WO1999015561A1 - Inhibiteurs des hematopoietines interleukine-3, interleukine-5 et facteur stimulant la proliferation des macrophages/granulocytes, et utilisation de ces inhibiteurs - Google Patents

Inhibiteurs des hematopoietines interleukine-3, interleukine-5 et facteur stimulant la proliferation des macrophages/granulocytes, et utilisation de ces inhibiteurs Download PDF

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WO1999015561A1
WO1999015561A1 PCT/SE1998/001687 SE9801687W WO9915561A1 WO 1999015561 A1 WO1999015561 A1 WO 1999015561A1 SE 9801687 W SE9801687 W SE 9801687W WO 9915561 A1 WO9915561 A1 WO 9915561A1
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peptide
lyn
peptide according
amino acid
peptides
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PCT/SE1998/001687
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English (en)
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Rafeul Alam
Tetsuya Adachi
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Astra Aktiebolag
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Priority to AU92890/98A priority Critical patent/AU9289098A/en
Publication of WO1999015561A1 publication Critical patent/WO1999015561A1/fr

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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • 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 peptide inhibitors of the haematopoietins: interleukin-3 (IL-3), interleukin-5 (IL-5) and granulocyte/macrophage colony-stimulating factor (GM- CSF) and to the use of such inhibitors in medical therapy.
  • IL-3 interleukin-3
  • IL-5 interleukin-5
  • GM- CSF granulocyte/macrophage colony-stimulating factor
  • receptors e.g. beta-adrenergic receptor agonists or antagonists
  • Such receptors are frequently coupled to multiple signalling pathways leading to different cellular functions.
  • receptor antagonists indiscriminately inhibit all receptor-associated functions. This type of complete receptor blockade is not always desirable since it frequently results in adverse side effects. Ideally, one would like to block only an unwanted signal from a receptor without interfering with other important signalling processes.
  • Interleukin-3 (IL-3), interleukin-5 (B -5) and granulocyte/macrophage colony-stimulating factor (GM-CSF) are three important growth and differentiation factors for myeloid progenitors.
  • EL-3 is the pluripotent stem cell growth factor.
  • GM-CSF promotes the growth and differentiation of granulocytes and macrophages whereas IL-5 stimulates the differentiation of eosinophils and basophils.
  • cytokines have a specific ⁇ receptor but they share a common ⁇ receptor ( ⁇ c).
  • ⁇ c receptor is the principal signalling receptor for these three cytokines and consequently all three haematopoietins have significant functional overlap, especially, with regard to their activity on eosinophils.
  • Asthma and allergic diseases are very common. Approximately 20% of the population has an allergic disease and 4% has asthma. In 1990, the cost of asthma medication alone was about $1.1 billion.
  • the symptomatic drugs such as beta-agonists, anti-cholinergics and methylxanthines do not inhibit eosinophilic inflammation.
  • the anti-inflammatory drugs for asthma include cromolyn sodium and corticosteroids. Cromolyn sodium is effective in only about 30% of asthmatic patients. Corticosteroids are a highly effective medicine for asthma but have many side effects. For these reasons, there is an intense effort in the industry to develop novel drugs for asthma that block eosinophilic inflammation.
  • the prior art is deficient in the lack of effective means of inhibiting the haematopoietins IL-3, IL-5 and GM-CSF.
  • the present invention fulfils this long-standing need and desire in the art.
  • a peptide inhibitor of the haematopoietins IL-3, IL-5 and GM-CSF signalling wherein said peptide inhibits the activation Lyn tyrosine kinase and thereby, blocks signal transduction of the haematopoietin ⁇ c receptor common to IL-3, IL-5 and GM-CSF
  • a pharmaceutical composition comprising a peptide inhibitor of the present invention and a pharmaceutically acceptable carrier.
  • a method of treating a condition involving increased production and function of eosinophils and other granulocytes comprising the step of administering a pharmacologically effective dose of the pharmaceutical composition of present invention.
  • the present invention is directed to a peptide inhibitor of the haematopoietins IL-3, IL-5 and GM-CSF signalling, wherein said peptide inhibits the activation of the Lyn tyrosine kinase and blocks signalling via the haematopoietin ⁇ c receptor common to IL-3, IL-5 and GM-CSF.
  • the peptide is an N-terminally modified peptide. More preferably, the peptide is modified by N-stearation.
  • the strategy described herein is to map the cytosolic region of the receptor for specific signal generating sites (e.g. tyrosine kinase binding sites) and design small peptides based upon the sequence information of the signal-generating site.
  • the next step is to modify the peptides in order to enable their cellular intemalisation and to improve their in vivo stability.
  • the modified peptides should function to block targeted cellular functions.
