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WO2009089362A1 - Procédés de traitement utilisant des agents pour réguler la fonction du récepteur fas dans des pathologies liées à la peau et aux cheveux - Google Patents

Procédés de traitement utilisant des agents pour réguler la fonction du récepteur fas dans des pathologies liées à la peau et aux cheveux Download PDF

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WO2009089362A1
WO2009089362A1 PCT/US2009/030454 US2009030454W WO2009089362A1 WO 2009089362 A1 WO2009089362 A1 WO 2009089362A1 US 2009030454 W US2009030454 W US 2009030454W WO 2009089362 A1 WO2009089362 A1 WO 2009089362A1
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fas
seq
peptide
fasl
mimetics
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Mark I. Greene
Ramachandran Murali
Franklin Pass
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University of Pennsylvania Penn
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University of Pennsylvania Penn
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C

Definitions

  • the present disclosure relates to use of therapeutic agents that act by disrupting or inhibiting signaling through cell surface receptors, including peptide mimetics that inhibit signaling through the Fas receptor. More specifically, disclosed are methods of using such peptide mimetics to treat Fas-related pathologies, including Fas Receptor and Fas Ligand related processes and reverse signaling that can be modulated and result in reduced pathology or conditions. Fas related pathologies that can be treated include, but are not limited to, hair loss and skin related diseases characterized by epidermal changes and/or pathologies and inflammation.
  • FAS CD95/APO-1 and its specific ligand (FASL/CD95L) are members of the tumor necrosis factor (TNF) receptor (TNF-R) and TNF families of proteins, respectively.
  • TNF tumor necrosis factor
  • F-R tumor necrosis factor receptor
  • FAS is a 45 kDa type I membrane protein expressed constitutively in various tissues, including spleen, lymph nodes, liver, lung, kidney and ovary.
  • FASL is a 40 kDa type II membrane protein, and its expression is predominantly restricted to lymphoid organs and perhaps certain immune-privileged tissues.
  • FASL can induce cytolysis of FAS-expressing cells, either as a membrane-bound form or as a 17 kDa soluble form, which is released through metalloproteinase-mediated proteolytic shedding.
  • Fas receptors and Fas ligands are capable of signaling for both apoptotic and non-apoptotic function (Wajant et al, Cytokine & Growth Fact. Rev., 14:53-66 (2003)).
  • FasL is a type II membrane protein, from which soluble molecules can be derived by proteolysis [Tanaka et al, EMBOJ. , ⁇ A: ⁇ 129-35 (1995); Kayagaki et al, J Exp Med., 182: 1777-83 (1995).
  • FasL assembles in homotrimers via its extracellular carboxy terminal domain. It has been shown that the Fas-stimulatory capacity of membrane-bound FasL is magnitudes higher compared to soluble FasL (Suda et al, J. Ex Med., 186:2045-2050 (1997); Tanaka et al, Nat Med, 4:31-36 (1998); Schneider et al, J Ex.
  • FasL Binding of FasL to Fas receptor elicits apoptotic signals either via classical pathways or via indirect pathways (Mundle & Raza., Trends. Immuno., 23:187-194 (2002)). Independently, Fas and FasL stimulation alone can induce cell proliferation (Aggarwal et al , FEBS Lett, 364:5-8 (1995); Freiberg et al, J Invest Dermatol, 108:215-219 (1997); Jelaska & Korn, J. Cell. Physiol, 175:19-29 (1998); Suzuki et al, J Immunol, 165:5537-5543 (2000); Suzuki et al, J. Exp.
  • the FASL/FAS system has been implicated in the control of the immune response and inflammation, the response to infection, neoplasia, and death of parenchymal cells in several organs. (Nagata et al supra; Biancone, L. et al., J Exp Med, 186:147-152 (1997); Krammer, P.H. Adv Immunol, 71 :163-210 (1999); Seino, K. et al, J Immunol, 161 :4484-4488 (1998)). Defects of the FASL/FAS system can limit lymphocyte apoptosis and lead to lymphoproliferation and autoimmunity.
  • FAS-FAS A role for FASL-FAS in the pathogenesis of rheumatoid arthritis, Sjogren's syndrome, multiple sclerosis, viral hepatitis, renal injury, inflammation, aging, graft rejection, HIV infection and a host of other diseases has been proposed.
  • FAS mediated apoptosis is an important component of tissue specific organ damage, such as liver injury that has been shown to be induced through the engagement of the FAS-FASL receptor system.
  • Fas-FasL interactions Previous studies have undertaken a structural analysis Fas-FasL interactions, using an interaction model of FasL and Fas created by computer assisted modeling and designed peptide mimetics based on the deduced secondary structural features of Fas, as described in publication US 20040132641 Al corresponding to U.S. Patent 7,288,519, which is incorporated herein by reference in its entirety for all purposes.
  • the mimetics were previously shown to be effective in inhibiting ⁇ -FGF induced corneal neovascularization in vivo, demonstrating that Fas- FasL interaction plays a significant role in regulating extension of new blood vessels into the cornea, and indicating that Fas is a therapeutic target for eye related pathologies.
  • the in vivo biological activity of the mimetics was also validated in a murine model of Fas-dependent Con A induced hepatitis injury. Accordingly, the mimetics are therefore also useful as therapy for fulminant hepatitis. Moreover, the understanding of Fas-FasL recognition features at the atomic level allowed design of mimetics that modulate Fas signaling functions and can thus be used to treat multiple Fas pathologies.
  • apoptosis a complex death program becomes initiated that ultimately leads to the fragmentation of the cell, finally breaking it up into membrane-enclosed bodies that are involved in a variety of diseases, including Hashimoto's thyroiditis and Helicobacter py /on-induced gastritis (Giordano, C, et al, 1997. Science, 275:960-963; Rudi, J. et al, 1998. J. Clin. Invest, 102:1506-1514.). Triggering of FAS either by agonistic antibodies or by its cognate ligand, FASL, induces apoptosis.
  • FADD FADD-associating protein with death domain
  • FADD is responsible for downstream signal transduction by recruitment of the cysteine protease, caspase- 8. Subsequently, a cascade of downstream caspases executes apoptotic cell death (Peter, M.E., and Krammer, P. H. 1998. Curr. Opin. Immunol, 10:545-551). Perhaps the best studied of the cells subject to this apoptotic pathway are lymphocytes.
  • FAS and its ligand are essential for normal homeostasis of the lymphoid system, as seen when defects in the FAS system result in lymphadenopathy, splenomegaly, or auto-immunity (Nagata, S., and Suda, T, 1995, Immunol. Today, 16:39-43).
  • This pathway is also implicated in the progression of tumors, since tumor cells that express high levels of FASL have been shown to escape an immune response by killing FAS-bearing lymphocytes (Hahne, M., et al, 1996. Science, 274:1363-1366.).
  • FAS-mediated keratinocyte death recently has been shown to be involved in another dermatosis, toxic epidermal necrolysis (TEN) (Viard, L, et al, 1998, Science, 282:490- 493).
  • TEN is a life-threatening drug-induced cutaneous reaction with extensive epidermal destruction.
  • AD and ACD in TEN keratinocytes kill themselves by expressing FASL and thus do not need the help of lymphocytes.
  • the mechanism for up-regulation of the killer ligand is unknown, inhibition of the FAS pathway by application of neutralizing FAS antibodies appears to be a promising therapeutic approach (Viard, L, et al, 1998, Science, 282:490-493).
  • the present inventors have identified that altering FAS function in keratinocytes and hair follicles has a beneficial effect and will therefore have therapeutic applications.
  • the designed mimetics that modulate FAS signaling functions can be used to treat FAS pathology in an animal model and in human diseases that include and are not limited to eczema, hair loss, skin inflammation, rosacea and any skin or hair disease that involves the FASL or the FAS receptor.
  • the mimetics were effective in inhibiting DNFB induced contact hypersensitive (CHS) allergy in vivo, demonstrating that FAS-FASL interaction plays a significant role in regulating CD4+ and CD8+ T cells (Suzuki and Fink., Proc. Natl.
  • the mimetics are therefore also useful as therapy for eczema and other skin related inflammatory pathologies in which T cells play a role.
