WO1994017095A1 - Analogues de motifs d'activation de tyrosine de recepteurs et leurs utilisations therapeutiques - Google Patents

Analogues de motifs d'activation de tyrosine de recepteurs et leurs utilisations therapeutiques Download PDF

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Publication number
WO1994017095A1
WO1994017095A1 PCT/US1994/001025 US9401025W WO9417095A1 WO 1994017095 A1 WO1994017095 A1 WO 1994017095A1 US 9401025 W US9401025 W US 9401025W WO 9417095 A1 WO9417095 A1 WO 9417095A1
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Prior art keywords
peptide
seq
tam
amino acid
amino acids
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PCT/US1994/001025
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English (en)
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Manfred Weigele
Guo Tao
Raji Sundaramoorthi
David C. Dalgarno
Lynne D. Zydowsky
Jeremy Green
Oluyinka M. Green
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Ariad Pharmaceuticals, Inc.
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Priority to AU60981/94A priority Critical patent/AU6098194A/en
Publication of WO1994017095A1 publication Critical patent/WO1994017095A1/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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • 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 peptides and analogs and derivatives thereof that are related to all or a portion of tyrosine activation motifs found in multichain immune recognition receptors.
  • the immune system consists of lymphocytes, macrophages, and other specialized cells.
  • An organism responds to antigen by virtue of having a large number of lymphocytes, each bearing receptors which recognize distinct antigens.
  • B lymphocytes are precursors of antibody-secreting cells, and T lymphocytes are involved in regulatory and effector functions.
  • natural killer cells mediate certain
  • T lymphocytes that have been sensitized to an antigen by virtue of their
  • Immunoglobulins are synthesized by B lymphocytes, and by their progeny, the plasma cells. B cells interact with antigen via the antibody molecules in their plasma membranes. Plasma cells secrete antibodies.
  • IgGs are the major component of serum immunoglobulin, and the predominant antibody involved in a secondary immune response.
  • IgM is the predominant antibody involved in a primary immune response.
  • IgD is a cell surface receptor present on mature B cells.
  • IgA is found in bodily secretions.
  • IgE is found in normal human serum at very low concentrations, and is
  • IgE appears to mediate local and systemic immediate hypersensitivity and anaphylactic reactions. Atopic individuals produce IgE in response to many environmental antigens. The event that initiates immediate hypersensitivity in such patients is the binding of antigen to IgE on the surface of mast cells. Binding of the Fc domain of the allergen-bound IgE to the extracellular domain of the FceRI receptor complex on mast cells and basophils induces
  • Crosslinking of the receptor causes mast cell activation and a cascade of intracellular signaling events which ultimately results in the release of lipid mediators of inflammation (leukotrienes, etc.), proinflammatory cytokines (interleukins, etc.) and of secretory granules (containing histamine, 5-hydroxytryptamine, hexosaminidase, etc.). This release causes the appearance of many of the symptoms of acute allergic response.
  • Cells respond to external stimuli through the binding of extracellular ligands to transmembrane receptor molecules. Binding of protein or peptide ligands to the extracellular domain of such a
  • membrane-spanning receptor results in a conformational change or aggregation of the receptor, and a
  • Intracellular signal transduction occurs via changes in the intermolecular associations of protein molecules arranged in a complex multi-step sequential pathway leading from the intracellular portion of the receptor to proteins involved directly in controlling gene expression, cytoskeletal
  • T and B cells are found on T and B cells, mast cells, macrophages, natural killer cells and platelets.
  • MIRRs constitute a family of immune cell receptors involved in the activation of cells involved in the immune system. MIRRs include the B cell antigen receptor complex (BCR); the T cell antigen receptor complex (TCR); Fc ⁇ RIII (FC7RIIIA in humans), a
  • MIRRs consist of polypeptide chains that bind antigen or Fc, associated with chains whose intracellular domains contain a related short sequence of 18-27 amino acids termed the tyrosine activation motif
  • TAM TAM
  • Figure 1 schematically depicts these MIRRs.
  • the TCR consists of the ⁇ (alpha) and ⁇ (beta) T cell antigen receptor antigen-binding chains (which, in a minority of T cells, are replaced by the ⁇ (gamma) and ⁇ (delta) heterodimer), the ⁇ , ⁇ and s (epsilon) chains of the CD3 complex, and a disulfide linked ⁇ (zeta) dimer or heterodimer of ⁇ and ⁇ (eta) chains.
  • the BCR consists of the heavy and light chains and at least one disulfide-linked heterodimer consisting of Ig- ⁇ (MB-1 gene product) and Iq- ⁇ (B29 gene product).
  • the Fc ⁇ RI receptor is a complex of three proteins, the ⁇ , ⁇ , and ⁇ subunits.
  • the ⁇ subunit of FceRI binds IgE, the ⁇ subunit contains four transmembrane regions, and the 7 subunit consists of a part of disulphide-linked chains (Fig. 2).
  • FceRI- ⁇ subunit residues lie on the cytoplasmic side of the cell membrane. This ⁇ subunit is shared by the FC7RIII MIRR, and contains a TAM motif. FceRI- ⁇ also contains a TAM motif (but not FceRI- ⁇ ), as do the CD3- ⁇ ,CD3- ⁇ and ⁇ components of the TCR (but not the CD3- ⁇ or TCR- ⁇ or TCR- ⁇ ), MB-1 and B29 in the BCR, and the envelope protein (gp30) of bovine leukemia virus (Reth, 1989, Nature 338:383-384; see also Fig. 3 herein).