  • Peptide inhibitors of the present invention may inhibit signalling of the haematopoietins IL- 3, IL-5 and GM-CSF in any of several ways, including inhibition of tyrosine phosphorylation, blocking the eosinophil survival promoting effects of E -5, inhibiting GM-CSF-stimulated proliferation of GM-CSF-dependent cells, inhibiting the growth and differentiation of eosinophils from bone marrow cells.
  • the peptide inhibitor of the present invention is useful in the treatment of conditions involving increased production and function of eosinophils and other granulocytes.
  • Representative examples of such conditions include asthma, allergic rhinitis, allergic conjunctivitis, idiopathic eosinophilic syndrome, eosinophilic pneumonia, allergic bronchopulmonary aspergillosis, Churg-Strauss syndrome, eosinophilic gastritis, Loeffler's syndrome and myelopoietic abnormalities such as myelodysplastic syndromes.
  • the peptide inhibitor corresponds to amino acid residues 450-465 of the ⁇ c receptor.
  • a particularly preferred peptide of this embodiment may have the sequence: stearate-YGYRLRRKWEEKIPNP-NH2.
  • the peptide inhibitor corresponds to amino acid residues 462-481 of the ⁇ c receptor.
  • a particularly preferred peptide of this embodiment may have the sequence: stearate-IPNPSKSHLFQNGSAELWPP-NH2.
  • the peptide inhibitor may comprise a D-basic amino acid residue at the end of the peptide; this is preferably the amino acid D-Arg
  • Figure 1 shows the mapping of the Lyn kinase binding site of ⁇ c receptor.
  • a previous publication indicated that the membrane proximal residues 450-517 of the ⁇ c receptor is critical for activation of Lyn, Fes and Jak2 kinases.
  • four overlapping and/or sequential peptides ⁇ c 450-465, ⁇ c 456-470, ⁇ c 462-481, ⁇ c 482-498 derived from this region were synthesised.
  • the peptides were biotinylated.
  • the ⁇ c 605-624 (pY612) peptide was used as a positive control because it binds to the SH2 domain of Lyn.
  • Lyn kinase exists in two molecular weight forms: p53 and p56.
  • the left lane containing the unprocessed cell lysate shows the position of the p53/p56 Lyn kinase.
  • Three overlapping peptides ( ⁇ c 450-465, ⁇ c 456-470, ⁇ bc 462-481), but not ⁇ c 482-498, FceRIb 29-48 and gpl30 658-677 peptides, bound to Lyn kinase.
  • Figure 2 shows the specific binding of ⁇ c 462-481 peptide to Lyn kinase.
  • TF-1 cell lysates were precleared with avidin-conjugated agarose and then incubated with or without the biotinylated ⁇ c 462-481 peptide or a mutated ⁇ c 462-481 peptide with Pro — > Ala substitutions. After incubation for 2 hours, the avidin-agarose conjugates were added and the bound proteins were separated. The proteins were electrophoresed and immunoblotted with the anti-Lyn antibody. The mutated ⁇ c 462-481 peptide did not bind to Lyn kinase.
  • FIG. 3 shows the effect of the stearated peptide on GM-CSF-induced H-thymidine incorporation.
  • TF-1 cells (a) or THP-1 cells (b) (105 cells/ml) were incubated with or without the N-stearated be 462-481 peptide for 3 hours.
  • the biotinylated ⁇ c 462-481 and stearated-IL-5a 316-335 peptides were used as controls.
  • the cells were further incubated with 5% FCS and GM-CSF (1 ng/ml) for 96 hours. During the last 6 hour incubation, 5
  • FIG. 4 shows the effect of the N-stearated ⁇ c 462-481 peptide on GM-CSF-induced tyrosine phosphorylation of proteins in eosinophils. Eosinophils (2 x 106 cells/ml) were incubated in the presence or absence of the N-stearated ⁇ c 462-481 peptide for 3 hours and then stimulated with or without GM-CSF (10 ng/ml) for 5 minutes. The biotinylated ⁇ c 462-481 and stearated-IL-5a 316-335 peptides were used as controls.
  • the lysates were subjected to electrophoresis and western blotting with anti- phosphotyrosine antibody.
  • the N-stearated ⁇ c 462-481 peptide inhibited GM-CSF- induced tyrosine phosphorylation of cellular proteins in eosinophils in a dose-dependent manner.
  • Figure 5 shows the effect of the N-stearated ⁇ c 462-481 peptide on GM-CSF-induced tyrosine phosphorylation of Lyn in eosinophils.
  • Eosinophils (5 x 106 cells/ml) were incubated in the presence or absence of the N-stearated ⁇ c 462-481, biotinylated ⁇ c 462- 481 and stearated-IL-5 ⁇ 316-335 peptides for 3 hours followed by the stimulation with or without GM-CSF (10 ng/ml) for 5 minutes.