  • Hair loss is a common side effect of many chemotherapeutic protocols and is one of the most distressing aspects of cancer therapy. Because of the rapid proliferation of hair matrix keratinocytes during hair shaft production, the hair follicle (HF) represents a "by-stander" target for many chemotherapeutic agents (Paus R, et al, N EnglJ Med, 341 :491-497 (1999); Botchkarev., J /rc vest. Dermatol. Symp. Proc, 8:72-75 (2003)).
  • p53 and cyclin-dependent kinase2 molecular signaling pathways are involved in mediating HF responses to chemotherapy (Botchkarev et al, Cancer Res., 60:5002-5006 (2000); Davis et al, Science, 291 :134-137 (2001)).
  • Botchkarev, et al, ⁇ Cancer Res., 60:5002-5006 (2000) showed that p53 is essential for triggering apoptotic cell death in the HF induced by cyclophosphamide (CYP) and that genetic loss of p53 results in a complete resistance of murine HF to chemotherapy.
  • CYP cyclophosphamide
  • Some embodiments include methods of treating a Fas-related pathology of the skin or hair comprising administering to a mammal suffering from said pathology a pharmaceutical composition comprising a cyclicized peptide having an amino acid sequence derived from a Fas surface domain Kp7 (CDEGHGL (SEQ ID NO: 20)) that interacts with FasL.
  • a pharmaceutical composition comprising a cyclicized peptide having an amino acid sequence derived from a Fas surface domain Kp7 (CDEGHGL (SEQ ID NO: 20)) that interacts with FasL.
  • Another embodiment includes the methods described above, wherein the amino acids in said mimetic that correspond to aspartic acid and glutamic acid moieties of said surface domain Kp7 (SEQ ID NO: 20) are independently selected from either aspartic acid and glutamic acid.
  • B 1 and B 2 are each independently a peptide of 1-6 amino acids each independently comprising at least one aromatic amino acid;
  • X 3 and X 4 are each aspartic acid or glutamic acid
  • X 5 and X 7 are each independently glycine or a bond
  • X 6 is glutamic acid, glutamine, histidine, lysine, leucine, or tyrosine;
  • X 8 is glutamine, leucine, phenylalanine or tyrosine; or a pharmaceutically acceptable salt form thereof.
  • FAS-related pathology is vitiligo, psoriasis, alopecia areata, toxic epidermal necrolysis, eczematous dermatitis, macropapular rashes, or rosacea.
  • Additional embodiments include the methods described above wherein the FAS- related pathology is inflammation, Lyell syndrome, eczema, toxic epidermal necrolysis, and drug-induced skin eruptions.
  • Still additional embodiments include the methods described above wherein said FAS-related pathology is a hair cell related disease condition selected from alopecia areata, alopecia totalis, alopecia universalis, ophiasis, androgenic alopecia, telogen effluvium, lichen planopilaris, chemotherapy induced hair loss, and radiation induced hair loss.
  • Other embodiments include the methods described wherein said chemotherapy induced hair loss is induced by treatment with cyclophosphamide, chlormethine, mustine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, vpl ⁇ , ifosfamide, irinotecan, mitozantrone, mitoxantrone, paclitaxel, topotecan, vinblastine, vincristine, gemcitabin, or carboplatin.
  • cyclicized peptide comprises a sequence selected from YCDEGHLCY (SEQ ID NO: 1); YCDEGLCY (SEQ ID NO: 2); YCDEGYFCY (SEQ ID NO: 3); YCDEGEYCY (SEQ ID NO: 4); YCDEHFCY (SEQ ID NO: 5); YCDEHGLCY (SEQ ID NO: 6); YCDEHGQCY (SEQ ID NO: 7); YCDEKFCY (SEQ ID NO: 8); and YCDEQFCY (SEQ ID NO: 9).
  • FIGS. IA-B depict the three dimensional structure of the binding site in FasL/Fas complex.
  • FIG. IA depicts the interaction between a FasL dimer and Fas peptide mimetics KpI, Kp2, Kp3, Kp4 and Kp7.
  • FIG. IB depicts a putative solution structure of peptide mimetic Kp7-6.
  • FIGS. 2A-B illustrate inhibition of FasL binding to Fas-receptor by exocyclic mimetics in a binding assay.
  • FIGS. 3A-B depict a surface plasmon resonance (biosensor) analysis of mimetic binding to immobilized FasL.
  • FIG. 4 shows the efficacy of Kp7-6 dissolved in propylene glycol (Fig. 4, left) as compared to Kp7-6 dissolved in ethanoholive oil (Fig, 4, right) in a standard mouse model (measuring contact hypersensitivity (CHS) to an epicutaneously applied hapten(s)) of allergic contact dermatitis in humans.
  • CHS contact hypersensitivity
  • FIG. 5 depicts the time scale for the hair cycle in female C57BL/6J mice and changes of skin pigmentation and skin thickness after depilation (FIG. 5 corresponds to Fig. 2(b) in Muller-Rover, S., B. Handjiski, et al, J Invest Dermatol, 117(1):3-15 (2001)).
  • FIG. 6 depicts images of the results of Kp7-6 treatment in a murine model of chemotherapy-induced hair loss and quantification of hair regrowth corresponding to each treatment.
  • FIG. 7 depicts results from a contact sensitivity experiment in mice, where, at 4 hours after challenge with DNFB (day 0), mice received treatment as indicated including topical administration with the NLOO 12Ac containing F2 cream (NLOO 12Ac (F2)) or administration by IP injection (NLOO 12Ac (IP)).
  • F2 topical administration with the NLOO 12Ac containing F2 cream
  • IP IP injection
  • FIG. 8 depicts results from another contact sensitivity experiment showing clear reduction of contact sensitivity in mice that received topical NLOO 12Ac treatment (8 mg/ml, in PBS/F2 solution) starting 2 hours after DNFB challenge, once on day 0 and twice on day 1 as compared to control mice that received PBS/F2 solution.
  • FIG. 9 depicts results from yet another contact sensitivity experiment in mice showing treatment with F2 cream formulation of an acetate salt of Kp7-6 two hours after challenge with DNFB reduces contact sensitivity.
  • FIG. 10 depicts statistically significant results from an additional contact sensitivity experiment in mice showing treatment with F2 cream formulation of an acetate salt of Kp7-6 thirty minutes after challenge with DNFB reduces contact sensitivity.
  • FIG. 11 depicts inhibition of FasL-induced cytolysis in Jurkat cells by Kp7-6 peptides, specifically the TFA salt of the Kp7-6 peptide, the acetate salt of the Kp7-6 peptide, and the lactam-bridged analog of the Kp7-6 peptide (i.e., YDDEHF (diaminopropionic acid) Y (SEQ ID NO: 21)).
  • Kp7-6 peptides specifically the TFA salt of the Kp7-6 peptide, the acetate salt of the Kp7-6 peptide, and the lactam-bridged analog of the Kp7-6 peptide (i.e., YDDEHF (diaminopropionic acid) Y (SEQ ID NO: 21)
  • the present invention is concerned with mimetics that are antagonists of the Fas receptor (Fas)-Fas ligand (FasL) signaling system and methods of using such mimetics.
  • the invention is based in part on the findings that sites on Fas that function in the binding of FasL can be identified by comparison to the TNF receptor and that peptide mimetics of the sites identified on Fas act to inhibit binding of FasL to Fas and inhibit Fas function, i.e., the mimetics are antagonists of FasL binding and of Fas signaling and function.
  • the mimetics are therefore useful in the treatment of Fas-related pathologies.
  • the invention provides exocyclic peptide mimetics comprising an amino acid sequence of a Fas surface domain that interacts with FasL or derived from an amino acid sequence of a Fas surface domain that interacts with FasL.
  • B 1 and B 2 are independently a peptide of 1-6 amino acids, at least one of which is a hydrophobic amino acid, an aromatic moiety, or heteroaromatic moiety;
  • X 3 is a hydrophilic amino acid or a bond
  • X 4 is an amino acid selected from aspartic acid or glutamic acid
  • X 5 is an amino acid selected from aspartic acid or glutamic acid
  • Xe is an amino acid selected from the group consisting of histidine, lysine, arginine, asparagine, or glutamine;
  • B 1 and B 9 independently are exocyclic portions of mimetics that are comprised of, e.g., amino acid residues.