  • TAM motif in signal transduction has been investigated for the TCR.
  • the consensus sequence shown above occurs in the ⁇ chain of the TCR.
  • Cell lines expressing a chimeric molecule in which the cytoplasmic tail of the ⁇ chain was attached to the unrelated CD8 receptor respond to antibody crosslinking stimuli (Romeo et al., 1992, Cell 68:889-897).
  • the present invention is directed to
  • TAMs tyrosine activation motifs
  • analogs and derivatives include peptides and hybrid molecules with both peptide and non-peptide portions.
  • a hybrid molecule is provided which contains an amino(N)- or carboxy(C)-terminal non- peptide helix inducer.
  • the hybrid molecule contains an internal non-peptide structure that maintains the helical character of the TAM mimic.
  • invention inhibit the activation of various cells with immune system function.
  • TAM Mimics are formulated for pulmonary
  • the invention provides inhalers containing compositions comprising a therapeutically or prophylactically effective amount of a TAM Mimic.
  • FIG. 1 Schematic diagram of four multichain immune recognition receptors (MIRRs): IgMR (the B cell antigen receptor complex), FC7RIII, EceRI, and TCR (the T cell antigen receptor complex).
  • IgMR the B cell antigen receptor complex
  • FC7RIII the B cell antigen receptor complex
  • EceRI the T cell antigen receptor complex
  • TCR the T cell antigen receptor complex
  • FceRI receptors Two FceRI receptors are depicted cross-linked by allergen binding.
  • Two IgMRs are depicted cross-linked by antigen binding.
  • TAM motifs are indicated by cross- hatching.
  • FIG. 1 A schematic view of the MIRR for the Fc region of IgE, called the FceRI receptor (after Keegan and Paul, 1992, Immunology Today 13 (2): 63-68).
  • FIG. 5 Computer-generated drawings.
  • the left side shows an idealized (computer-generated) drawing of an alpha-helical peptide.
  • 7 amino acids i.e., an 11 angstrom stretch
  • spacer molecule like that present in a compound of formula (V).
  • N-terminal and C-terminal helix inducers are depicted.
  • Compound I and Compound III were developed by Paul Bartlett (Bartlett et al., "Intuitive- and Computer- Assisted Approaches to the Design of Conformationally Restrained Peptides and Their Mimics," October 28-29, 1991, reprinted from Proceedings of The Robert A.
  • the present invention is directed to peptides related to all or a portion of tyrosine activation motifs (TAMs), as well as analogs and derivatives thereof (collectively termed herein “TAM Mimics”).
  • TAMs tyrosine activation motifs
  • analogs and derivatives include peptides and hybrid molecules with both peptide and non-peptide portions.
  • a hybrid molecule is provided which contains an amino (N)- or carboxy(C) -terminal non- peptide helix inducer.
  • the hybrid molecule contains an internal non-peptide structure that maintains the size and general shape of the TAM mimic.
  • the TAM Mimics of the invention retain the conformational information of TAM motifs required for biological activity, however, preferably incorporate sequence changes that provide the TAM Mimic with enhanced ability to cross lipid bilayers.
  • the TAM motif plays a critical role in the activation of the immune functions of T cells
  • TAM motif within receptor complexes appears to interact with target proteins that transmit the activation signal within the cytoplasm to bring about cellular immune responses.
  • the TAM Mimics of the invention mimic the structure of these TAM motifs. While Applicants do not intend to be bound by any specific mechanism, Applicants believe that the TAM Mimics function by binding to the target protein, mimicking the interaction with the receptor-associated TAM motif, and thus preventing the natural activation of this target. Stimulation of mast cells, B
  • lymphocytes T lymphocytes, and macrophages is
  • the TAM Mimics of the invention thus act as inhibitors of the signal transduction events mediated by the various MIRRs containing TAM sequences, by competitively inhibiting the interaction of such molecules with an effector or regulatory molecule via their TAM motifs, and thus preventing the natural sequence of activation in MIRRs.
  • TAM Mimics of the invention can also be used to study and elucidate the signal transduction mechanism in cells expressing the MIRRs. 5.1. TAM MIMICS
  • TAM Mimics of the invention include peptides and hybrid molecules, as detailed more fully in the subsections below.
  • the TAM Mimic is a peptide.
  • a TAM Mimic of the invention comprises or consists of all or a portion of the sequences shown in Figure 3 (SEQ ID NOS:2-12).
  • a TAM Mimic comprises or consists of the sequence
  • a TAM mimic comprises or consists of the following sequences:
  • DGGYMTLNPRAPTDDDKNTYLTLP (part of SEQ ID NO: 2)
  • DQLYQPLKDREDDQYSHLQ (part of SEQ ID NO: 5)
  • DQVYQPLRDRDDAQYSHLG (part of SEQ ID NO: 6)
  • ENLYEGLNLDDCSMYEDIS (part of SEQ ID NO: 9, 10)
  • EHTYEGLNIDQTATYEDIV (part of SEQ ID NO: 12)
  • a TAM Mimic peptide comprises or consists of the ⁇ or ⁇ TAM motif, or a portion thereof, present in the human FceRI complex, or one of the three TAM motifs, or a portion thereof, present in the human CD3 zeta chain; such TAM motifs are as follows:
  • CD3 ⁇ TAM motif #1 CD3 ⁇ TAM motif #1:
  • CD3 ⁇ TAM motif #2 CD3 ⁇ TAM motif #2:
  • CD3 ⁇ TAM motif #3 CD3 ⁇ TAM motif #3:
  • TAM motifs from different receptor subunits appear to specifically activate distinct biological responses in different cell types.
