injection did not cause any adverse effects and was used in further studies (see below).
• Example 9 Subcutaneous Administration of C1 in OVA model
• OVA ovalbuproliferative factor
• mice were euthanased and BALF was obtained by cannulating the trachea and flushing the airways with two 0.8 ml volumes of Hank's Balanced Salt Solution (HBSS).
• HBSS Hank's Balanced Salt Solution
• Recovered cells were treated with erythrocyte lysis buffer, cytospins prepared and stained with
• May-Grunwald Giemsa and differential leukocyte counts performed based on morphological criteria (min 200 cells counted/slide). Statistical analysis was performed using a student's unpaired t-test. Results were compared to historical data.
• the endpoints of this study include Bronchoalveolar lavage inflammatory infiltrates, Local (peribronchial lymph node) and systemic (spleen) antigen specific cytokine production (IL-5 and IL-13) and Airway hyperreactivity.
• BALF was obtained by cannulating the trachea and flushing the airways with two 0.8 ml volumes of Hank's Balanced Salt Solution (HBSS). Recovered cells were treated with erythrocyte lysis buffer, cytospins prepared and stained with May-Grunwald Giemsa and differential leukocyte counts performed based on morphological criteria (min 200 cells counted/slide). Statistical analysis was performed using a student's unpaired t-test.
• HBSS Hank's Balanced Salt Solution
• Peribronchial lymph nodes (PBLN) and spleens were filtered through 70 ⁇ m nylon mesh, treated with erythrocyte lysis buffer and cultured at 37°C/5% CO2 in 96-well plates at 1 x 10 6 cells/well in animal cell culture medium (ACCM; 0.1 mM sodium pyruvate, 2 mM L-glutamine, 20 mM HEPES, 100 U/ml penicillin/streptomycin, 50 ⁇ M 2-ME and 10% FBS in RPMH 640). Cells were either unstimulated (media only), or antigen-stimulated (200 ⁇ g/ml OVA).
• IL-5 BD Pharmingen
• IL-13 R&D Systems
• Airway hyperreactivity to inhaled-methacholine representative of the large (Transpulmonary resistance, RL) and small (dynamic compliance, Cdyn) airways was determined.
• Animals were anesthetized by intraperitoneal injection of ketamine-xylazine and tracheostomised with insertion of a polyethylene cannula (i.d. 0.813 mm).
• the tracheal tube was connected to a ventilation port within the plethysmograph chamber, and this port was connected to a rodent ventilator (HSE Minivent Type 845, Hugo Sachs Elektronik, Harvard, Germany). Mice were mechanically ventilated at a rate of 170 breaths per minute with a stroke volume of 175 ⁇ l.
• mice were challenged with saline, followed by increasing concentrations of methacholine (0.625, 1.25, 2.5, 5.0 and 10.0 mg/ml). Aerosols were generated with an ultrasonic nebuliser (Buxco, Aeroneb Laboratory Nebulizer) and delivered to the inspiratory line. Each aerosol was delivered for a period of 5 minutes, during which pressure and flow data were continuously recorded, and specialist software (BioSystemXA, Buxco Electronics, Inc.) was used to calculate pulmonary resistance and compliance. Peak values were taken as the maximum response to the concentration of methacholine being tested, and were expressed as the percentage change over the saline control. Statistical analysis was performed using a two-way ANOVA.
• Table 7 is a summary of the effect of peptide administration on a spectrum of parameters associated with allergic airway disease. Mice were treated with linear C1 peptide, cyclic C1 peptide, core peptide or methylprednisolone subcutaneously during OVA challenge. Statistically significant differences compared to mice given OVA/OVA alone ('allergic mice') are summarised.
• Example 12 Treatment of NOD mice with C1 peptide NOD mice were fasted for at least 1.5 h prior to blood glucose level (BGL) measurements.
• mice were treated with intraperitoneal (i.p.) injections of 0.1 mg C1 and BGLs were taken. Control mice received 0.2 mL of water. Any mice that displayed a BGL of higher than
• mice 5 One other mouse (mouse 5) developed diabetes.
• mice 7/21 mice developed diabetes.
• Molecules and agents of the present invention may be used for the treatment or prevention of various disease states and conditions. Such molecules and agents may be administered alone, although it is more typical that they be administered as a pharmaceutical composition.
• a composition for intramuscular injection could be prepared to contain 1 ml sterile buffered water, and 1 mg of a suitable agent or molecule.
• composition for intravenous infusion may comprise 250 ml of sterile Ringer's solution, and 5 mg of a suitable agent or molecule.
• a composition suitable for administration by injection may also be prepared by mixing 1% by weight of a suitable agent or molecule in 10% by volume propylene glycol and water. The solution is sterilised by filtration.
• Example 13(b) - Capsule Composition A composition of a suitable agent or molecule in the form of a capsule may be prepared by filling a standard two-piece hard gelatin capsule with 50 mg of the agent or molecule, in powdered form, 100 mg of lactose, 35 mg of talc and 10 mg of magnesium stearate.
• Example 13(c) - Eye Drop Composition A typical composition for delivery as an eye drop is outlined below: Suitable agent or compound 0.3 g
• methyl and propyl hydroxybenzoates are dissolved in 70 ml purified water at 75 0 C, and the resulting solution is allowed to cool.
• the suitable agent or molecule is then added, and the solution sterilised by filtration through a membrane filter (0.22 ⁇ m pore size), and aseptically packed into sterile containers.
• Example 13(d) - Composition for Inhalation Administration For an aerosol container with a capacity of 20-30 ml: a mixture of 10 mg of a suitable agent or compound with 0.5-0.8% by weight of a lubricating agent, such as polysorbate 85 or oleic acid, is dispersed in a propellant, such as freon, and put into an appropriate aerosol container for either intranasal or oral inhalation administration.
• a suitable agent or compound with 0.5-0.8% by weight of a lubricating agent such as polysorbate 85 or oleic acid
• a propellant such as freon
• a typical composition for delivery as an ointment includes 1.0g of a suitable agent or molecule, together with white soft paraffin to 100.0 g, dispersed to produce a smooth, homogeneous product.
• Example 13(f) - Topical Cream Composition A typical composition for delivery as a topical cream is outlined below: Suitable agent or molecule 1.0 g Polawax GP 200 25.O g
• the polawax, beeswax and lanolin are heated together at 6O 0 C, a solution of methyl hydroxybenzoate is added and homogenisation achieved using high speed stirring. The temperature is then allowed to fall to 5O 0 C. The agent or molecule is then added and dispersed throughout, and the composition is allowed to cool with slow speed stirring.