  • Z 2 , X 3 , X 4 , X 5 , X 6 , X 7 and Z 8 comprise the cyclicized portion of mimetics of formula (I).
  • Z 2 and Z 8 are further linking moieties, preferably linking amino acids.
  • Fas mimetics of the invention are conformationally restrained peptides.
  • such conformationally restrained peptides are cyclicized peptides comprising a cyclicized portion, one or more exocyclic region, one or more linking moiety and an active region.
  • Another embodiment is a method using a mimetic of formula (I) comprising an amino sequence selected from the group consisting of YCDEGHLCY (SEQ ID NO: 1), YCDEGLCY (SEQ ID NO: 2), YCDEGYFCY (SEQ ID NO: 3), YCDEGEYCY (SEQ ID NO: 4), YCDEHFCY (SEQ ID NO: 5), YCDEHGLCY (SEQ ID NO: 6), YCDEHGQCY (SEQ ID NO: 7), YCDEKFCY (SEQ ID NO: 8) and YCDEQFCY (SEQ ID NO: 9), wherein the cysteine residues of said amino acid sequence are joined by a covalent bond, to form a cyclic peptide.
  • YCDEGHLCY SEQ ID NO: 1
  • YCDEGLCY SEQ ID NO: 2
  • YCDEGYFCY SEQ ID NO: 3
  • YCDEGEYCY SEQ ID NO: 4
  • a mimetic of formula (I) comprises an amino sequence selected from the group consisting of YCDEHFCY (SEQ ID NO: 5), YCDEKFCY (SEQ ID NO: 8) and YCDEQFCY (SEQ ID NO: 9), wherein the cysteine residues of said amino acid sequence are joined by a covalent bond, to form a cyclic peptide.
  • a method using a mimetic comprising an amino acid sequence selected from the group consisting of YCNSTVCY (SEQ ID NO: 10), YCDKAEHFCY (SEQ ID NO: 11), YCNTRTQNTCY (SEQ ID NO: 12), YCQEKEYCY (SEQ ID NO: 13), and YCQERKEYCY (SEQ ID NO: 14).
  • Some embodiments include a method of treating a Fas-related pathology of the skin or hair comprising administering to a mammal suffering from said pathology a pharmaceutical composition comprising an cyclicized peptide having an amino acid sequence derived from a Fas surface domain Kp7 (SEQ ID NO: 20) that interacts with FasL.
  • amino acids in said mimetic that correspond to aspartic acid and glutamic acid moieties of said surface domain Kp7 are independently selected from either aspartic acid and glutamic acid.
  • B 1 and B 2 are each independently a peptide of 1-6 amino acids each independently comprising at least one aromatic amino acid;
  • X3 and X 4 are each aspartic acid or glutamic acid
  • X 5 and X 7 are each independently glycine or a bond
  • Xe is glutamic acid, glutamine, histidine, lysine, leucine, or tyrosine;
  • Xg is glutamine, leucine, phenylalanine or tyrosine; or a pharmaceutically acceptable salt form thereof.
  • FAS-related pathology is vitiligo, psoriasis, alopecia areata, toxic epidermal necrolysis, eczematous dermatitis, macropapular rashes, or rosacea.
  • FAS-related pathology is inflammation, Lyell syndrome, eczema, toxic epidermal necrolysis, and drug- induced skin eruptions.
  • FAS-related pathology is a hair cell related disease condition selected from alopecia areata, alopecia totalis, alopecia universalis, ophiasis, androgenic alopecia, telogen effluvium, lichen planopilaris, chemotherapy induced hair loss, and radiation induced hair loss.
  • Some mimetics provided in the invention are in the range of about 5 to about 100 amino acids in length. Additional mimetics provided in the invention are in the range of about 5 to about 50 amino acids in length, or in the range of about 10 to about 50 amino acids in length. Other mimetics provided in the invention are in the range of about 5 to about 40 amino acids in length, or in the range of about 10 to about 40 amino acids in length. Other mimetics provided in the invention are in the range of about 5 to about 30 amino acids in length, or in the range of about 10 to about 30 amino acids in length. Other mimetics provided in the invention are in the range of about 5 to about 20 amino acids in length, or in the range of about 10 to about 20 amino acids in length. Some mimetics provided in the invention are in the range of about 5 to about 10 amino acids in length.
  • amino acid residues used in the present invention may be recited by their full name or by reference to either the three letter or single letter amino acid code (see, e.g., Table 5.1 of Mathews & van Holde, Biochemistry, Second Edition (Benjamin/Cumings Publishing Company, Inc., New York) at page 131).
  • the mimetics that are encompassed within the scope of the invention are partially defined in terms of amino acid residues of designated classes.
  • the amino acids may be generally categorized into three main classes: hydrophilic amino acids, hydrophobic amino acids and cysteine-like amino acids, depending primarily on the characteristics of the amino acid side chain. These main classes may be further divided into subclasses.
  • Hydrophilic amino acids include amino acids having acidic, basic or polar side chains. Hydrophobic amino acids include amino acids having aromatic or apolar side chains. Apolar amino acids may be further subdivided to include, among others, aliphatic amino acids.
  • the definitions of the classes of amino acids as used herein are as follows:
  • hydrophobic amino acid refers to an amino acid having a side chain that is uncharged at physiological pH and that is repelled by aqueous solution.
  • examples of genetically encoded hydrophobic amino acids include He, Leu and VaI.
  • examples of non- genetically encoded hydrophobic amino acids include t-BuA.
  • aromatic amino acid refers to a hydrophobic amino acid having a side chain containing at least one ring having a conjugated pi-electron system (aromatic group).
  • aromatic group may be further substituted with substituent groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfanyl, nitro and amino groups, as well as others.
  • substituent groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfanyl, nitro and amino groups, as well as others.
  • genetically encoded aromatic amino acids include phenylalanine, tyrosine and tryptophan.
  • Non-genetically encoded aromatic amino acids include phenylglycine, 2- naphthylalanine, ⁇ -2-thienylalanine, l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 4- chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine and 4-fluorophenylalanine.
  • hydrophilic amino acid refers to an amino acid having a side chain that is capable of bonding to solvent molecules in an aqueous solution.
  • hydrophilic amino acids include Ser and Lys.
  • non-encoded hydrophilic amino acids include Cit and hCys.
  • heteroaromatic moiety refers to an aromatic moiety wherein one or more of the ring carbon atoms is replaced with another atom such as, for example, nitrogen, oxygen or sulfur.
  • Typical heteroaromatic moieties include, but are not limited to, pyran, pyrazole, pyridine, pyrrole, pyrazine, pyridazine, pyrimidine, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, selenophene, thiophere, tellurophene, xanthene, and the like.
  • mimetics refers to compounds which mimic the activity of a peptide.
  • Mimetics may themselves be peptides.
  • Mimetics may also be non-peptides and/or may comprise amino acids linked by non-peptide bonds, e.g., without limitation, psi bonds (see, e.g., Benkirane, N., et al, J. Biol. Chem., 271 :33218-33224 (1996)).
  • U.S. Pat. No. 5,637,677 and its parent applications contain detailed guidance on the production of mimetics.
  • Preferred mimetics are "conformationally constrained" peptides. Also preferred are cyclic mimetics.
  • constrained peptides and “conformationally constrained peptides” are used interchangeably and are meant to refer to peptides which, e.g., through intramolecular bonds, are conformationally stable, or substantially conformationally stable, and remain in a restricted conformation, or substantially restricted conformation.
  • exocyclic amino acid residue refers to an amino acid residue that is linked directly or indirectly to a cyclicized peptide but which is not within the portion of the peptide that makes up the circularized structure.
  • exocyclic portion refers to an amino acid sequence having one or more amino acid residues which are linked to cyclicized peptide but which are not within the portion of the peptide that makes up the circularized structure.
  • linking moiety refers to a molecular component or functional group which is capable of forming bonds with three amino acids.