  • T cell receptor is not expected to be as active in activating mast cells as in activating T cells
  • the ⁇ subunit TAM motif from the IgE mast cell receptor is not expected to be as active in activating T cells as in activating mast cells.
  • SEQ ID NO:1 other than X are invariant residues that are shared by all members of the TAM family. These shared residues must be important for a common
  • TAM Mimics are also provided which specifically interfere with activation or immune responses in specific immune cells, by virtue of containing sequences of variant residues (e . g . , residue numbers 2-8, 10-11, 13-14, 16-22, and/or 24-25 in SEQ ID N0:1) which are identical or substantially the same as those sequences present in MIRRs (see Fig. 3).
  • TAM Mimics which mimic FceRI 7 or ⁇ TAM motifs.
  • a TAM mimic comprises one of the following sequences, related to the FceRI 7 TAM motifs:
  • dashed lines represent potential salt bridges between the indicated residues; solid lines represent cyclization (covalent linkage) of the indicated residues.
  • SEQ ID NO: 16 is via disulfide bond formation.
  • the envisioned salt bridges and the cyclizations are preferably present in the indicated sequences because they are expected to increase the conformational stability of the peptide.
  • SEQ ID NO: 13 is the actual sequence contained in the C-terminal region of human FceRI- ⁇ .
  • a TAM Mimic comprises or consists of the sequence:
  • TAM Mimics of the invention which have a sequence more homologous to a TAM motif found in an FceRI ⁇ or ⁇ chain than to other TAM motifs are preferred for inhibition of mast cell (or basophil cell) activation.
  • TAM Mimics which have a sequence more homologous to a TAM motif found in FC7RIII (or FC7RIIIA) ⁇ or ⁇ chain are preferred for inhibition of macrophage or natural killer cell activation.
  • TAM Mimics which have a sequence more homologous to a TAM motif found in the B cell antigen receptor complex (MB-1 or B29) are preferred for inhibition of B cell activation.
  • TAM Mimics which have a sequence more homologous to a TAM motif found in the CD3- ⁇ , CD3- ⁇ , or ⁇ chain of the T cell antigen receptor complex are preferred for inhibition of T cell activation.
  • TAM Mimics of the invention can be derived from TAM motifs of proteins of human or other animal origin, including but not limited to mammals such as cows, horses, pigs, sheep, goats, rats, mice, dogs, chickens, rabbits, etc.
  • the peptide TAM Mimics of the invention have a sequence in the range of 15-39 amino acids. In a specific aspect, the peptides have a sequence in the range of 17-25 amino acids. In a preferred aspect, the peptides contain 19 amino acids.
  • the TAM mimic peptides of the invention preferably contain naturally-occurring amino acids.
  • the most common naturally-occurring amino acids are shown in Table I:
  • TAM mimic peptides can contain non-natural amino acids or cyclic peptides.
  • Non- classical amino acids include but are not limited to the D-isomers of the common amino acids, ⁇ -amino isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ - alanine, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, N ⁇ -methyl amino acids, and amino acid analogs in general.
  • the amino acid can be the D (dextrorotary) or L
  • the peptide may be prepared by methods that are known in the art.
  • solid phase peptide synthesis consists of coupling the carboxyl group of the C-terminal amino acid to a resin and successively adding N-alpha protected amino acids.
  • the protecting groups may be any known in the art. Before each new amino acid is added to the growing chain, the protecting group of the previous amino acid added to the chain is removed.
  • the coupling of amino acids to appropriate resins is described by Rivier et al., U.S. Patent No. 4,244,946. Such solid phase syntheses have been described, for example, by
  • TAM Mimics of the invention also include hybrid molecules that comprise peptides bound to amino- or carboxy-terminal helix inducers.
  • such hybrid molecules are TAM Mimic peptides, in which two amino acid residues at the N-terminus and/or C-terminus of the peptide are replaced respectively by an N-terminal or C-terminal helix inducer, or a helix inducer of 0-3 residues can be added.
  • Such helix inducers are nonpeptide organic structures that function as helix nucleating modules. Examples of N-terminal and C-terminal helix inducers, respectively in C-terminal or N-terminal linkage to a peptide amino or carboxyl group respectively, are shown in Figure 6.
  • the invention thus provides a compound of formula (IIe), wherein X is OH or an amino-terminally linked peptide having a
  • X is a hydrogen atom or a carboxy-terminally linked peptide having a sequence in the range of 17-39 amino acids and comprising the sequence V Y T G L S T R N Q E T Y E T L K (part of SEQ ID NO: 13).
  • hybrid peptide (la) is produced, containing helix inducer (lb) replacing the N-terminal DG in the sequence of the TAM Mimic Peptide TAM-1 (see Section 6) having the sequence:
  • the C-terminus of compound (la) is the free acid, an ester (OR, where R is preferably an alkyl of 1-4 carbon atoms), or an amide.
  • R is preferably an alkyl of 1-4 carbon atoms
  • hybrid peptide (la) may be prepared according to Scheme I.
