WO2009089362A1 - Methods of treatment using agents for regulating fas receptor function in skin and hair related pathologies - Google Patents

Abstract

Exocyclic peptide mimetics that modulate FAS and FASL biological activity in skin related pathology in vivo. The mimetics are useful, e.g., for treating FAS and FASL-related pathologies of the skin and hair.

Description

METHODS OF TREATMENT USING AGENTS FOR REGULATING FAS RECEPTOR FUNCTION IN SKIN AND HAIR RELATED PATHOLOGIES

TECHNICAL FIELD

[0001] 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.

CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application 61/010,334, filed January 8, 2008.

BACKGROUND

[0003] 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. (Nagata, S. et al,. Science, 267:1449-1456 (1995). Interaction between FAS and FASL triggers a cascade of subcellular events that results in a definable cell death process in FAS-expressing targets. FAS is a 45 kDa type I membrane protein expressed constitutively in various tissues, including spleen, lymph nodes, liver, lung, kidney and ovary. (Leithauser, F. et al, Lab Invest, 69:415-429 (1993); Watanabe-Fukunaga, R. et al, J Immunol 148:1274-1279 (1992)). FASL is a 40 kDa type II membrane protein, and its expression is predominantly restricted to lymphoid organs and perhaps certain immune-privileged tissues. (Suda, T. et al, Cell, 75:1169-1178 (1993); Suda, T. et al, J Immunol, 154:3806-3813 (1995)). In humans, 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. (Kayagaki, N. et al, JExp Med, 182:1777-1783 (1995); Mariani, S.M. et al, Eur J Immunol, 25:2303-2307 (1995)).

[0004] Fas receptors and Fas ligands (FasL) are capable of signaling for both apoptotic and non-apoptotic function (Wajant et al, Cytokine & Growth Fact. Rev., 14:53-66 (2003)). Like other proteins of this ligand superfamily, 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). Chemical cross-linking (Tanaka et al, EMBOJ., 14:1129-35 (1995)) and sequence homologies between FasL and other members of the TNF ligand superfamily(TNFSF), whose crystal structure have been resolved (Fesik SW, Cell, 103:273-283 (2000)) suggest that 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. Med, 187:1205-1213 (1998)). Although soluble FasL fails to activate Fas properly, it nevertheless binds to this receptor. Similar differential bioactivity of soluble and membrane-bound variants of the same ligand of the TNFSF has also been observed for TNF [Grell et al, Cell, 83:793-801 (1995); Grell et al, Proc. Natl. Acad. ScL, 95:570-575 (1998)), TRAIL (Wajant et al, Oncogene, 20:4101-4106 (2001)), and CD40L (Haswell et al, Eur. J. Immuno., 31 :3094-3100 (2001)). Thus, receptor binding of a TNFSF does not necessarily result in receptor activation.

[0005] 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. Med.; 187: 123-8 (1998)) Membrane bound TNF superfamily members including FasL has been show to "reverse-signal" via their membrane attach cytoplasmic tail and thus they also possess a "bi-directional" signaling (Sun & Fink, J. Immuno., 179:4307-4312 (2007)). These studies suggest that small molecules, such as Kp 7 and mimetics thereof, which bind to both Fas and FasL can regulate Fas receptor signaling in a tissue-specific manner can be used to treat a variety of autoimmune pathologies.

[0006] 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. 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. (Famularo, G., et al., Med. Hypotheses, 53:50-62 (1999)). 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. (Kakinuma, C. et al., Toxicol Pathol, 27: 412-420 (1999); Famularo, G., et al., Med Hypotheses, 53: 50-62 (1999); Martinez, O.M. et al., Int Rev Immunol, 18:527-546 (1999); Kataoka, Y. et al, Immunology, 103:310-318 (2001); Chung, CS. et al, Surgery, 130:339-345 (2001); Doughty, L. et al, Pediatr Res, 52:922-927 (2002)). Consequently, the FASL-FAS pathway represents an important general target for therapeutic intervention.