  • the lysates of esinophils were immunoprecipitated with anti-Lyn antibody.
  • the immunocomplex was subjected to electrophoresis and western blotting with anti-phosphotyrosine antibody. Lyn activation after GM-CSF stimulation was significantly inhibited in the presence of the N-stearated ⁇ c 462-481 peptide.
  • FIG. 6 shows the results of eosinophil survival assay.
  • Eosinophils 106 cells/ml
  • the biotinylated ⁇ c 462-481 and stearated-IL-5 ⁇ 316-335 peptides were used as controls.
  • the cells were further cultured with or without GM-CSF ( 1 ng/ml) or (b) IL-5 ( 1 ng/ml) for 72 hours, and then the viability of eosinophils was counted.
  • Figure 7 shows the effect of the stearated peptide on ECP release from eosinophils. Eosinophils (106 cells/ml) were incubated with or without the N-stearated ⁇ c 462-481 peptide for 3 hours. The biotinylated ⁇ c 462-481 and stearated-IL-5 ⁇ 316-335 peptides were used as controls.
  • FIG 8 shows the binding of SHPTP2 (SHP-2) to a phosphotyrosine based ITIM motif- containing peptide (peptide 1) derived from IL-5 ⁇ cR (residues 605-624).
  • the controls were a nonphosphorylated F612 peptide (peptide 2) and a ⁇ c450-465 peptide (peptide 3).
  • the peptides were biotinylated and used in a peptide binding assay using the HL-60 cell lysate.
  • the bound proteins were precipitated with streptavidin-agarose followed by immunoblotting with anti-SHPTP2.
  • the first lane, (L: cell lysate) shows the position of the 70 kD SHPTP2.
  • the identity of the upper band is unknown.
  • the buffer control is shown in the last lane (0).
  • SHPTP2 binds to the phosphorylated ⁇ c 605-624 but not to the ⁇ c 450-465 or mutated
  • Figure 9 shows the effect of the ⁇ c 462-481 peptide on Jak2 kinase activation.
  • TF-1 cells (5 x 106 cells/ml) were incubated in the presence or absence of the N-stearated ⁇ c 462-481, biotinylated ⁇ c 462-481 and stearated-IL-5 ⁇ 316-335 peptides for 3 hours followed by the stimulation with or without GM-CSF (10 ng/ml) for 5 minutes.
  • the cell lysates were immunoprecipitated with an anti-Jak2 antibody.
  • the immune-complex was subjected to electrophoresis and western blotting with an anti-phosphotyrosine antibody.
  • the Jak2 activation after GM-CSF stimulation was not inhibited by the N-stearated ⁇ c 462-481 peptide.
  • Figure 10 shows the effect of the ⁇ c 462-481 peptide on activation of ERK2 MAP kinase by GM-CSF.
  • TF- 1 cells were incubated with or without the N-stearated ⁇ c 462-481 peptide for 3 hours. The cells were then stimulated with GM-CSF (10 ng/ml) for 5 minutes. The cell lysate was immunoprecipitated with an anti-ERK2 antibody. The immune complex was separated, electrophoresed and western blotted with anti- phosphotyrosine antibody (left panel). GM-CSF stimulated tyrosine phosphorylation of ERK2 MAP kinase which was not affected by pre-treatment with the N-stearated peptide. The membrane was stripped and reprobed with the anti-ERK2 antibody which shows equal amount of the ERK protein in each lane (right panel).
  • Figure 11 shows the effect of the ⁇ c 462-481 peptide on anti-IgE-induced histamine release from basophils.
  • FIG. 12 shows the effect of the stearated peptide on H-thymidine incorporation of T cells (a) and B cells (b).
  • Purified T cells and B cells (5 x 106 cells/ml) were incubated with or without the N-stearated ⁇ c 450-465 peptide for 3 hours in the presence of 10%
  • T cells were stimulated with Con A (5 mg/ml), whereas the B cells were stimulated with immobilised anti-IgM antibody (10 (g/ml). The cells were further
  • ECP or eosinophil cationic protein shall refer to a granular protein of eosinophilic leukocytes.
  • GM-CSF or granulocyte-macrophage colony-stimulating factor shall refer to a growth factor for stem cells.
  • haematopoietins shall refer to cytokines that stimulate the growth of blood cells from bone marrow cells.
  • IL or interleukin shall refer to specific cytokines that are proteins or glycoproteins made by leukocytes and other cells and modulate the function of a variety of cells.
  • Lyn and Jak2 shall refer to tyrosine kinases that catalyse the phosphorylation of tyrosine residues on target proteins.