  • 3,5-Dimethoxybenzoic acid is converted to its acid chloride by treatment with oxalyl chloride in
  • the cis-acid (9) is coupled to L-valine benzyl ester using bromo-tris-pyrrolidino- phosphonium hexafluorophosphate and diisopropyl ethylamine in dichloromethane at 0°C to r.t. for 16 h to give a 1:1 mixture of diastereomers 10L and 10D.
  • Hydrogenolysis of the mixture using hydrogen and a catalytic amount Pd/C followed by HPLC on a reverse phase column provides the desired diastereamer 11L along with diastereomer 11D.
  • Coupling of 11L to TAM peptide 16-mer followed by removal of side chain protecting groups gives the desired hybrid peptide (la).
  • the above-described synthetic scheme has been carried out as described to yield the hybrid peptide (la).
  • hybrid peptide (IIa) is produced, containing helix inducer (lIb) replacing the N-terminal DG in the sequence of the TAM Mimic peptide TAM-1 (see Section 6.2).
  • N-terminal helix inducer lib can be synthesized from L-aspartic acid and L-pyroglutamic acid, according to Scheme II.
  • L-pyroglutamic acid was first protected as its methyl ester by treatment with thionyl chloride in methanol at 0°C to room temperature (r.t.) for 20 h and then treated with triethyloxonium
  • TAM Mimics of the invention also include hybrid molecules with helix replacements to achieve gradual reduction of the peptide character of the molecules.
  • peptide hybrids ol the invention are TAM Mimic peptides in which the peptide sequence corresponding to amino acid numbers 16-22 in the consensus sequence (SEQ ID NO:1) (see e.g. Fig. 3) are replaced by a phenothiazine ring structure.
  • Compounds of the invention thus include peptide-like hybrid molecules of the formula (V) where R 1 is a sequence of at least 4, and is preferably 7, amino acids, wherein R 1 comprises a C-terminal leucine, and a tyrosine in the fourth position counting from a C to N-terminal direction.
  • R 1 corresponds to a portion of SEQ ID NO:1, amino-terminal to SEQ ID NO:1 residue number 16, except that the amino acid
  • R 1 has a sequence in the range of 4-25 amino acids.
  • R 2 is a sequence of at least 4 amino acids, and is most preferably 5 amino acids, comprising an amino- terminal Tyr, and Leu or Ile in the fourth position counting in the N- to C-terminal direction.
  • R 2 has a sequence in the range of 4-25 amino acids, and preferably 4-6 amino acids.
  • R 3 is Hydrogen or C 1 -C 6 alkyl. 2 is S, SO, or SO 2 .
  • TAM Mimics of the invention which are peptide hybrids and which mimic the FceRI ⁇ TAM, are of structure (V) in which R 3 is H; Z is S; R 2 is YETLK-NH 2 (part of SEQ ID NO: 17); and R 1 is DGVYTGL (part of SEQ ID NO: 17), QKVYDKL (part of SEQ ID NO:19), or QKVYCKL (part of SEQ ID NO:20).
  • the phthalimide group is removed with hydrazine producing the free benzyl amine which is subsequently protected as its BOC (tert- butoxycarbonyl) derivative (30) by reaction with 2- (tert-butoxycarbonyloxyamino)-2-phenylacetonitrile.
  • BOC tert- butoxycarbonyl
  • Metallation of (30) with n-butyllithium at -78°C followed by quenching with carbon dioxide produces the carboxylic acid (31).
  • Peptide coupling with the carboxyl end of (31) followed by deprotection of the amine followed by a second peptide coupling with the amino end of (31) yields the desired peptide-like hybrid molecules.
  • TAM Mimics are further characterized as described below prior to in vitro and in vivo testing of
  • TAM Mimic peptides The conformational preferences and membrane-penetrating properties of the TAM Mimic peptides can be investigated both
  • Mimic peptide can be predicted based on four
  • amphiphilic moment (iii) the electric dipole of the peptide; and (iv) the net charge of the peptide.
  • the lipid-induced helix-forming potential of a TAM Mimic can be assessed by CD (circular dichroism) spectroscopy in the presence of lipid-mimicking solvents (e.g., trifluorethanol) and lipid vesicles.
  • CD circular dichroism
  • NMR nuclear magnetic resonance
  • NMR has been used extensively to study the three-dimensional structure of biologically active peptides in solution or bound to micelles or
  • TAM Mimics The membrane transport properties of TAM Mimics can be studied by size exclusion
  • Lipid vesicles are prepared,
  • TAM Mimics incubated with TAM Mimics, then chromatographed to separate free TAM Mimic.
  • the vesicle contents are then assayed to determine the amount of TAM Mimic present in the lipid vesicle fraction (determinable by HPLC or amino acid analysis).
  • a fluorescence assay for peptide transport can be used; the assay uses a water-stable fluorogenie reagent that becomes trapped inside the vesicles during formation. This
  • fluorogenie reagent reacts with TAM Mimic molecules that have crossed the lipid bilayer, producing a fluorescent derivative. 5.5. DEMONSTRATION OF THERAPEUTIC UTILITY
  • TAM Mimics are tested in vitro and then preferably in vivo for the desired therapeutic
  • TAM Mimic inhibitory to mast cell
  • histamine release can be detected in in vitro assays as indications of mast cell or basophil activation (see e.g., Stephan et al., 1992, J. Biol. Chem. 267:5434). Histamine release can be assayed, for example, by commercially available radioimmunoassay (e.g., AMAC Inc., Westbrook, Maine, Cat. No. 1302).