  • linking amino acid residue refers to an amino acid residue that is a linking moiety.
  • active region refers to the amino acid sequence of the portion of a mimetic that directly interacts with a receptor or receptor ligand, wherein the interaction is characterized by an affinity between the active region of the mimetic and the receptor or receptor ligand.
  • Fas mimetic refers to a peptide mimetic that is derived from a Fas peptide; i.e., a Fas mimetic of the invention is a compound that mimics a Fas peptide (a peptide derived from Fas).
  • Preferred Fas mimetics are mimetics of a peptide derived from and/or corresponding to a Fas surface domain.
  • Further preferred are Fas mimetics that are mimetics of a peptide derived from and/or corresponding to a Fas surface loop domain.
  • Exemplary Fas peptides are provided, infra, in Table 1.
  • Particularly preferred Fas mimetics of the invention are mimetics of Fas Kp7surface domain peptides.
  • Fas mimetics are preferably derived from human or mouse Fas.
  • Fas mimetics are derived from human Fas, e.g., without limitation, human Fas.
  • mimetics are derived from human Fas with the mature amino acid sequence:
  • Fas mimetics are capable of interacting with (e.g., binding to) either Fas, FasL or both Fas and FasL.
  • Fas mimetics may therefore modulate Fas activity (in particular, Fas mediated signaling), for example as antagonists, agonists, or inverse agonists.
  • Fas mediated signaling for example as antagonists, agonists, or inverse agonists.
  • a Fas mimetic of the invention may inhibit binding of FasL to Fas, and may thereby inhibit Fas activity.
  • a Fas mimetic of the invention may bind to and activate Fas.
  • surface domain refers to a domain of a protein comprising one or more solvent accessible amino acid.
  • a surface domain make include, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 solvent accessible amino acids.
  • a surface domain may also include greater than 20 solvent accessible amino acids.
  • each amino acid in a surface domain is a solvent accessible amino acid.
  • Solvent accessible amino acids and hence surface domains may be identified using methods well known in the art (see e.g. , Jones et al., J. MoI. Biol, 272:121-132 (1997) and Samanta, U. et al, Prot. Eng., 15:659-667 (2002)).
  • Solvent accessibility of an amino acid is expressed as a value from 0.0 (buried) to 1.0 completely accessible.
  • a surface domain comprises at least one amino acid with a solvent accessibility of at least about 0.3. More preferably, a surface domain comprises at least one amino acid with a solvent accessibility of at least about 0.4, 0.5, 0.6 or 0.7. More preferably, a surface domain comprises at least one amino acid with a solvent accessibility of at least about 0.8, 0.9 or 0.95. Further preferred is where a surface domain comprises about 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids with a solvent accessibility of at least 0.3, or comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids with a solvent accessibility of at least about 0.3.
  • a surface domain comprises about 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids with a solvent accessibility of at least about 0.4, 0.5, 0.6, or 0.7, or comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids with a solvent accessibility of at least about 0.4, 0.5, 0.6, or 0.7. Further preferred is where a surface domain comprises about 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids with a solvent accessibility of at least about 0.8, 0.9, or 0.95, or comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids with a solvent accessibility of at least about 0.8, 0.9, or 0.95.
  • a surface domain consists of about 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids with a solvent accessibility of at least about 0.3, or consists of at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids with a solvent accessibility of at least about 0.3. Further preferred is where a surface domain consists of about 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids with a solvent accessibility of at least about 0.4, 0.5, 0.6, or 0.7, or consists of at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids with a solvent accessibility of at least about 0.4, 0.5, 0.6, or 0.7.
  • a surface domain consists of about 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids with a solvent accessibility of at least about 0.8, 0.9, or 0.95, or consists of at least about 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids with a solvent accessibility of at least about 0.8, 0.9, or 0.95.
  • treatment refers to administering an active agent to lessen the severity or the likelihood of the re-occurrence of a pre-existing condition.
  • treatment encompasses, for example and without limitation, ameliorating at least one symptom associated with a condition or reducing the rate of occurrence or re-occurrence of a condition.
  • the term "preventing” refers to the lessening of the likelihood of the occurrence of a condition.
  • a "subject in need of treatment of or a "subject suffering from” a condition is a mammal (e.g., human) that manifests at least one symptom of a condition or that is at risk of developing or re-developing the particular condition to be treated.
  • a "therapeutically effective amount" of an agent is an amount sufficient to ameliorate at least one symptom associated with a pathological, abnormal or otherwise undesirable condition, an amount sufficient to prevent or lessen the probability that such a condition will occur or re-occur, or an amount sufficient to delay worsening of such a condition.
  • a "Fas-related pathology” is a pathological condition that can be treated by increasing or decreasing activity (i.e., signaling) of Fas.
  • a "Fas-related pathology” is preferably treated with an antagonist of Fas activity. More preferably, said antagonist binds to one or both of Fas or FasL to lower Fas activity.
  • a "Fas- related pathology” may be treated with an agonist of Fas activity. Examples of "Fas-related pathologies" include, without limitation, pathologies related to lymphocyte apoptosis leading to abnormal lymphoproliferation and autoimmunity.
  • Additional examples include, without limitation, aging and cell death (e.g., in the skin and other organs), visual loss, rheumatoid arthritis, Sjogren's syndrome, multiple, viral hepatitis, renal injury, HIV infection, and angiogenesis. (See also Famularo, G., et al., Med. Hypotheses 53:50-62 (1999)).
  • the inhibition of FasL and Fas antigen interactions has also been used to prevent transplantation rejections.
  • Mimetics of the invention which inhibit the binding of Fas with FasL may be used to augment immune responses in certain settings.
  • a “metabolite” of a compound disclosed herein is a derivative of a compound which is formed when the compound is metabolized.
  • active metabolite refers to a biologically active derivative of a compound which is formed when the compound is metabolized.
  • metabolized refers to the sum of the processes by which a particular substance is changed in the living body. In brief, all compounds present in the body are manipulated by enzymes within the body in order to derive energy and/or to remove them from the body. Specific enzymes produce specific structural alterations to the compound.
  • cytochrome P450 catalyses a variety of oxidative and reductive reactions while uridine diphosphate glucuronyltransferases catalyze the transfer of an activated glucuronic-acid molecule to aromatic alcohols, aliphatic alcohols, carboxylic acids, amines and free sulfhydryl groups. Further information on metabolism may be obtained from The Pharmacological Basis of Therapeutics, 9th Edition, McGraw-Hill (1996), pages 11-17.
  • Metabolites of the compounds disclosed herein can be identified either by administration of compounds to a host and analysis of tissue samples from the host, or by incubation of compounds with hepatic cells in vitro and analysis of the resulting compounds. Both methods are well known in the art.
  • a "prodrug" of a compound disclosed herein is an inactive substance that converts into an active form of the disclosed compounds in vivo when administered to a mammal.
  • the compounds of the present invention are related to mimetics of Fas as disclosed above, including all enantiomers, diastereomers, crystalline forms, hydrates, solvates or pharmaceutically acceptable salts thereof, as well as active metabolites of these mimetics having the same type of activity.
  • An antagonist of Fas is a substance which diminishes or abolishes the effect of a ligand (agonist) which typically activates Fas receptor, e.g., FasL.
  • the antagonist may be, for example, a chemical antagonist, a pharmacokinetic antagonist, an antagonist by receptor block, a non-competitive antagonist or a physiological antagonist.
  • the antagonist is a chemical antagonist or an antagonist by receptor block.
  • the antagonist is preferably a Fas mimetic. Further preferred, the antagonist is a mimetic of Fas peptide KpI, Kp2,
  • a chemical antagonist is a substance wherein the antagonist binds the ligand in solution so the effect of the ligand is lost.
  • a pharmacokinetic antagonist is one which effectively reduces the concentration of the ligand at its site of action, for example, by increasing the rate of metabolic degradation of the active ligand.
  • Antagonism by receptor-block involves two important mechanisms: reversible competitive antagonism and irreversible, or non-equilibrium competitive antagonism. Reversible competitive antagonism occurs when the rate of dissociation of the antagonist molecules is sufficiently high such that, on addition of the ligand, displacement of chemical antagonist molecules from the receptors effectively occurs.