  • MAP kinase mitogen-activated protein kinase
  • Ras shall refer to a small GTP binding protein that activates the MAP kinase pathway.
  • SH2 domain or "src homology domain” shall refer to a structural motif that binds to proline-rich residues.
  • SH3 or “src homology” shall refer to domain or structural motif that binds to phosphorylated tyrosine residues.
  • SHP-2/Syp/SHPTP2 shall refer to a tyrosine phosphatase that dephosphorylates tyrosine residues of target proteins.
  • TF-1 shall refer to a cell line that is dependent upon GM-CSF for its continuous growth.
  • the term 'variant' shall mean a peptide having a deletion, substitution addition or chemical modification of one or more amino acids such that it has an ability to inhibit the activity of IL-3, IL-5 or GM-CSF of not less than 50% of that of the corresponding non-variant peptide which has the same amino acid sequence as the variant peptide but for that deletion, substitution, addition or chemical modification. More preferably the variant has 60%,70%,80%,90%,100%,110%, of the inhibitory activity of the non- variant and most preferably, greater than 120%.
  • compositions may be prepared using the peptide inhibitors of the present invention.
  • the pharmaceutical composition comprises the peptide inhibitors of the present invention and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier e.g., a pharmaceutically acceptable carrier.
  • the peptide inhibitors of the present invention are administered to the human patient or other mammal in therapeutically effective amounts, i.e., amounts that effectively inhibit function of the haematopoietins. They will normally be administered parenterally, preferably intravenously, but other routes of administration will be used as appropriate.
  • the dose and dosage regimen will depend upon the nature of the disease, the characteristics of the particular inhibitor, e.g., its therapeutic index, the patient, the patient's history and other factors.
  • the amount of the peptide inhibitor administered will typically be in the range of about 1 to about 100 mg/kg of patient weight.
  • the schedule will be continued to optimise effectiveness while balanced against negative effects of treatment. See Remington's Pharmaceutical Science, 17th Ed. (1990) Mark Publishing Co., Easton, Penn.; and Goodman and Gilman's: The Pharmacological Basis of Therapeutics 8th Ed (1990) Pergamon Press; which are incorporated herein by reference.
  • the peptide inhibitors will most typically be formulated in a unit dosage injectable form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle.
  • a pharmaceutically acceptable parenteral vehicle are preferably non-toxic and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as fixed oils and ethyl oleate may also be used. Liposomes may be used as carriers.
  • the vehicle may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives.
  • IL-5 is the single most important cytokine that regulates growth, differentiation and function of eosinophils.
  • the present invention develops inhibitors of IL-5 as well as other haematopoietins (IL-3 and GM-CSF).
  • Critical signalling molecules were identified that are involved in eosinophil growth and differentiation.
  • Lyn tyrosine kinase is an important signalling molecule. The Lyn-binding site was mapped on ⁇ c receptor which is common to EL-3, EL-5 and GM-CSF. Peptides encompassing the Lyn-binding site were used to interfere with IL-5 and GM-CSF signalling.
  • Two N-terminally modified peptides blocked Lyn kinase activation by the haematopoietins. Further, they inhibited tyrosine phosphorylation of many other signalling molecules.
  • the peptides blocked the eosinophil survival promoting effects of IL-5 and inhibited GM-CSF-stimulated proliferation of factor-dependent cell lines but not autonomous tumour cell lines. Finally, the peptides inhibited the growth and differentiation of eosinophils from bone marrow cells. Based upon these observations, these peptides are useful in the treatment of diseases wherein eosinophils play an important role.
  • the present invention is the first mapping of the Lyn-binding site of ⁇ c receptor.
  • the Lyn- binding peptide was modified by N-stearation.
  • the modified peptides inhibit eosinophil growth, differentiation and survival in a specific manner.
  • the peptides may be used to treat allergic and other eosinophilic inflammatory conditions.
  • the inhibitors described herein are useful for treatment of diseases involving increased production and function of eosinophils and other granulocytes.
  • Diseases with increased production and function of eosinophils asthma, allergic rhinitis, allergic conjunctivitis, idiopathic eosinophilic syndrome, eosinophilic pneumonia, allergic bronchopulmonary aspergillosis, Churg-Strauss syndrome, eosinophilic gastritis, Loeffler's syndrome.
  • Diseases with myelopoietic abnormalities include myelodysplastic syndromes.
  • ⁇ c peptides that bind to Lyn kinase in vitro were designed.
  • the peptides was modified by N-stearation and used to inhibit IL-5 and GM-CSF activity.