  • cytokines e.g., interleukins
  • phosphatidylinositol hydrolysis or tyrosine phosphorylation can be detected in in vitro assays as indications of mast cell or basophil activation (see e.g., Stephan et al., 1992, J. Biol. Chem. 267:5434). Histamine release can be assayed, for example, by commercially available radioimmunoassay (e.g., AMAC Inc., Westbrook, Maine, Cat. No. 1302).
  • Basophilic cell lines such as RBL-2H3 (Stephan et al., supra ) , KU812 (Matsson et al., 1989, Int. Arch. Allergy Appl. Immunol. 88:122-125; Valent et al., 1990, J. Immunol. 145:1885-1889), etc. can be employed in such in vitro assays.
  • the release of histamine from basophils is an in vitro assay of immediate hypersensitivity (Ishizaka & Ishizaka, 1975, Prog. Allergy 19:60).
  • cytotoxicity assays measuring natural killer lysis of target cells such as K562 erythromyeloid leukemia cells. Macrophage activation can be assayed by measuring macrophage phagocytic activity, induction of macrophage cytoxicity, or induction of macrophage Class II MHC cell surface expression, or by observing morphological changes associated with activation (see e . g . , Wright and Meyer, 1985, J. Exp. Med.
  • T cell activation can be assayed by T cell proliferation in vitro, or by measuring expression of cell surface interleukin-2 receptor (IL-2R), which increases upon activation of T cells (Waldman et al., 1984, J. Exp. Med. 160:1450-1466).
  • IL-2R cell surface interleukin-2 receptor
  • B cell activation can be assayed by measuring B cell proliferation in vitro .
  • TAM Mimics demonstrated to have the desired activity in vitro can be tested in vivo for the desired inhibitory activity.
  • such compounds can be tested in suitable animal model systems prior to testing in humans, including but not limited to rats, mice, chicken, cows, monkeys, rabbits, etc.
  • suitable model systems are also used to demonstrate therapeutic utility (see infra) .
  • an animal model system for rheumatoid arthritis is that consisting of animals of the autoimmune MRL/l mouse strain (Murphy, E.D. and Roths, J.B., 1978, in Genetic Control of Autoimmune Disease. Rose, N.R., et al . , eds., Elsevier/North- Holland, New York, pp. 207-219), that develop a spontaneous rheumatoid arthritis-like disease (Hang et al., 1982, J. Exp. Med. 155:1690-1701).
  • mice used as animal models for asthma include mice, rats, guinea pigs, rabbits, ponies, dogs, sheep, and primates.
  • TAM Mimics have therapeutic and prophylactic utility in the modulation of functions mediated by MIRRs, in particular in diseases or disorders involving the immune system or inflammation (inflammatory and immune disorders). TAM Mimics which inhibit lymphocytes are important therapeutically, because lymphocytes initiate autoimmune and alloimmune diseases.
  • TAM Mimics which inhibit an immune or inflammatory response and thus are useful according to the invention are most preferably identified by use of known convenient in vitro assays, e .g . , based on their ability to inhibit activation of cells of the immune system assayed in vitro , or in vivo assays (see
  • TAM Mimics which inhibit activation of T cells, B cells, and/or macrophages in vitro are preferred for treatment (or prevention) of immune disorders such as but not limited to autoimmune diseases and transplant rejection.
  • TAM Mimics which inhibit activation of macrophages and/or natural killer cells in vitro are preferred for treatment (or prevention) of immune complex diseases such as but not limited to glomerulonephritis and other autoimmune diseases.
  • TAM Mimics which inhibit activation of mast cells in vitro are preferred for treatment (or
  • the invention provides methods of treating or preventing diseases and disorders associated with undesirable or inappropriate immune system activity or inflammation by administration to a subject of an effective amount of a TAM Mimic of the invention.
  • the subject is preferably an animal
  • animals including but not limited to animals such as cows, pigs, chickens, etc., and is preferably a mammal, and most preferably human.
  • inflammatory/ immune response include but are not limited to the following:
  • Inflammatory arthritis e.g., rheumatoid arthritis, seronegative spondyloarthritites (Behcets disease, Reiter's syndrome, etc.), juvenile rheumatoid arthritis, vasculitis, psoriatic arthritis,
  • SLE Systemic lupus erythematosus
  • Inflammatory dermatoses e.g., psoriasis, dermatitis herpetiformis, eczema, necrotizing and cutaneous vasculitis, bullous diseases.
  • autoimmune disorders In addition to the autoimmune disorders SLE and rheumatoid arthritis, disorders such as glomerulonephritis, juvenile onset diabetes, multiple sclerosis, allergic conditions, autoimmune thyroiditis, allograft rejection (e . g . , rejection of transplanted organs such as kidney, heart, pancreas, bowel, or liver), and graft-versus- host disease can be treated.
  • SLE and rheumatoid arthritis disorders such as glomerulonephritis, juvenile onset diabetes, multiple sclerosis, allergic conditions, autoimmune thyroiditis, allograft rejection (e . g . , rejection of transplanted organs such as kidney, heart, pancreas, bowel, or liver), and graft-versus- host disease can be treated.