[0007] Monoclonal anti-FASL antibody, naturally occurring decoy receptors and recombinant soluble FAS protein are well-recognized potential candidate antagonists for clinical studies. (Hashimoto, H. et al, Arthritis and Rheumatism, 41 :657-662 (1998); Kanda, Y. et al, Bone Marrow Transplantation, 22:751-754 (1998); Kato, K. et al, British Journal of Haematology, 103:1164-1166 (1998); Maggi, CA. Pharmacological Research, 38:1-34 (1998); Poulaki et al, Drug Resist update, 4:233-42 (2001)). Attempts to neutralize FASL with antibodies have been examined in a variety of animal models. (Okuda, Y. et al, Biochem Biophys Res Commun, 275:164-168 (2000)). While antibodies and soluble receptors/ligands have a long half life and are highly specific, they also have important limitations: (i) commercial- scale production may be either difficult or costly, (ii) conformational stability may vary with the environment of the body fluids, (iii) antibodies may be excluded from certain compartments e.g., the brain, due to failure to cross the blood/brain barrier, and (iv) they may lead to the development of neutralizing antibodies, etc. (Cho, M.J. et al, Trends Biotechnol, 14:153-158 (1996)). [0008] Many disadvantages of large macromolecules can be overcome by creating small molecular inhibitors that are targeted to surface receptors or their ligands. Peptidomimetics that are constructed to resemble secondary structural features of the targeted protein represent an approach to overcome some of the limitations of macromolecules and can mimic inhibitory features of large molecules such as antibody (Park, B. W. et al, Nat Biotechnol, 18:194-198 (2000)) and soluble receptors. (Takasaki, W. et al, Nat Biotechnol, 15:1266-1270 (1997); Hasegawa, A. et al, Proc. Natl Acad. Sci. USA 101: 6599-6604. (2004)). Recently several peptidomimetics that inhibit ligand-receptor binding and that mediate potent biological effects have been described. (Park et al., supra; Takasaki, W. et al., Nat Biotechnol, 15:1266-1270 (1997)). It is also possible to develop small peptides from the structural features of FASL and decoy proteins such as Met (Zou et al., Nat. Med, 13:1078-1085 (2007). These peptides represent novel small molecular tools that can act with potency comparable to or equivalent to the natural antagonist. (Takasaki, W. et al., Nat Biotechnol, 15:1266-1270 (1997); Wrighton, N.C. et al, Nat Biotechnol, 15:1261-1265 (1997); Zou et al, Nat. Med, 13:1078-1085 (2007)).

[0009] 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 β-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.

[0010] Several studies suggest that FAS receptor complex mediate apoptosis in keratinocytes (Sayama, et al, 1994. J. Invest. Dermatol, 103:330-334; Trautmann et al, JCI, 2000, and references therein). Trautmann et al provide evidence that another pathway, induction of apoptosis of keratinocytes via activation of the FAS receptor system, also disrupts the epidermal barrier, and showed that infiltrating T lymphocytes are the direct cause of keratinocyte death through this novel pathway. Cell death by apoptosis is a tightly regulated process that enables removal of unnecessary, aged, or damaged cells. During 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. Ligand binding causes trimerization of FAS, and the trimerized cytoplasmic region transduces the signal by recruiting the adapter molecule FADD (FAS-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.). Trautmann et al, now identifies the keratinocyte as an additional target of FAS -induced apoptosis and provides evidence that this form of cell death contributes to the pathogenesis of eczematous dermatitis. Keratinocytes normally express low levels of FAS, but IFN-gamma upregulates FAS on these cells (Matsue, H., et al, 1995. Arch. Dermatol. Res., 287:315-320; Sayama, et al, 1994. J. Invest. Dermatol, 103:330-334). The authors showed that cultured keratinocytes are driven into apoptosis upon co-incubation with autologous activated T lymphocytes. Addition of a blocking FAS-Fc fragment inhibited apoptosis, indicating that keratinocyte death is due to triggering of the FAS system. Secretion of IFN-g by T lymphocytes, which promotes FAS upregulation in keratinocytes, is a crucial early step in this pathway. In this case keratinocyte apoptosis occurs only in association with an inflammatory reaction; but it is important to mention that the inflammatory infiltrate is not the consequence but the cause of apoptosis.

[0011] 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. Unlike in AD and ACD, in TEN keratinocytes kill themselves by expressing FASL and thus do not need the help of lymphocytes. Although 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).

[0012] These and other studies suggest that inhibition of FAS function will be useful in the treatment of TEN or any skin condition that involves inflammation such as eczema.

[0013] Accordingly, the present inventors have identified that altering FAS function in keratinocytes and hair follicles has a beneficial effect and will therefore have therapeutic applications. As disclosed herein, 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. Acad. ScL, 97:1707-1712 (2000)). Moreover, FASL can also stimulate T cell activation. Accordingly, the mimetics are therefore also useful as therapy for eczema and other skin related inflammatory pathologies in which T cells play a role.