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Medicinal Chemistry (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Public Health (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Animal Behavior & Ethology (AREA)
• Veterinary Medicine (AREA)
• Pharmacology & Pharmacy (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Engineering & Computer Science (AREA)
• Diabetes (AREA)
• Pulmonology (AREA)
• Dermatology (AREA)
• Immunology (AREA)
• Endocrinology (AREA)
• Communicable Diseases (AREA)
• Biochemistry (AREA)
• Cardiology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Molecular Biology (AREA)
• Heart & Thoracic Surgery (AREA)
• Genetics & Genomics (AREA)
• Obesity (AREA)
• Oncology (AREA)
• Biophysics (AREA)
• Emergency Medicine (AREA)
• Hematology (AREA)
• Neurology (AREA)
• Otolaryngology (AREA)
• Neurosurgery (AREA)
• Biomedical Technology (AREA)
• Transplantation (AREA)
• Physical Education & Sports Medicine (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Peptides Or Proteins (AREA)

Priority Applications (4)

Applications Claiming Priority (2)

Publications (2)

Family

ID=42197769

Family Applications (1)

Country Status (5)

Cited By (2)

Families Citing this family (3)

Citations (1)

Family Cites Families (3)

• 2009
  • 2009-11-24 EP EP09827064A patent/EP2358740A4/en not_active Withdrawn
  • 2009-11-24 JP JP2011536707A patent/JP2012509848A/ja active Pending
  • 2009-11-24 AU AU2009317889A patent/AU2009317889A1/en not_active Abandoned
  • 2009-11-24 WO PCT/AU2009/001530 patent/WO2010057275A1/en active Application Filing
  • 2009-11-24 US US13/130,718 patent/US20120077732A1/en not_active Abandoned

Patent Citations (1)

Non-Patent Citations (3)

Also Published As

Similar Documents

Legal Events