  • Non-competitive antagonism describes the situation where the antagonist exerts its blocking action at some point in the signal transduction pathway leading to the production of a response by the ligand.
  • mimetics are preferably antagonists of Fas.
  • mimetics are selective antagonists of Fas.
  • a selective antagonist of Fas is one which antagonizes Fas, but antagonizes other receptors of the TNF family of receptors only weakly or substantially not at all.
  • Additional embodiments include mimetic antagonists, which are those that selectively antagonize Fas receptor at low concentration, for example, those that cause a level of antagonism of 50% or greater at a concentration of 1000 mM or less.
  • Selective Fas mimetic antagonists can thus typically exhibit at least a 10-fold, in some embodiments a 100- fold and in other embodiments a 1000-fold greater activity for Fas than at other TNF receptors.
  • Fas mimetics preferably have one or more of the following properties:
  • Mimetics preferably exhibit antagonist potency (measured as IC 50 ) between 1 nM and 500 mM.
  • potency may be measured by determining the antagonist activity of mimetics in vivo or in vitro, including cell extracts or fractions of extracts. Inhibitory potency may also be determined using, as non-limiting examples, native or recombinant Fas, and/or soluble Fas. Fas binding may be determined using methods that are well known to those skilled in the art, such as ELISA and proliferation by MTT (Hansen et ah, J. Immunol. Methods, 199:203-210 (1989))
  • Preferred mimetics exhibit at least about 10-fold greater antagonist potency for
  • Fas compared to other TNF receptors. More preferred are compounds that exhibit about 100- fold greater antagonist for Fas, compared to other TNF receptors. Most preferred are compounds that exhibit about 1000-fold greater antagonist for Fas, compared to other TNF receptors.
  • Mimetics preferably bind to FasL and inhibit the interaction of FasL and Fas.
  • the binding affinity of mimetics to FasL can be described by different parameters, e.g., k on k off and K D .
  • the mimetics have affinities for FasL represented by values of kon greater than 10 M "1 s "1 , k o ff of less than 10 "3 s "1 or K D of less than 10 "1 M. More preferably, mimetics have affinities for FasL represented by values of k on greater than 10 M " s " , k o ff of less than 10 "4 s "1 or K D of less than 10 "5 M. Most preferably, mimetics have affinities for FasL represented by values of k on greater than 10 3 M "1 s "1 , k off of less than 10 "5 s "1 or K D of less than 10 "6 M.
  • mimetics having one or more of these properties are candidates for use in treatment of Fas-related pathologies in mammals and especially in humans.
  • Mimetics with one or more of the above properties can further be tested for biological activity using assays that measure Fas function in vitro or in vivo.
  • Measurement of mimetic biological activity can be determined in vitro, e.g., by measuring inhibition of Fas-L induced ctyotoxicity or by inhibition of Fas-L induced apoptosis in cell culture, as described below in Examples 4 and 5, respectively.
  • mimetic biological activity may also be measured by directly or indirectly measuring signaling aspects of Fas activity, such as caspase activity, NF-.kappa.B activation, and/or other downstream mediators of Fas signaling.
  • a useful animal model for measurement of Fas activity in vivo is the Con A- induced hepatitis model described previously.
  • Other models and methods for measuring Fas activity in vivo are known in the art and/or are described herein.
  • a mimetic is designed based on a known region of FAS.
  • the mimetic mimics an extracellular domain of an Fas, more preferably a cystine knot region of Fas.
  • the mimetic is based on the Fas Kp7 domain.
  • Example 1 describes one embodiment in which an interaction model of FasL and Fas was created by computer assisted modeling, and used to design peptide mimetics based on deduced secondary structural features of Fas.
  • a skilled user may define optimal interactions of Fas surfaces that are consistent with either available biological data or structural surface complementarity.
  • Peptide mimetics can then be designed and synthesized for all secondary structures involved in such interactions, typically corresponding to about five mimetic compounds. Each compound may be modified as described, e.g., in U.S. Pat. Nos. 6,100,377 and 6,265,535. The mimetic compounds thus obtained can then be tested, e.g., using methods described in this application, for their activity as Fas agonists or antagonists.
  • the mimetics disclosed herein and in references incorporated herein may be prepared using virtually any art-known technique for the preparation of peptides and peptide analogues.
  • the peptides may be prepared in linear or non-cyclized form using conventional solution or solid phase peptide syntheses and cyclized using standard chemistries.
  • the chemistry used to cyclize the peptide will be sufficiently mild so as to avoid substantially degrading the peptide.
  • Suitable procedures for synthesizing the peptides described herein as well as suitable chemistries for cyclizing the peptides are well known in the art.
  • Formation of disulfide linkages is generally conducted in the presence of mild oxidizing agents.
  • Chemical, enzymatic or photolytic oxidation agents may be used.
  • Various methods are known in the art, including those described, for example, by Tarn, J. P. et al, 1979, Synthesis, 955-957; Stewart et al, 1984, Solid Phase Peptide Synthesis, 2d Ed., Pierce Chemical Company Rockford, III; Ahmed et al, 1975, J. Biol. Chem., 250:8477-8482; and Pennington et al., 1991 Peptides, 1990 164-166, Giralt and Andreu, Eds., ESCOM Leiden, The Netherlands.
  • the peptide is composed entirely of gene-encoded amino acids, or a portion of it is so composed, the peptide or the relevant portion may also be synthesized using conventional recombinant genetic engineering techniques. The isolated peptides, or segments thereof, are then condensed, and oxidized, as previously described, to yield a cyclic peptide.
  • a polynucleotide sequence encoding a linear form of the peptide is inserted into an appropriate expression vehicle, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation.
  • an appropriate expression vehicle i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation.
  • the expression vehicle is then transfected into a suitable target cell which will express the linear form of the cyclic peptide.
  • the expressed peptide is then isolated by procedures well-established in the art.
  • microorganisms such as bacteria transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing an appropriate coding sequence; yeast or filamentous fungi transformed with recombinant yeast or fungi expression vectors containing an appropriate coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an appropriate coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing an appropriate coding sequence; or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing an appropriate coding sequence; yeast or filamentous fungi transformed with recombinant yeast or fungi expression vectors containing an appropriate coding sequence; insect cell systems infected with recomb
  • the expression elements of the expression systems vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used in the expression vector. For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage X, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used; when cloning in insect cell systems, promoters such as the baculovirus polyhedron promoter may be used; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g., heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV) may be used; when cloning in
  • sequences encoding the peptides of the invention may be driven by any of a number of promoters.
  • promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al, 1984, Nature, 310:511-514), or the coat protein promoter of TMV (Takamatsu et al, 1987, EMBO J.
  • plant promoters such as the small subunit of RUBISCO (Coruzzi et al, 1984, EMBOJ., 3:1671-1680; Brogue et al, 1984, Science, 224:838- 843) or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B (Gurley et al, 1986, MoI Cell Biol, 6:559-565) may be used.
  • These constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc.
  • Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express the foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • a coding sequence may be cloned into non-essential regions (for example the polyhedron gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedron promoter).
  • Successful insertion of a coding sequence will result in inactivation of the polyhedron gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedron gene).
  • a number of viral based expression systems may be utilized.
  • a coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing peptide in infected hosts, (e.g., See Logan et ah, 1984, Proc.
  • the vaccinia 7.5 K promoter may be used, (see, e.g., Mackett et ah, 1982, Proc. Natl. Acad. Sci. USA, 79:7415-7419; Mackett et al, 1984, J. Virol, 49:857-864; Panicali et al, 1982, Proc. Natl. Acad. Sci. USA, 79:4927-4931).
  • peptides and peptide analogues disclosed herein and in references incorporated by reference herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography and the like.
  • the actual conditions used to purify a particular peptide or analogue will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those having skill in the art.
  • any antibody which specifically binds the peptides or peptide analogues may be used.
  • various host animals including but not limited to rabbits, mice, rats, etc., may be immunized by injection with a linear or cyclic peptide.
  • the peptide may be attached to a suitable carrier, such as BSA, by means of a side chain functional group or linkers attached to a side chain functional group.