[0014] Hair loss (alopecia) 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)). In particular, 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.

[0015] Specifically, it was demonstrated by genetic studies that alopecia is a FAS related pathology (Freyschmidt-Paul et al, Invest. Derm., 8:104-108 (2003)). Furthermore, Sharov et al, (Cancer Res., 64:6266-6270 (2004)) demonstrated that FAS is up-regulated in HF keratinocytes after cyclophosphamide treatment, FAS ligand-neutralizing antibody partially inhibits HF response to cyclophosphamide in wild-type mice, and FAS knockout mice show significant retardation of cyclophosphamide-induced HF involution associated with reduced FAS-associated death domain and caspase-8 expression. [0016] The blockade of FAS signaling as a part of complex local therapy for inhibiting keratinocyte apoptosis and hair loss induced by chemotherapy, as disclosed herein, represents a novel approach. These studies suggest that an induction of apoptosis in FAS-expressing cells of the hair follicles by FASL-expressing cells of the perifollicular infiltrate resulting in hair loss. Accordingly, the mimetics are therefore also useful as therapy for skin related pathologies that include alopecia and chemotherapy- and or radiotherapy-induced hair loss.

[0017] The citation and/or discussion of a reference in this section, and throughout this specification, shall not be construed as an admission that such reference is prior art to the present invention. Numerous references, including patents, patent applications and various publications, are cited and discussed in the background and the rest of the specification. The citation and/or discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any such reference is "prior art" to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety and to the same extent as if each reference was individually incorporated by reference.

SUMMARY

[0018] 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.

[0019] 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.

[0020] Other embodiments include this method of treating a Fas related pathology, wherein said mimetic is a cyclic peptide of formula (II):

wherein B₁ and B₂ are each independently a peptide of 1-6 amino acids each independently comprising at least one aromatic amino acid;

Z=Z is a cysteine=cysteine disulfide bridge or an aspartate=diaminopropionic acid lactam bridge;

X₃ and X₄ are each aspartic acid or glutamic acid;

X₅ and X₇ are each independently glycine or a bond;

X₆ is glutamic acid, glutamine, histidine, lysine, leucine, or tyrosine; and

X₈ is glutamine, leucine, phenylalanine or tyrosine; or a pharmaceutically acceptable salt form thereof.

[0021] Other embodiments include the methods described above, wherein the FAS- related pathology is vitiligo, psoriasis, alopecia areata, toxic epidermal necrolysis, eczematous dermatitis, macropapular rashes, or rosacea.

[0022] 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.

[0023] 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.

[0024] 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, vplβ, ifosfamide, irinotecan, mitozantrone, mitoxantrone, paclitaxel, topotecan, vinblastine, vincristine, gemcitabin, or carboplatin.

[0025] Other embodiments include the methods described above wherein, in Formula (II), Z=Z is a cysteine=cysteine disulfide bridge. Other embodiments include the methods described above wherein, in Formula (II), Z=Z is a cysteine=cysteine disulfide bridge and wherein the 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).

[0026] Other embodiments include the methods described above wherein, in Formula

(II), Z=Z is an aspartate=diaminopropionic acid lactam bridge. Other embodiments include the methods described above wherein, in Formula (II), Z=Z is an aspartate=diaminopropionic acid lactam bridge and wherein the cyclicized peptide is prepared from a peptide comprising the sequence YDDEHF(diaminopropionic acid)Y (SEQ ID NO: 21).

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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.

[0028] FIGS. 2A-B illustrate inhibition of FasL binding to Fas-receptor by exocyclic mimetics in a binding assay.

[0029] FIGS. 3A-B depict a surface plasmon resonance (biosensor) analysis of mimetic binding to immobilized FasL.

[0030] 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.

[0031] 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)).

[0032] 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.

[0033] 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)).

[0034] 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.

[0035] 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. [0036] 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.

[0037] 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)).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0038] 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.

[0039] Hence, 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.

[0040] Certain embodiments provide a method of using a Fas mimetic represented by formula (I):

wherein B₁ and B₂ 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;

X₃ is a hydrophilic amino acid or a bond;

X₄ is an amino acid selected from aspartic acid or glutamic acid;

X₅ 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;

X₇ is an aromatic or heteroaromatic moiety; — is a covalent linkage comprising an amide, substituted amide or an isostere of amide thereof; and = is a covalent linkage, or a pharmaceutically acceptable salt, metabolite or prodrug thereof.