  • TAM Mimics of the invention including but not limited to those associated with hemolytic anemia, blood transfusion, certain
  • necrotizing enterocolitis necrotizing enterocolitis, granulocyte-transfusion- associated syndromes, Reynaud's syndrome, or other central nervous system inflammatory disorders.
  • a TAM Mimic which inhibits mast cell activation is administered to treat (or prevent) a type I allergic reaction such as one or more of the following listed in Table 1 (see generally, Terr, 1987, in Basic & Clinical Immunology, 6th Ed., ch. 24, Stites et al. (eds), Appleton & Lange, Norwalk,
  • Urticaria or Angioedema increased cutaneous
  • the invention provides methods of treatment
  • the TAM Mimic is purified.
  • subject is preferably an animal, including but not
  • microcapsules expression by recombinant cells
  • TAM Mimic-encoding nucleic acid as part of a retroviral or other vector, etc. Since preferred TAM Mimics of the invention are permeable to the cell membrane, a preferred mode of delivery is via
  • pulmonary administration as detailed more fully in Section 5.7.1 infra .
  • methods of introduction include but are not limited to intradermal,
  • TAM Mimics may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • TAM Mimics which are used for inhibition of mast cell activation, e . g . , for therapy of asthma or allergy, the preferred route of
  • administration is nasal or via a bronchial aerosol.
  • invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application (e.g., for skin conditions such as
  • psoriasis by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or
  • gelatinous material including membranes, such as sialastic membranes, or fibers.
  • compositions comprise a therapeutically (or prophylactically) effective amount of a TAM Mimic, and a
  • Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the carrier and composition can be sterile.
  • the formulation should suit the mode of administration.
  • the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a
  • solubilizing agent and a local anesthetic to ease pain at the site of the injection.
  • solubilizing agent and a local anesthetic to ease pain at the site of the injection.
  • ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by:
  • infusion it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the TAM Mimics of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartarlc acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the
  • Optionally associated with such container (s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of
  • a TAM Mimic is administered by pulmonary
  • bronchial aerosol is employed. This mode of administration is particularly preferred where the TAM Mimic inhibits mast cell activation and thus is useful in treating IgE-related disorders.
  • pulmonary administration is particularly preferred where the TAM Mimic inhibits mast cell activation and thus is useful in treating IgE-related disorders.
  • treatment e.g., of allergy or asthma.
  • Pulmonary administration can be
  • nebulizer Melt, Inc., St. Louis, Missouri
  • Acorn II nebulizer Marquest Medical Products
  • Such devices typically entail the use of formulations suitable for dispensing from such a device, in which a propellant material may be present.
  • Ultrasonic nebulizers tend to be more efficient than jet nebulizers in producing an aerosol of respirable size from a liquid (Smith and Spino, "Pharmacokinetics of Drugs in Cystic Fibrosis,"
  • a nebulizer may be used to produce aerosol particles, or any of various physiologically
  • acceptable inert gases may be used as an aerosolizing agent.
  • Other components such as physiologically acceptable surfactants (e.g., glycerides), excipients (e.g., lactose), carriers, and diluents may also be included.
  • FmocTyr ('Bu)OH [Abbreviations: Acm, acetamidomethyl; Boc, tert-butoxycarbonyl; 'Bu, tert-butyl; Fmoc,
  • NMP N-methylpyrrolidone
  • DMF N,N-dimethylformamide
  • Deprotection of the Fmoc group is effected using ca. 20% piperidine in NMP. At the end of each synthesis the amount of peptide present is assayed by
  • dibenzofulvene-piperidine adduct (formed by cleavage of the N-terminal Fmoc group) is recorded at 301 nm.
  • w is the weight of the peptide-resin sample (in mg).
  • N-terminal Fmoc group is cleaved using 20% piperidine in DMA, then acetylated using acetic anhydride and pyridine in DMA.
  • the peptide resin is thoroughly washed with DMA, CH 2 Cl 2 and finally diethyl ether.
  • the air-dried peptide resin is treated with ethylmethyl-sulfide (EtSMe), ethanedithiol (EDT), and thioanisole (PhSMe) for approximately 20 min. prior to addition of 95% aqueous trifluoracetic acid (TFA).
  • EtSMe ethylmethyl-sulfide
  • EDT ethanedithiol
  • PhSMe thioanisole
  • TAM-1 19-mer TAM Mimic peptide, termed "TAM-1", having the TAM native sequence shown was synthesized:
  • TAM-2 TAM Mimic peptide
  • Non-consensus amino acids were changed relative to the TAM-1 peptide sequence, with a view toward achieving a higher degree of helicity.
  • TAM-4 TAM Mimic peptide
  • the peptide contained two cysteine residues linked via a disulfide bridge to help lock the peptide into the presumed desired conformation.
  • the crude peptide (385 mg) was initially partially purified by gel filtration on Sephadex G-10 eluted with 10% aqueous acetic acid to give 300 mg material.
  • Peptide (73 mg) was dissolved in degassed 50% aqueous acetic acid (2.5 mL) and mercuric acetate (91 mg) in 50% aqueous acetic acid (1.0 mL) was added. The mixture was stirred under N 2 for 3 h, then
  • TAM-5 TAM Mimic peptide
  • TAM-6 TAM Mimic peptide
  • NMR nuclear magnetic resonance
  • the purified TAM-1 was prepared both as a 3 mM solution in 30% (v/v) deuterated trifluoroethanol (30% TFE) and as a 3 mM solution in the presence of deuterated
  • DPC dodecylphosphatidylcholine
  • the NMR analysis of the secondary structure of the TAM-1 peptides was based on an analysis of the relative intensities of interproton NOE data
  • ⁇ -hexosaminidase from basophils is measured as an in vitro assay of basophil activation.