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum bacilli Calmette-Guerin
  • Monoclonal antibodies to a peptide may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein, 1975, Nature 256:495-497, the human B-cell hybridoma technique, Kosbor et al., 1983, Immunology Today 4:72; Cote et al, 1983, Proc. Natl. Acad. Sci. USA, 80:2026-2030 and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)).
  • Antibody fragments which contain deletions of specific binding sites may be generated by known techniques.
  • fragments include but are not limited to F(ab') 2 fragments, which can be produced by pepsin digestion of the antibody molecule and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries may be constructed (Huse et al., 1989, Science, 246:1275- 1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for the cyclic peptide of interest.
  • the antibody or antibody fragment specific for the desired cyclic peptide can be attached, for example, to agarose, and the antibody-agarose complex is used in immunochromatography to purify cyclic peptides of the invention. See, Scopes, 1984, Frotein Purification: Principles and Practice, Springer- Verlag N. Y., Inc., NY, Livingstone, 1974, Methods Enzymology: Immunoaffinity Chromatography of Proteins, 34:723-731.
  • compositions comprising the compounds of the invention may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the active peptides or peptide analogues into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the compounds of the invention may be formulated as solutions, gels, ointments, creams, suspensions, etc., as are well-known in the art.
  • Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral or pulmonary administration.
  • the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • the solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • the compounds can be readily formulated by combining the active peptides or peptide analogues with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • suitable excipients include fillers such as sugars, such as lactose, sucrose, mannitol and sorbitol; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulating agents; and binding agents.
  • disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • solid dosage forms may be sugar-coated or enteric-coated using standard techniques.
  • suitable carriers, excipients or diluents include water, glycols, oils, alcohols, etc. Additionally, flavoring agents, preservatives, coloring agents and the like may be added.
  • the compounds may take the form of tablets, lozenges, etc., formulated in conventional manner.
  • the compounds for use according to the present application are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneous Iy or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • Liposomes and emulsions are well known examples of delivery vehicles that may be used to deliver peptides and peptide analogues of the invention.
  • Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
  • the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the therapeutic agent.
  • sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
  • the mimetics may contain charged side chains or termini, they may be included in any of the above-described formulations as the free acids or bases or as pharmaceutically acceptable salts.
  • Pharmaceutically acceptable salts are those salts which substantially retain the antimicrobial activity of the free bases and which are prepared by reaction with inorganic acids. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base forms.
  • the compounds of the invention will generally be used in an amount effective to achieve the intended purpose.
  • the compounds of the invention, or pharmaceutical compositions thereof are administered or applied in a therapeutically effective amount.
  • therapeutically effective amount is meant an amount effective ameliorate or prevent the symptoms, or prolong the survival of, the patient being treated. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art.
  • a therapeutically effective dose can be estimated initially from in vitro assays.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of test compound that inhibits 50% of Fas:Fas-L binding interactions). Such information can be used to more accurately determine useful doses in humans.
  • Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the compounds which are sufficient to maintain therapeutic effect.
  • Usual patient dosages for administration by injection range from about 0.01 to about 25 mg/kg/day, preferably from about 0.1 to about 10 mg/kg/day and more preferably from about 0.5 to about 5 mg/kg/day. Also preferred are total daily dosages from about 25 to about 1000 mg per day, preferably from about 100 to about 750 mg per day and more preferably from about 250-500 mg per day.
  • Therapeutically effective serum levels may be achieved by administering multiple doses each day.
  • the effective local concentration of the compounds may not be related to plasma concentration.
  • One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.
  • the amount of compound administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing health professional.
  • the therapy may be repeated intermittently while symptoms are detectable or even when they are not detectable.
  • the therapy may be provided alone or in combination with other drugs.
  • the drugs that may be used in combination with the mimetics of the invention include, but are not limited to, anti-inflammatories, steroids and antimetabolites (for example, methotrexate).
  • Drugs used "in combination" are administered as part of a common method of treating a given pathology. Drugs used in combination may be administered in single or separate dosage forms. Separate dosage forms may be administered at the same or different times.
  • a therapeutically effective dose of the compounds described herein will provide therapeutic benefit without causing substantial toxicity.
  • Toxicity of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD 5O (the dose lethal to 50% of the population) or the LD 1O o (the dose lethal to 100% of the population).
  • the dose ratio between toxic and therapeutic effect is the therapeutic index.
  • Compounds which exhibit high therapeutic indices are preferred.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human.
  • the dosage of the compounds described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • mice C57BL/6 (B6) mice were purchased from CLEA JAPAN Inc. (Tokyo, Japan). All mice used were maintained under specific pathogen- free conditions in our animal facility. For most experiments, eight-week-old mice were purchases.
  • Fas/FasL complex was optimized using rigid body minimization using XPLOR 3.1 (Brunger, A. T. X-PLOR. Version 3.1. A System for X-ray Crystallography and NMR. (Yale University Press, New Haven, Conn., 1992)) and INSIGHT.
  • Mimetics were designed from essential sequences of Fas-FasL interactions. About five to seven amino acid sequences of Fas, have been used to mimic the conformation of loops KpI to Kp7as shown in Table 1, infra.
  • Peptide synthesis and cyclization Peptides were synthesized by solid-phase methods, deprotected, and released from the resin using anhydrous HF. Peptides were lyophilized and further purified by HPLC utilizing a Cl 8 column and then relyophilized. Peptides were more than 95% pure by HPLC analysis and mass spectrometry.
  • peptides containing internal cysteine residues were refolded and oxidized as described previously. (Takasaki, W. et al. Nat Biotechnol 15, 1266-1270 (1997)). Briefly, peptides were dissolved at 100 ⁇ g/ml in distilled water adjusted to pH 8.0 by (NH 4 ) 2 CO 3 and stirred at 4° C until 95% formation of intramolecular disulfide bonds had been confirmed by DTNB (Sigma Biochemical Co., St. Louis, Mo.). The cyclized peptides were lyophilized and analyzed for purity by HPLC. These peptides showed greater than 90% purity by HPLC analysis.
  • Peptides containing internal lactam bridges may be prepared using appropriate protecting groups and cyclizing on the resin.
  • the lactam-bridged analog of Kp 7-6, YDDEHF(diaminopropionic acid)Y (SEQ ID NO: 21) was synthesized on the resin using standard Fmoc method using Fmoc-Dap (Ivdde) and Fmoc-Asp (Odmab). Cyclization was carried out on the resin.
  • the cyclised peptide resin was cleaved using TFA (trifluoroacetic acid) and scavengers.
  • the crude material was purified using a TFA buffer system to yield the peptide at a purity of approximately 98%.
  • Fas-Fc fusion protein 250 ng/ml diluted in PBS was immobilized onto 96 well ELISA plate (Costar, High Wycombe, UK) by incubating overnight at 4° C. After blocking with PBS containing 1% skim milk overnight at 4° C and subsequent washing with PBS containing 0.05% Tween 20 (PBS-Tw), Flag-tagged soluble FasL (100 ng/ml)/peptide solution preincubated in PBS containing 1% skim milk for one h at 37° C was added onto the Fas-Fc coated wells.
  • the plate was washed with PBS-Tw, and anti-FLAG(M2)-HRP antibody 1 :2500 in PBS containing 1% skim milk was added. After 1 h incubation at room temperature, the plate was washed with PBS-Tw, and the enzyme reaction was started by adding the substrate solution (0.1M sodium acetate buffer (pH 5.0) containing 100 ⁇ g/ml of TMBZ and 0.005% (v/v) H 2 O 2 ) and stopped with 2N H 2 SO 4 . The absorbance at 450 nm was measured with an ELISA reader.
  • substrate solution 0.1M sodium acetate buffer (pH 5.0) containing 100 ⁇ g/ml of TMBZ and 0.005% (v/v) H 2 O 2
  • Biosensor analysis All experiments were carried out on a BIAcore 3000 instrument (Biacore A G, Uppsala, Sweden) at 25° C using PBS, pH 7.4, containing 0.005% surfactant P20 (Biacore A G) as the running buffer. FasL-Flag, Fas-Fc or TNFRI-Fc was immobilized on research-grade CM5 sensor chips (Biacore AG) using standard N-ethyl-N- dimethylaminopropyl carbodiimid/N-hydroxysuccinimide coupling.