  • the structure of the peptides are as follows:
  • peptide ⁇ c 450-465 Stearate-YGYRLRRKWEEKIPNP-NH2 peptide ⁇ c 462-481 : Stearate-IPNPSKSHLFQNGS AELWPP-NH2
  • a peptide capable of blocking signal transduction via the haematopoietin ⁇ c receptor common to IL-3,IL-5 and GM-CSF is preferably one which has been derived from the sequence of the haematopoietin ⁇ c receptor common to IL-3,IL-5 and GM-CSF, or is a variant thereof.
  • a peptide capable of inhibiting IL-3.IL-5 or GM-CSF which preferably acts by binding to Lyn tyrosine kinase.
  • a peptide comprising or more preferably consisting of a variant of the amino acid sequence of residues 450-465 or 462-481, of the haematopoietin ⁇ c receptor and having not less than 50% of the inhibitor activity of a peptide comprising or consisting of the non-variant amino acid sequence.
  • the peptides stearate- YGYRLRRKWEEKIPNP-NH2 or stearate- IPNPSKSHLFQNGSAELWPP-NH 2 .
  • a peptide according to the present invention for use in medical therapy.
  • a peptide of the present invention in the preparation of a medicament for use in the therapy of conditions displaying increased production or function of granulocytes or in the therapy of conditions requiring any one of inhibition of tyrosine phosphorylation; blocking of the eosinophil survival promoting effects of interleukin-5 and granulocyte/macrophage colony-stimulating factor; or inhibition of the growth and differentiation of eosinophils from bone marrow cells.
  • a peptide according of the present invention in the preparation of a medicament for use in the therapy of asthma, allergic rhinitis, allergic conjunctivitis, idiopathic eosinophilic syndrome, eosinophilic pneumonia, allergic bronchopulmonary aspergillosis, Churg-Strauss syndrome, eosinophilic gastritis, Loeffler's syndrome or myelodysplastic syndromes.
  • a pharmaceutical formulation comprising a peptide of the present invention, and a pharmaceutically acceptable carrier therefor.
  • a method of treating a condition involving increased production or function of eosinophils or other granulocytes comprising the step of administering a pharmacologically effective dose of a peptide of the present invention.
  • Lyn is a member of the src family of tyrosine kinases and physically associates with the IL- 5 ⁇ c receptor [Pazdrak et al., J Exp Med 1995;181: 1827-34].
  • EL-5 activates Lyn kinase and transduces signals via the Ras-MAP kinase pathway. Lyn kinase plays a critical role in EL- 5-stimulated eosinophil survival.
  • the present invention maps the Lyn-binding site on ⁇ c receptor of BL-3/GM-CSF/IL-5. The membrane-proximal ⁇ c 450-517 region is important for Lyn binding. In order to identify the exact binding site of Lyn, four biotinylated peptides were designed and synthesised:
  • the peptides were biotinylated.
  • the ⁇ c 605-624 (pY612) peptide was used as a positive control because it binds to the SH2 domain of Lyn.
  • Control peptides were derived from FceRIb (amino acids 29-48) and gpl30 (amino acids 658-677) and their sequences were similar to that of ⁇ c 462-481.
  • the binding of the biotinylated peptides to Lyn was studied in a solution binding assay using TF-1 cell lysate as a source of Lyn.
  • the detection of the peptide-bound Lyn kinase was performed by western blotting.
  • Proline residues have been implicated in hydrophobic interactions (e.g. SH3 binding) among various proteins. These peptides had two to four Pro residues in their sequences. In order to determine the role of the Pro residues, experiments were performed with a Pro — Ala substituted peptide.
  • GM-CSF-dependent cell lines were incubated in the presence or absence of the N-stearated ⁇ c 462-481 peptide or the control peptides for 3 hours and then stimulated with or without GM-CSF (10 ng/ml) for 5 minutes.
  • TF-1 cells After lysing TF-1 cells, the lysates were subjected to electrophoresis and western blotting with anti-phosphotyrosine antibody. For thymidine incorporation study, TF-1 cells were incubated with 5% FCS and GM-CSF (1 ng/ml) for 96 hours and their radioactivity was counted.
  • the N-stearated ⁇ c 462-481 peptide but not the biotinylated ⁇ c 462-481 peptide nor a control N-stearated peptide derived from IL-5 ⁇ receptor (IL-5 ⁇ 316-335), inhibited GM- CSF-induced tyrosine phosphorylation of cellular proteins in eosinophils ( Figure 4) and 3H-thymidine incorporation in TF-1 cells ( Figure 3).
  • EXAMPLE 3 Effect of the N-stearated Lyn-binding peptide on GM-CSF/IL-5 signalling in eosinophils.
  • the capability of the peptide to interfere with the signal transduction of GM- CSF/IL-5 was examined by the two parameters.