  • a cell line, RBL-2H3 (from the laboratory of Dr. Siraganian, National Institutes of Health), of rat basophilic leukemia (RBL) cells is used in such an in vitro assay, according to the protocol described below (see also Barsumian et al., 1981, Eur. J.
  • BSA bovine serum albumin
  • Beta-hex buffer Solution 1 0.2 M Na 2 PO 4 (sodium phosphate dibasic ANHYDROUS) - 14.2 g/500 ml distilled H 2 O Note: If there is no anhydrous, use the sodium phosphate dibasic HEPTAHYDRATE (7H 2 O), but use 26.7 grams.
  • Beta-hex buffer (from above): 90 ml Distilled H 2 O: 135 ml p-nitrophenyl-N-acetyl-beta-D-glucosaminide
  • Beta-hex STOP solution glycine 15.0 g/ liter bring to pH 10.7 with 10 N NaOH (will need to add -30 ml of NaOH)
  • the mast cell/basophil degranulation assay can also be performed using permeabilized cells, according to the protocol described below (see also Cunha-Melo et al., 1989, J. Immunol. 143:2617-2625; Ali et al., 1989, J. Immunol. 143:2626-2633; Ali et al., 1989, Biochim. Biophys. Acta 1010:88-99).
  • LiCl (10 to 20 mM) for phosphoinositide hydrolysis experiments LiCl (10 to 20 mM) for phosphoinositide hydrolysis experiments.
  • RBL-2H3 cells (0.2 x 10 6 / well, in growth medium) are plated in 24 well tissue culture plate and incubated overnight.
  • the cells are permeabilized by exposure to
  • streptolysin 0 (0.1 to 0.3 units/ml, 200-500 ⁇ l/well) for 5 to 10 min.
  • concentration of streptolysin 0 and the time required for permeabilization depends on the number of passages the cells have been cultured for and may vary with batch of streptolysin 0.
  • a useful starting point is to permeabilize cells with 0.25 I.U. /ml streptolysin for 10 min.
  • Streptolysin O solution is prepared just before permeabilization by dilution of the stock solution (10 I.U. /ml) into prewarmed KG buffer. After the cells are permeabilized, remove buffer by aspiration, add fresh buffer (without toxin) and perform experiment as desired to measure cell
  • activation e.g., measure phosphoinositide hydrolysis, or ⁇ -hexosaminidase or histamine release.
  • Modifications of the permeabilization procedure can be made for measuring degranulation.

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Abstract

L'invention concerne des peptides représentant tout ou partie de motifs d'activation de tyrosine (TAM), ainsi que leurs analogues et dérivés (appelés ici collectivement 'imitateurs de TAM'). Ces analogues et dérivés comprennent notamment des peptides et des molécules hybrides présentant des parties à la fois peptidiques et non peptidiques. Dans un mode de réalisation spécifique, on a obtenu une molécule hybride contenant un inducteur d'hélice non peptidique amino(N)- ou carboxy(C)-terminal. Dans un autre mode de réalisation spécifique, la molécule hybride contient une structure non peptidique interne maintenant le caractère hélicoïdal de l'imitateur de TAM. Les imitateurs de TAM de l'invention inhibent l'activation de diverses cellules par leur fonction de système immun. L'invention concerne également des procédés thérapeutiques basés sur l'activité inhibitrice de la fonction immune des immitateurs de TAM, ainsi que des compositions pharmaceutiques. Dans un mode de réalisation préféré de l'invention, des immitateurs de TAM sont formulés pour une administration pulmonaire, ainsi, par exemple, l'invention permet d'obtenir des inhalateurs contenant des compositions comprenant une dose thérapeutiquement ou profilactiquement efficace d'un imitateur de TAM.