  • Immobilization was performed in 10 mM sodium acetate buffer at pH 4.5 for FasL-Flag and Fas-Fc, and at pH 4.0 for TNFRI-Fc. After coupling, excess N-hydroxysuccinimide groups were inactivated with ethanolamine. For binding studies, about 1500 resonance units (RU) of FasL-Flag, Fas-Fc and TNFRI-Fc were coupled to the chips. Surface plasmon resonance (SPR) measurements were carried out at a flow rate of 20 ml min "1 . Data were analyzed with the BIA evaluation 3.0 software (Biacore AG). The sensograms give values for the relative response in resonance units (RU) after background subtraction versus time in seconds. The association phase injection time was 300 seconds followed by dissociation buffer.
  • SPR Surface plasmon resonance
  • Cytotoxicity assay Twenty microliters of Jurkat cells at 1 x 10 5 cells/ml were plated in 96-well U-bottom plates. FasL-Flag (120 ng/ml in culture medium) was preincubated with equal volume of peptide sample in PBS for 1 hour at 37°C, and 20 ⁇ l of mixture was added to each well. After an incubation period of 24 hours, each culture was pulsed with 1 ⁇ Ci of ⁇ - thymidine for 24 hours before harvesting on glass fiber filters. Incorporation of the ⁇ - thymidine obtained with culture medium alone and with 30 ng/ml of FasL was used as reference for 100% survival and 0% survival, respectively.
  • Results are expressed as mean.+-.SE, and analyzed by the Student's t test or analysis of variance (ANOVA) where appropriate. Post hoc comparisons were performed using Scheffe test. A 95% confidence interval was used to define statistical significance.
  • Fas a member of TNF superfamily, shares significant structural homology with the TNF receptor.
  • the structure of the TNF receptor contains distinct "cystine-knot" repeating domains. (Naismith, J. H. et al Structure 4, 1251-1262 (1996)). Loop structures in the first three domains as well as ⁇ -turns in proteins are considered to mediate roles in molecular recognition and binding. (Leszczynski, J. F. et al, Science, 234:849-855 (1986)).
  • cystine -knot peptide mimetic sites of protein-protein interaction that might be disrupted or influenced by small molecules were identified.
  • FIG. IA A molecular model of the Fas and FasL complex (FIG. IA) was developed using the crystal structure of TNF receptor complex in our methodology as well as other already published models.
  • the overall features of the receptor-ligand interaction were noted to be very similar to that of TNF receptor ligand complex. See, U.S. Pat. No. 6,265,535.
  • Fas-FasL contact sites predicted by the molecular model are consistent with the mutation analysis data. (Beltinger, C. et al, British Journal ofHaematology, 102:722- 728 (1998)).
  • the Fas-FasL structural model suggested five surfaces where FasL can bind to Fas, designated KpI, Kp2, Kp3, Kp4 and Kp7 (FIG. IB), compared to such three sites identified in the TNF receptor (Takasaki, W. et al. Nat Biotechnol 15, 1266-1270 (1997)).
  • the amino acids in the loops KpI, 2, 3, 4 and 7 adopt well defined conformations (i.e., adopt statistically allowed conformations) as judged by Ramachandran plots (Ramachandran, G. N. et al., Biopolymers, 6:1255-1262 (1968); Ramachandran, G. N. et al, Adv Protein Chem, 23:283-438 (1968)) and profile analyses. (Zhang, K. Y. et al, Protein Sci, 3:687-695 (1994)).
  • Peptide analogs were designed from the various loop structures. Peptido- mimetics were cyclized and constrained with cysteine disulfide bridges or lactam bridges. Each mimetic was optimized for its ability to mimic the binding conformation of the loop and for its ring size which was determined to be critical to reduce the inherent flexibility of mimetics. Specific features optimized included conformational mimicry between the loop structure and the mimetic, hydropathic value of the mimetics, dissociation rate (as measured from surface plasmon resonance; see e.g., Example 3, infra), stability and solubility.
  • One set of mimetics selected for biological assay is shown in Table 1.
  • Fas mimetic activity was evaluated in an assay that measured FasL-Flag binding (100 ng/ml) to Fas-Fc fusion protein immobilized onto plastic plates.
  • the first generation mimetics Kp 1-1, 2-2, 3-2,4-2 and 7-2 were designed from different deduced binding sites of Fas to FasL and screened using a binding inhibition assay comprising 300 ⁇ M of peptide and 20 nM of soluble Fas receptor (FIG. 2A).
  • the aspartic and glutamic acids in Kp7 loop appear to represent the most relevant residues involved in the interaction.
  • the particular acidic amino acid at each position is not critical.
  • either an aspartic acid or a glutamic acid residue can be present at either position. Modification of other residues of Kp 7 led to some improvement of inhibitory activities as seen with the Kp7 series (FIG. 2A).
  • FIG. 3A shows the sensogram result obtained with these different Kp7-6 concentrations.
  • the k on and k off rate constants were estimated to be 6.85 x 10 1 M " 1 s "1 and 7.65 x 10 ⁇ 4 s "1 , respectively, and a K D of value of 1.12 x 10 ⁇ 5 M was obtained from the ratio of the dissociation/association rate constants.
  • the k o ff value is considered as an important indicator in the development of therapeutics with biological activity (Benveniste, M. et al Br J Pharmacol 104, 207-221 (1991); Yiallouros, I. et al, Biochem J, 331 :375-379 (1998)), and generally correlates with potent biological effects. (Moosmayer, D. et al, J Interferon Cytokine Res, WAlX-Ml (1996)). Although the K D of the Kp7-6-FasL interaction showed less affinity than that noted for Fas-FasL interactions (Starling, G. C.
  • CHS contact hypersensitivity
  • a standard CHS assay was employed in which groups of 6-7 C57BL/6 female mice age 7-9 weeks were shaved on their abdomen and painted with 25 ⁇ l of either 0.5% 2,4-Di- nitro-1-Fluorobenzene (DNFB) dissolved in acetone:olive oil (4:1) or vehicle alone. Five days later mice were challenged by application of lO ⁇ l of 0.2% DNFB on each side of one ear. The degree of ear swelling, which is a surrogate measure for the degree of immune response, was measured over 4-5 days using an engineer's micrometer. Kp7-6 (0.2%) was applied topically only once either to the abdomen 10 minutes after sensitization or to the ear 10 minutes after challenge depending on the experiment. Kp7-6 was dissolved either in 10% Propylene glycol or in ethanol: olive oil (1 :4).
  • DNFB 2,4-Di- nitro-1-Fluorobenzene
  • Kp7-6 dissolved in propylene glycol ( Figure 4, left) was compared with Kp7-6 dissolved in ethanoholive oil ( Figure 4, right).
  • Kp7-6 produced a statistically insignificant reduction in CHS.
  • the reduction in CHS was observable on day 1 but became more pronounced on subsequent days. This was unexpected given that Kp7-6 was only applied on day 0.
  • Kp7-6 may act by inhibiting the signals that initiate the inflammatory response or by any other process which involves FAS or FASL.
  • Kp7-6 was able to limit hair loss in the standard mouse chemotherapy induced hair loss model when applied topically to the backs of mice that had their hair removed (depilated). Moreover, Kp7-6 applied topically to the skin of mice that had their hair removed (depilated) but were not treated with chemotherapy also had accelerated hair re-growth. Therefore agents which alter FAS or FASL related processes may have significant benefit in treating hair loss that is induced by any chemotherapy agent that is known to lead to hair loss in humans.
  • Kp7-6 species, and other mimetics of the present invention having an amino acid sequence derived from a Fas surface domain Kp7, sequence CDEGHGL (SEQ ID NO: 20), also promote accelerated hair growth and may be useful in treating hair loss in humans caused by hormonal or aging processes.