[0041] In formula (I), B₁ and B₉ independently are exocyclic portions of mimetics that are comprised of, e.g., amino acid residues. Z₂, X₃, X₄, X₅, X₆, X₇ and Z₈ comprise the cyclicized portion of mimetics of formula (I). Z₂ and Z₈ are further linking moieties, preferably linking amino acids.

[0042] In some embodiments, Fas mimetics of the invention are conformationally restrained peptides. In other embodiments, 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.

[0043] Independent preferred moieties for the formula (I) are as follows: Z₂ is cysteine; Z₈ is cysteine; X₃ is a bond; X₄ is aspartic acid; X₅ is glutamic acid; X₇ is phenylalanine; and X₇ is an aromatic amino acid.

[0044] The following are also illustrative embodiments for formula (I): B₁ is tyrosine; B₉ is tyrosine; both B₁ and B₉ are tyrosine; B₁ is --R₁-R₂, where R₁ is an aromatic amino acid linked to Z₂ and R₂ is a peptide of 1-5 amino acids; B₉ is -Rs-R₄, where R₃ is an aromatic acid linked to Z₈ and R₄ is a peptide of 1-5 amino acids.

[0045] 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. Additionally, 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.

[0046] Also provided is 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). [0047] 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.

[0048] Also provided is the method 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.

[0049] Also provided is the method of treating a Fas related pathology, wherein said mimetic is a cyclic peptide of formula (II):

wherein B₁ and B₂ are each independently a peptide of 1-6 amino acids each independently comprising at least one aromatic amino acid;

Z=Z is a cysteine=cysteine disulfide bridge or an aspartate=diaminopropionic acid lactam bridge;

X3 and X₄ are each aspartic acid or glutamic acid;

X₅ and X₇ are each independently glycine or a bond;

Xe is glutamic acid, glutamine, histidine, lysine, leucine, or tyrosine; and

Xg is glutamine, leucine, phenylalanine or tyrosine; or a pharmaceutically acceptable salt form thereof.

[0050] Also provided are the methods described above, wherein the FAS-related pathology is vitiligo, psoriasis, alopecia areata, toxic epidermal necrolysis, eczematous dermatitis, macropapular rashes, or rosacea.

[0051] Also provided are the methods described above wherein the FAS-related pathology is inflammation, Lyell syndrome, eczema, toxic epidermal necrolysis, and drug- induced skin eruptions.

[0052] Also provided are 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. [0053] Also provided are the methods described wherein said chemotherapy induced hair loss is induced by treatment with cyclophosphamide, chlormethine, mustine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, vplβ, ifosfamide, irinotecan, mitozantrone, mitoxantrone, paclitaxel, topotecan, vinblastine, vincristine, gemcitabin, or carboplatin.

[0054] Also provided are the methods described above wherein in Formula (II) Z=Z is a cysteine=cysteine disulfide bridge. Other embodiments include the methods described above wherein in Formula (II) Z=Z is a cysteine=cysteine disulfide bridge and wherein the 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).

[0055] Also provided are the methods described above wherein in Formula (II) Z=Z is an aspartate=diaminopropionic acid lactam bridge. Other embodiments include the methods described above wherein in Formula (II) Z=Z is an aspartate=diaminopropionic acid lactam bridge and wherein the cyclicized peptide is prepared from a peptide comprising the sequence YDDEHF(diaminopropionic acid)Y (SEQ ID NO: 21).

[0056] 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.

Definitions:

[0057] 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:

[0058] The term "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.

[0059] The term "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). The aromatic group may be further substituted with substituent groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfanyl, nitro and amino groups, as well as others. Examples of genetically encoded aromatic amino acids include phenylalanine, tyrosine and tryptophan. Commonly encountered non-genetically encoded aromatic amino acids include phenylglycine, 2- naphthylalanine, β-2-thienylalanine, l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 4- chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine and 4-fluorophenylalanine.

[0060] The term "hydrophilic amino acid" refers to an amino acid having a side chain that is capable of bonding to solvent molecules in an aqueous solution. Examples of genetically encoded hydrophilic amino acids include Ser and Lys. Examples of non-encoded hydrophilic amino acids include Cit and hCys.

[0061] The term "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.

[0062] Additional examples of amino acids and related compounds are found in U.S. Pat. No. 6,265,535.

[0063] As used herein, the term "mimetic" 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.

[0064] As used herein, the terms "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.

[0065] As used herein, the term "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.

[0066] As used herein, the term "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.

[0067] As used herein, the term "linking moiety" refers to a molecular component or functional group which is capable of forming bonds with three amino acids.