PCT/US1994/001025 1993-01-29 1994-01-28 Analogues de motifs d'activation de tyrosine de recepteurs et leurs utilisations therapeutiques WO1994017095A1 (fr)

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WO1997004010A1 (fr) * 1995-07-20 1997-02-06 Syntex (U.S.A) Inc. AGENTS DE TRANSDUCTION DE SIGNAUX DU LOCUS DU GENE Vα-1 RECEPTEUR DE LYMPHOCYTES T
EP0769957A1 (fr) * 1995-04-13 1997-05-02 Milkhaus Laboratory, Inc. Procedes de traitement des deficits moteurs
EP0804218A1 (fr) * 1994-03-17 1997-11-05 National Jewish Center For Immunology And Respiratory Medicine Produit et procede de regulation des voies de transduction de signaux
US5723462A (en) * 1996-04-26 1998-03-03 Neurogen Corporation Certain fused pyrrolecarboxamides a new class of GABA brain receptor ligands
US5976819A (en) * 1995-11-21 1999-11-02 National Jewish Medical And Research Center Product and process to regulate actin polymerization in T lymphocytes
US6080873A (en) * 1996-01-19 2000-06-27 Neurogen Corporation Fused pyrrolecarboxamides; a new class of GABA brain receptor ligands
WO2000063372A1 (fr) * 1999-04-16 2000-10-26 Celltech Therapeutics Limited Molecules de signalisation synthetiques
US6211365B1 (en) 1996-01-19 2001-04-03 Neurogen Corporation Fused pyrrolecarboxamides; a new class of GABA brain receptor ligands
WO2001032709A2 (fr) * 1999-11-01 2001-05-10 Celltech R & D Limited Polypeptides a motifs de signalisation primaire non naturelle
US6353109B2 (en) 1996-03-22 2002-03-05 Neurogen Corporation Certain fused pyrrolecarboxamides; a new class of GABA brain receptor
US7109351B1 (en) 1999-08-31 2006-09-19 Neurogen Corporation Fused pyrrolecarboxamides; GABA brain receptor ligands
EP2145884A1 (fr) * 2007-04-02 2010-01-20 Banyu Pharmaceutical Co., Ltd. Dérivé d'indoledione
US20200190141A1 (en) * 2017-12-28 2020-06-18 Avixgen Inc. Peptide for inhibiting skin inflammation and composition for preventing or treating skin inflammation containing the same

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Title
ANNUAL REPORTS IN MEDICINAL CHEMISTRY, Volume 24, issued 1989, B.A. MORGAN et al., "Approaches to the Discovery of Non-Peptide Ligands for Peptide Receptors and Peptidases", pages 243-252. *
PROC. NATL. ACAD. SCI. USA, Volume 84, Number 24, issued December 1987, H. SATO et al., "Close Linkage to the Mouse and Human CD3Gamma- and Delta-Chain Genes Suggest that their Tanscription is Controlled by Common Regulatory Elements", pages 9131-9134. *

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0804218A1 (fr) * 1994-03-17 1997-11-05 National Jewish Center For Immunology And Respiratory Medicine Produit et procede de regulation des voies de transduction de signaux
EP0804218A4 (fr) * 1994-03-17 1997-11-12
EP0769957A4 (fr) * 1995-04-13 1999-11-03 Milkhaus Lab Inc Procedes de traitement des deficits moteurs
EP0769957A1 (fr) * 1995-04-13 1997-05-02 Milkhaus Laboratory, Inc. Procedes de traitement des deficits moteurs
WO1997004010A1 (fr) * 1995-07-20 1997-02-06 Syntex (U.S.A) Inc. AGENTS DE TRANSDUCTION DE SIGNAUX DU LOCUS DU GENE Vα-1 RECEPTEUR DE LYMPHOCYTES T
US5976819A (en) * 1995-11-21 1999-11-02 National Jewish Medical And Research Center Product and process to regulate actin polymerization in T lymphocytes
US6211365B1 (en) 1996-01-19 2001-04-03 Neurogen Corporation Fused pyrrolecarboxamides; a new class of GABA brain receptor ligands
US6080873A (en) * 1996-01-19 2000-06-27 Neurogen Corporation Fused pyrrolecarboxamides; a new class of GABA brain receptor ligands
US6720339B2 (en) 1996-03-22 2004-04-13 Neurogen Corporation Certain fused pyrrolecarboxamides; a new class of GABA brain receptor ligands
US6353109B2 (en) 1996-03-22 2002-03-05 Neurogen Corporation Certain fused pyrrolecarboxamides; a new class of GABA brain receptor
US6096887A (en) * 1996-04-26 2000-08-01 Neurogen Corporation Certain fused pyrrolecarboxamides; a new class of GABA brain receptor ligands
US5723462A (en) * 1996-04-26 1998-03-03 Neurogen Corporation Certain fused pyrrolecarboxamides a new class of GABA brain receptor ligands
WO2000063372A1 (fr) * 1999-04-16 2000-10-26 Celltech Therapeutics Limited Molecules de signalisation synthetiques
US7109351B1 (en) 1999-08-31 2006-09-19 Neurogen Corporation Fused pyrrolecarboxamides; GABA brain receptor ligands
WO2001032709A2 (fr) * 1999-11-01 2001-05-10 Celltech R & D Limited Polypeptides a motifs de signalisation primaire non naturelle
WO2001032709A3 (fr) * 1999-11-01 2002-05-10 Celltech R&D Ltd Polypeptides a motifs de signalisation primaire non naturelle
EP2145884A1 (fr) * 2007-04-02 2010-01-20 Banyu Pharmaceutical Co., Ltd. Dérivé d'indoledione
EP2145884A4 (fr) * 2007-04-02 2010-11-03 Banyu Pharma Co Ltd Dérivé d'indoledione
US8106086B2 (en) 2007-04-02 2012-01-31 Msd K.K. Indoledione derivative
US20200190141A1 (en) * 2017-12-28 2020-06-18 Avixgen Inc. Peptide for inhibiting skin inflammation and composition for preventing or treating skin inflammation containing the same
AU2018353934B2 (en) * 2017-12-28 2021-04-01 Avixgen Inc. Peptide for inhibiting skin inflammation and composition for preventing or treating skin inflammation containing the same
EP3708574A4 (fr) * 2017-12-28 2021-11-03 Avixgen Inc. Peptide permettant d'inhiber une inflammation cutanée et composition le contenant pour la prévention ou le traitement d'une inflammation cutanée
US11208433B2 (en) 2017-12-28 2021-12-28 Avixgen Inc. Peptide for inhibiting skin inflammation and composition for preventing or treating skin inflammation containing the same

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