  • mice were purchased from Jackson Labs (Bar Harbor, ME). Age upon arrival was 42 days, or 6 weeks. After a one week acclimation period, all 45 mice (then 7 weeks old), were sedated with 135 mg/kg of ketamine in sterile water, delivered via IP administration. Once animals were sedated, a commercial body wax remover was used to depilate hair from the dorsal area of the mouse. Depilated area covered from the scapula to the rear hip area and about 3 A inch across the back. None of the mice experienced lesions or irritations from the waxing.
  • mice were returned to cages for one week.
  • mice were divided into 5 treatment groups (9 mice per group): Group 1 : Pre and Post chemotherapy treatment with Kp7-6
  • Group 2 Pre and Post chemotherapy treatment with Vehicle only
  • Group 5 Wax only (Control-no chemotherapy, no Kp7-6, no Vehicle)
  • mice in Group 1 received two IP injections per day (one AM and one PM injection) of lOO ⁇ l of Kp7-6 in 10% DMSO/PBS at 6mg/kg mouse, and mice in Group 2 received the same volume of Vehicle only.
  • all mice except the negative control, Group 5) received cyclophosphamide (CYP) treatment at 150 mg/kg mouse via IP injection.
  • mice in Groups 1 and 3 received 2 IP injections of Kp7-6 (one AM and one PM injection), whereas mice in Groups 2 and 4 received vehicle alone. All mice that received Kp7-6 received the same total amount of Kp7-6.
  • mice in groups 1 and 2 were euthanized via CO 2 , photographed and the dorsal skin harvested into Zamboni's fixative.
  • mice in Groups 3 and 4 were treated as above with either 2 injections of Kp7-6 or vehicle.
  • Groups 3 and 4 were euthanized on day 16 after a total of 6 days of treatment (post-CYP) with either the vehicle or Kp7.
  • Mice were photographed and dorsal skin harvested into Zamboni's fixative for 24 hours after which all samples were transferred to cryopreservative with azide for extended storage. Images were captured with a Canf ⁇ eld Scientific Rodent Imaging System and are shown in Figure 6. Hair growth density of the depilated are was calculated by thresholding and counting percent area of hair growth (pixels) /depilated area (pixels) for each group using Photoshop; select data is shown in Figure 6.
  • mice [0174] Visual inspection of mice, post euthanization, by 3 blind observers, revealed a noticeable difference in hair growth between the vehicle-treated mice and the Kp7-6 treated mice as follows:
  • mice treated with the Kp7-6 peptide had more hair in the depilated area compared to mice that received vehicle only.
  • the animals treated with Kp7-6 before and after cyclophosphamide had an even higher percentage of hair coverage compared to the animals treated with vehicle. (Groups 2 and 4).
  • Example 6 In vivo suppression of contact sensitivity by NLOOHAc, an acetate salt of the peptide of Kp7-6
  • mice (> 6 weeks old) were used. One week after the mice were delivered to the animal facility, the abdomens of mice were shaved (10 mm x 10 mm). 2. Sensitization: The skin of Balb/c mice was sensitized with 20 ⁇ l of DNFB (1%, in 4:1 acetone-olive oil). The sensitizing fluid was discharged, using a pipetteman, to the shaved area on the abdomen. The mice were held for 20 seconds to allow evaporation of the sensitizing fluid.
  • DNFB 1%, in 4:1 acetone-olive oil
  • NLOO 12Ac was applied to the challenge site on the ear.
  • NLOO 12Ac is an acetate salt of the peptide of Kp7-6, normally used, including supra, as a TFA salt.
  • mice were assigned to each group. At 4 hours after challenge with DNFB (day 0), mice received treatment as indicated in Figure 7.
  • NLOO 12Ac (F2) treatment indicates topical administration with the NLOO 12Ac containing F2 cream
  • NL0012Ac IP indicates administration by IP injection.
  • F2 topical administration with the NLOO 12Ac containing F2 cream
  • IP NL0012Ac
  • HCV Hydrocortisone Valerate, the positive control
  • the control group did not fully develop contact sensitivity, possibly due to the interference from HCV applied to the other ear of the same animal.
  • NL0012Ac administered by IP injection did not show activity, but the "NLOO 12Ac (F2)" group showed some activity as compared with the control and "NL0012Ac IP.” groups.
  • application of the NL0012Ac F2 solution was found to slightly increase ear thickness. This interference was subtracted during the analysis of the data.
  • mice received topical NLOO 12Ac treatment (8 mg/ml, in PBS/F2 solution) starting 2 hours after DNFB challenge, once on day 0 and twice on day 1. At the same time, the control mice received PBS/F2 solution.
  • NL0012Ac clearly reduced the contact sensitivity induced by DNFB.
  • Separate control groups which were treated with PBS/F2 solution, were used for the different time points.
  • mice were 11-13 weeks old. Unexpectedly strong contact sensitivity was encountered with these mice. At only 30 minutes post DNFB challenge, redness and swelling in the ear was observed, while in the previous experiments, described supra, only slight redness in the ear was observed ( Figures 9 and 10). When the first NLOO 12Ac treatment (in PBS/F2) was applied at 2 hr, no suppression of contact sensitivity was observed. However, the group of mice treated with the topical application of NL0012Ac-F2 cream showed reduced contact sensitivity (Figure 9), suggesting the topical F2 cream is better than the PBS-diluted F2.
  • FasL-sensitive Jurkat cells were stimulated with soluble FasL-Flag fusion protein in the presence of various concentrations of the various Kp7-6 mimetics.
  • the inhibitory effects of the mimetics on Fas-mediated cytotoxicity were consistent with the results of the FasL-binding inhibition in Example 2. As shown in Figure 11, the inhibitory effects of the
  • Kp7-6 mimetic on Fas-mediated cytoxicity were also consistent with the results of the FasL- binding inhibition in Example 2.
  • Kp7-6 showed a dose-dependent inhibitory activity.
  • a concentration of 1 mg/ml of the lactam-bridged analog of Kp7-6 i.e., a mimetic comprising the sequence YDDEHF(diaminopropionic acid)Y (SEQ ID NO: 21) protected more than 90% cells from Fas-mediated cytotoxicity (Figure 11). Cytotoxicity of the lactam-bridged analog of Kp7-6 was compared to the cytoxicity of the acetate salt of the Kp7-6 peptide and the TFA salt of the Kp7-6 peptide ( Figure 11). The cyclic peptides alone did not mediate any cytotoxicity for Jurkat cells in the range of concentration tested (data not shown).

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Abstract

L'invention concerne des substances mimétiques peptidiques exocycliques qui modulent l'activité biologique de FAS et FASL dans une pathologie liée à la peau in vivo. Les substances mimétiques sont utiles, par exemple pour traiter des pathologies liées au FAS et au FASL de la peau et des cheveux.
PCT/US2009/030454 2008-01-08 2009-01-08 Procédés de traitement utilisant des agents pour réguler la fonction du récepteur fas dans des pathologies liées à la peau et aux cheveux Ceased WO2009089362A1 (fr)

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US8680047B2 (en) 2002-05-23 2014-03-25 The Trustees Of The University Of Pennsylvania Fas peptide mimetics and uses thereof
KR20200067745A (ko) * 2018-12-04 2020-06-12 주식회사 시그넷바이오텍 Fas 신호전달 억제용 펩티드를 포함하는 비만, 지방간 또는 지방간염의 예방 또는 치료용 약학적 조성물

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8680047B2 (en) 2002-05-23 2014-03-25 The Trustees Of The University Of Pennsylvania Fas peptide mimetics and uses thereof
KR20200067745A (ko) * 2018-12-04 2020-06-12 주식회사 시그넷바이오텍 Fas 신호전달 억제용 펩티드를 포함하는 비만, 지방간 또는 지방간염의 예방 또는 치료용 약학적 조성물
KR102173183B1 (ko) 2018-12-04 2020-11-03 주식회사 시그넷바이오텍 Fas 신호전달 억제용 펩티드를 포함하는 비만, 지방간 또는 지방간염의 예방 또는 치료용 약학적 조성물
CN113164549A (zh) * 2018-12-04 2021-07-23 斯格尼特生物技术有限公司 包含针对Fas信号传导的抑制肽的用于预防或治疗肥胖症、脂肪肝或脂肪性肝炎的药物组合物

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