[0068] As used herein, the term "linking amino acid residue" refers to an amino acid residue that is a linking moiety.

[0069] As used herein, the term "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.

[0070] The term "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.

[0071] Fas mimetics are preferably derived from human or mouse Fas. In some embodiments, Fas mimetics are derived from human Fas, e.g., without limitation, human Fas. In other embodiments, mimetics are derived from human Fas with the mature amino acid sequence:

RLSSKSWAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCP PGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGH GLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLT SNTKCKEEGSRSNLGWLCLLLLPIPLIVWVKRKEVQKTCRKHRKENQG SHESPTLNPETVAINLSDVDLSKYITTIAGVMTLSQVKGFVRKNGVNEA KIDEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAYDTLIKDLKKANLC TLAEKIQTIILKDITSDSENSNFRNEIQSLV (SEQ ID NO: 15) [0072] In some embodiments, 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. For example, in certain embodiments a Fas mimetic of the invention may inhibit binding of FasL to Fas, and may thereby inhibit Fas activity. In other embodiments (that are preferably practiced either in the absence of or at very low concentrations of FasL) a Fas mimetic of the invention may bind to and activate Fas.

[0073] As used herein, the term "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. Preferably, 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)).

[0074] Solvent accessibility of an amino acid is expressed as a value from 0.0 (buried) to 1.0 completely accessible. Preferably, 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. 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 0.4, 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.

[0075] Also 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 0.3, 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. 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 0.8, 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.

[0076] As used herein, the term "treatment" refers to administering an active agent to lessen the severity or the likelihood of the re-occurrence of a pre-existing condition. Hence, "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.

[0077] As used herein, the term "preventing" refers to the lessening of the likelihood of the occurrence of a condition.

[0078] As used herein, 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.

[0079] As used herein, 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.

[0080] As used herein, 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. Alternatively, in certain embodiments, 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.

[0081] A "metabolite" of a compound disclosed herein is a derivative of a compound which is formed when the compound is metabolized. The term "active metabolite" refers to a biologically active derivative of a compound which is formed when the compound is metabolized. The term "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. For example, 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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. In a preferred embodiment, 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,

Kp3, Kp4, or Kp7. [0086] 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. Of course the ligand cannot evict a bound antagonist molecule, or vice versa. Irreversible or non-equilibrium competitive antagonism occurs when the antagonist dissociates very slowly, or not at all, from the receptor with the result that no change in the antagonist occupancy takes place when the ligand is applied. Thus, the antagonism is insurmountable. 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.

[0087] In some embodiments, mimetics are preferably antagonists of Fas. In other embodiments, 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.

[0088] Fas mimetics preferably have one or more of the following properties:

(1) Significant Inhibition of FasL Binding to Fas:

[0089] Mimetics preferably exhibit antagonist potency (measured as IC₅₀) between 1 nM and 500 mM. Without limiting the present disclosure, as described in more detail below, 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))

(2) Selectivity:

[0090] 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. (3) Binding to FasL:

[0091] 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. In preferred embodiments, the mimetics have affinities for FasL represented by values of kon greater than 10 M^"1 s^"1, k_off 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_off 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 10³ M^"1 s^"1, k_off of less than 10^"5 s^"1 or K_D of less than 10^"6 M.

[0092] Accordingly, mimetics having one or more of these properties are candidates for use in treatment of Fas-related pathologies in mammals and especially in humans.

[0093] 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.

[0094] 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. Alternatively, 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.

[0095] 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.

Design of Mimetics:

[0096] According to preferred embodiments a mimetic is designed based on a known region of FAS. In preferred embodiments, the mimetic mimics an extracellular domain of an Fas, more preferably a cystine knot region of Fas. Most preferably, the mimetic is based on the Fas Kp7 domain.

[0097] Identification of Fas peptides on which to base mimetics can be performed using computer modeling and structural analysis. For instance, Example 1, infra, 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. In such an embodiment, 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.

[0098] Reducing a macromolecule to a small molecule with similar function is a general chemical problem. There have been some attempts to design mini-proteins by transplanting functional units onto suitable scaffolds (Vita et al, Biopolymers, 47: 93-100 (1998)) and minimizing antibodies to single chain antibodies (Magliani et al, Nature BiotechnoL, 15:155-158 (1997)).