  • GM-CSF-induced activation of Lyn kinase in eosinophils in the presence of the peptide was determined. The activation was investigated by immunoprecipitation with anti-Lyn antibody followed by anti- phosphotyrosine immunoblotting.
  • an assessment of eosinophil stimulation by GM-CSF or IL-5 using an eosinophil survival assay and the ECP release method was performed.
  • N-stearated ⁇ c 462-481 peptide inhibited GM-CSF-induced tyrosine phosphorylation of cellular proteins in eosinophils in a dose-dependent manner (Figure 4).
  • Lyn activation after GM-CSF stimulation was significantly inhibited in the presence of the N-stearated ⁇ c 462-481 peptide ( Figure 5).
  • Eosinophil survival induced by GM-CSF was also inhibited by this stearated peptide ( Figure 6).
  • ECP release from eosinophils was not affected by the N-stearated ⁇ c 462-481 peptide ( Figure 7).
  • Lyn kinase in myeloid cell differentiation, the effect of the N- stearated Lyn-binding peptide on murine bone marrow cells was studied.
  • BALB/c mice were sacrificed and the femurs were removed.
  • the bone marrow cavity was washed with saline to obtain cells.
  • the bone marrow cells (2 x 105 cells/ml) were incubated for 1 week with 10 ng/ml of murine IL-3 and EL-5 and then harvested. The total cell count was obtained, and the rest of the cells were used for cytospin preparations.
  • the cytospin preparations were stained with Wright's stain for a differential count of bone marrow cells.
  • IL-3 and EL- 5 stimulated the proliferation of bone marrow cells and the cell count increased nearly four-fold. Further, 56% of the cells differentiated with eosinophilic lineage (eosinophilic myelocytes and mature eosinophils).
  • the effect of N-stearated ⁇ c 462-481 peptide on the bone marrow cell differentiation was investigated. When the cells were pretreated with the peptide, the total cell number and the percentage of cells in eosinophilic lineage were reduced by nearly 50%. TABLE I shows the effect of the N-stearated ⁇ c 462- 481 peptide on the proliferation and differentiation of murine bone marrow stem cells.
  • Lyn-binding peptides do not bind or activate these molecules ( Figures 8-10). Lyn is predominantly expressed in myeloid cells and B cells.
  • Lyn knockout mice have decreased myelopoiesis and dysregulated immunoglobulin production. Further, the IgE-dependent mast cell activation is impaired (Hibbs et al.
  • Lyn has an N-terminal unique domain, followed by an SH3 domain, SH2 domain and the tyrosine kinase domain. Lyn can interact with receptors via three different binding sites: unique domain, SH3 and SH2 domains.
  • unique domain unique domain
  • SH3 unique domain
  • Small peptides are generally susceptible to amino- and carboxypeptidases.
  • the lipopeptide of the present invention is N-terminally acylated which will protect it from aminopeptidases. Further, the peptide has C-terminal amide which may make it partially resistant to carboxypeptidases. Formation of another ester C-terminal modification can be used to improve lipophilicity.
  • the D-amino acid residues are known to be resistant to peptidases.
  • a number of peptide dmgs have been designed using this principle as is well known in the art (e.g., bradykinin antagonists, neuropeptide antagonists, etc.). Additional modifications include introduction of pseudopeptide bonds (resistant to peptidases). Cyclization of peptides is also known to increase their stability.
  • the present invention involves the concept that signal specific inhibitors such as the be 462-481 peptide will inhibit only the unwanted signal(s) of a receptor keeping other signalling processes intact. This is in contrast to receptor antagonists which block all receptor-associated signalling processes indiscriminately.
  • signal specific inhibitors such as the be 462-481 peptide will inhibit only the unwanted signal(s) of a receptor keeping other signalling processes intact. This is in contrast to receptor antagonists which block all receptor-associated signalling processes indiscriminately.
  • receptor antagonists which block all receptor-associated signalling processes indiscriminately.
  • the therapeutic concentration of many currently used dmgs is within 10-20 mg/ml, e.g., theophylline. Others require much higher concentrations, e.g., ⁇ -lactam antibiotics, aspirin. Thus, usage of 10-20 mM concentrations does not necessarily indicate that the peptide will be toxic.
  • mice for ⁇ c receptor have eosinopenia but no other haematological or immunological abnormalities. Unlike humans, mice have an additional receptor for EL-3. Thus, double knockout mice were created by crossing ⁇ c knockout mice with IL-3 knockout mice. Haematopoiesis and immunologic studies were performed. The results were similar. Eosinopenia was the only haematological abnormality noted in these mice (Nishinakamura et al., Blood 88: 2458-2464, 1996). This study suggests the ⁇ c receptor is critical only for eosinopoiesis but not for other myeloid lineages.
  • cytokines such as G-CSF, M-CSF, IL-8 may regulate important aspects of myeloid cell haematopoiesis. From these studies one can conclude that a blockade of the ⁇ c receptor by a signal specific inhibitor may not have toxic effects on the growth and differentiation of neutrophils.
  • the delivery of peptide of the present invention can be accomplished in a variety of conventional ways as well as using other technologies.
  • Technology in the filed of nasal and inhalation delivery of drags is rapidly changing.
  • the delivery of proteins with large porous particles such as poly(lactic acid-co- glycolic acid) (PLGA) orpoly(lactic acid-co-lysine-graft-lysine) (PLAL-Lys) may prolong their half-life and provides steady delivery.
  • the strategy described herein is to map the cytosolic region of the receptor for specific signal generating sites (e.g. tyrosine kinase binding sites) and design small peptides based upon the sequence information of the signal-generating site.
  • the next step is to modify the peptides in order to enable their cellular intemalisation and to improve their in vivo stability.
  • the modified peptides should function to block targeted cellular functions.
  • IL-5 activates multiple signalling pathways in eosinophils including the activation of Lyn kinase, the Jak2-Stat and the Raf-MAP kinase pathways. Lyn and Jak2 kinases are important for eosinophil growth, differentiation, and survival whereas the Raf-MAP kinase pathway is critical for eosinophil degranulation.
  • the Lyn-binding site of IL-5 ⁇ c receptor was mapped and small peptides which bind Lyn in vitro were designed. Modified Lyn binding peptides enter the cells and act as competitive inhibitors, and block Lyn-specific eosinophil functions without interfering with other functions of IL-5 ⁇ c receptor.
  • Lyn signal specific inhibitors By tmncation mutations, various regions of EL-5R ⁇ c have been found responsible for distinct signalling processes. For example, the membrane proximal 450- 517 residues are important for activation of Lyn, Jak2, c-myc, and pim-1, whereas the distal 626-763 residues are critical for the activation of Ras-MAP kinase pathway as well as c-fos and c-jun. Based upon this observation, overlapping peptides encompassing residues 450-498 were synthesised.
  • IL-5 signal specific inhibitors based upon the Lyn-binding site of the _EL-5R ⁇ c.
  • the general strategy for developing signal specific inhibitors involves 5 steps.
  • the first step is to identify a critical signalling molecule that associates with the receptor.
  • the second step is to map the signalling molecule-binding site of the receptor.
  • the third step is to design small peptide(s) based upon the binding site and study the in vitro binding activity.
  • the fourth step is to N-acylate the peptide for cellular intemalisation and examine the specificity of signalling inhibition and biologic effects in vitro.
  • the fifth step is to modify the peptide for increased stability.
  • the peptides bc450-465 and bc462-481 are N-stearated to enhance cellular intemalisation. All peptides and their modification can be obtained from Quality Controlled Biochemicals, Hopkinton, MA. The intemalisation of N-stearated peptides and their effect on cytosolic signalling (protein tyrosine phosphorylation) are studied using eosinophils and a GM-CSF- dependent cell line TF-1. Further, the peptides are tested for inhibition of Lyn and other kinases/signalling molecules (e.g.
  • Controls include peptides without N-stearation and N-stearated peptides of similar length obtained from the IL-5 receptor b chain, the IgE receptor b chain, and the IL-6/LIF receptor common chain gpl30.
  • the effect of the N-stearated peptides on haematopoietin function is studied in the following assays: 1) eosinophil survival; 2) proliferation (thymidine incorporation) of GM-CSF-dependent cell lines; 3) IL-3 and EL-5-stimulated differentiation of eosinophils from stem cells; and 4) GM-CSF-stimulated degranulation (release of eosinophil cationic protein ECP as measured by a commercially available RIA) of eosinophils. Further the effects of the N-stearated peptides on the following Lyn kinase-dependent cell functions are shown; 5) IgE-mediated histamine release from basophils; 6) Anti-IgM-stimulated B cell proliferation.
  • Lyn physically associates EL5R ⁇ c. Lyn is important for eosinophil growth and survival but not necessary for eosinophil degranulation (ECP release) and upregulation of adhesion molecule CD1 lb. There is concern about the importance of secondary and tertiary stmctures of the receptor in Lyn binding which may be lost in a small peptide. However, recent experience with SH2 and SH3 domain-binding small peptides suggests that the tertiary stmcture is not essential. N-acylation of small peptides causes their intemalisation through the lipid membranes as demonstrated by spin label electron spin resonance and 2H NMR.
  • N-myristoylation of a protein kinase C substrate analogue causes its intemalisation and specific inhibition of the kinase. Because of the bigger size of these peptides, they are N-stearated.
  • Peptides from receptors Fc(RI( and gpl30) that are known to activate Lyn can be used as controls.
  • the control peptides have equal numbers of similarly spaced proline residues.
  • D-amino acids are relatively resistant to digestion by peptidases. For this reason, L-amino acid residues are replaced systematically with D-amino acid residues except the critical proline residues. Additional ways to improve stability include C(-alkyl amino acid or cyclopropyl amino acid substitutions.
  • the modified peptides are biotinylated and tested for Lyn binding by western blotting according to the method described in Figure 2. Then their serum half-life is assessed by serial measurement (ELISA) of the serum concentration of biotin following injection into the mice. Simultaneously, the kinetics and "half-life" of Lyn inhibition in blood granulocytes are monitored as a measure of in vivo effects of signal specific inhibitors.
  • EL-5-induced peripheral blood eosinophilia in mice Mice are pre-treated with increasing concentrations of signal specific inhibitors or a stearated control peptide. At the time of maximal serum concentration, the mice are injected with BL-5. Peripheral blood eosinophilia are monitored every 30 minutes for 4 hours. Next, an allergen-induced airway eosinophilia in a mouse model of asthma is used. Eosinophils constitute up to 60% of the bronchoalveolar lavage cells upon allergen inhalation challenge.
  • the immunised mice are pre-treated intrabronchially with increasing concentrations of signal specific inhibitors or a stearated control peptide. Thirty minutes later the mice undergo allergen inhalation change. Bronchoalveolar lavage are performed 12, 24 or 48 hours later. The total and differential cell count are obtained. Further, the lung tissue are analysed histologically for inflammatory cell influx. The bronchoalveolar lavage cells and inflammatory influx into the airways in signal specific inhibitors- and control peptide- treated mice are compared.

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Abstract

La présente invention se rapporte à des inhibiteurs peptidiques des hématopoïétines que sont l'interleukine-3, l'interleukine-5 et le facteur stimulant la prolifération des macrophages/granulocytes. Ces peptides inhibent l'activation de la Lyn tyrosine kinase et bloquent par conséquent la transduction des signaux via le récepteur βc d'hématopoïétine commun à l'interleukine-3, à l'interleukine-5 et au facteur stimulant la prolifération des macrophages/granulocytes. L'invention se rapporte également à une méthode de traitement des troubles associés à une augmentation de la production et du fonctionnement des éosinophiles et d'autres granulocytes, consistant à administrer une dose pharmacologiquement efficace d'une composition pharmaceutique contenant ces inhibiteurs.
PCT/SE1998/001687 1997-09-23 1998-09-21 Inhibiteurs des hematopoietines interleukine-3, interleukine-5 et facteur stimulant la proliferation des macrophages/granulocytes, et utilisation de ces inhibiteurs WO1999015561A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009006688A1 (fr) * 2007-07-11 2009-01-15 Medvet Science Pty Ltd Régulation des taux d'il-4 et d'il-13 par blocage de la liaison de haute affinité par l'il-3, l'il-5 et le gm-csf à leur récepteur commun

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997028190A1 (fr) * 1996-01-30 1997-08-07 Medvet Science Pty. Ltd. Antagonistes et agonistes de la cytokine

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997028190A1 (fr) * 1996-01-30 1997-08-07 Medvet Science Pty. Ltd. Antagonistes et agonistes de la cytokine

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JBC -- RAO AND MUFSON, Volume 270, No. 12, March 1995, PADMINI RAO et al., "A Membrane Proximal Domain of the Human Interleukin-3 Receptor beta Subunit that Signals DNA Synthesis in NIH 3T3 Cells Specifically Binds a Complex of Src and Janus Family Tyrosine Kinases and Phosphatidylino.....", pages 6886-6893. *
JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 272, No. 22, May 1997, HEATHER BONE et al., "SHP1 and SHP2 Protein-Tyrosine Phosphatases Associate with Betac After Interleukin-3-Induced Receptor Tyrosine Phosphorylation", pages 14470-14476. *
JOURNAL OF IMMUNOLOGY, Volume 155, No. 4, August 1995, YAN LI et al., "Association Between Lyn Protein Tyrosine Kinase (p53/56lyn) and the beta Subunit of the Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) Receptors in a GM-CSF-Dependent Human Megakaryocytic Leukemia....", pages 2165-2174. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009006688A1 (fr) * 2007-07-11 2009-01-15 Medvet Science Pty Ltd Régulation des taux d'il-4 et d'il-13 par blocage de la liaison de haute affinité par l'il-3, l'il-5 et le gm-csf à leur récepteur commun

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