[0099] It is now established that conformation constrained peptides are more bioactive than unconstrained ones. In recent years, the chemistry of peptide cyclization by solid phase synthesis and other methods has improved dramatically (Burgess et al., (1996) in Solid phase syntheses of peptidomimetics (American Chemical Society, Washington D. C.) pp. ORGN-157; Goodman & Shao, Pure Appl. Chem., 68:1303-1308 (1996); Hanessian et al., Tetrahedron 53:12789-12854 (1997); Koskinen & Hassila, Acta Chem. Scand., 50:323-327 (1996); Kuhn et al, Tetrahedron, 53:12497-12504 (1997); Wύd et al, Tetrahedron Lett., 37:4091-4094 (1996); Zuckermann, Curr. Opin. Struct. Biol, 3:580-584 (1993)). This provides additional opportunities to develop peptides into peptidomimetics.

[0100] General principles to create cyclic peptide mimetics that have been adopted and in some cases (such as eptifitimide (Integrilin) a glycoprotein lib Ilia inhibitor (COR Therapeutics)) have become clinically available. Aromatic residues placed at the termini of cyclically constrained small peptides increase the activity of mimetics (Takasaki et al, Nat. Biotechnol, 15:1266-1270 (1997); Zhang et al, Nature Biotechnol, 14:472-475 (1996)). Employing such modifications has allowed creation of high affinity mimetics of antibody mimics based on CDRs (Park et al, Nature Biotechnol, 18:194-198 (2000)), CD4 (Zhang et al, Nature Biotechnol, 15:150-154 (1997)), IL4 receptor loop mimetics, anti-CD3 antibody mimics, and TNF cystine knot mimetic that affect TNFα binding to its receptor (Takasaki et al , Nature Biotechnol, 15:1266-1270 (1997)). Chemical Synthesis:

[0101] 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. For example, the peptides may be prepared in linear or non-cyclized form using conventional solution or solid phase peptide syntheses and cyclized using standard chemistries. Preferably, 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.

[0102] Formation of disulfide linkages, if desired, 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. An additional alternative is described by Kamber et al., 1980, HeIv Chim Acta, 63:899-915. A method conducted on solid supports is described by Albericio, 1985, Int. J. Peptide Protein Res. 26:92-97. Any of these methods may be used to form disulfide linkages in the peptides of the invention.

Recombinant Synthesis:

[0103] If 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.

[0104] For recombinant production, 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. The expression vehicle is then transfected into a suitable target cell which will express the linear form of the cyclic peptide. Depending on the expression system used, the expressed peptide is then isolated by procedures well-established in the art. Methods for recombinant protein and peptide production are well known in the art (see, e.g., Maniatis et al, 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N. Y.; and Ausubel et al, 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y.). [0105] A variety of host-expression vector systems may be utilized to express the peptides described herein. These include, but are not limited to, 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.

[0106] 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 mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5 K promoter) may be used; when generating cell lines that contain multiple copies of expression product, SV40-, BPV- and EBV-based vectors may be used with an appropriate selectable marker.

[0107] In cases where plant expression vectors are used, the expression of sequences encoding the peptides of the invention may be driven by any of a number of promoters. For example, viral 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. 6:307-311) may be used; alternatively, 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. For reviews of such techniques see, e.g., Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.

[0108] In one insect expression system that may be used to produce the peptides of the invention, 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). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed, (e.g., see Smith et ah, 1983, J. Virol., 46:584; Smith, U.S. Pat. No. 4,215,051). Further examples of this expression system may be found in Current Protocols in Molecular Biology, Vol. 2, Ausubel et ah, eds., Greene Publish. Assoc. & Wiley Interscience.

[0109] In mammalian host cells, a number of viral based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, 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. Natl. Acad. Sci. USA, 81 :3655- 3659). Alternatively, 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).

[0110] Other expression systems for producing linear or non-cyclized forms of the cyclic peptides of the invention will be apparent to those having skill in the art.

Purification of the Peptides and Peptide Analogues:

[0111] The 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.

[0112] For affinity chromatography purification, any antibody which specifically binds the peptides or peptide analogues may be used. For the production of antibodies, 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. Various 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.

[0113] 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)). In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al, 1984, Proc. Natl. Acad. Sci. USA, 81 :6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985, Nature, 314:452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce cyclic peptide-specifϊc single chain antibodies.

[0114] Antibody fragments which contain deletions of specific binding sites may be generated by known techniques. For example, such 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. Alternatively, 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.

[0115] 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.

Formulation and Routes of Administration:

[0116] The compounds disclosed herein, and in references incorporated by reference herein, may be administered to a subject per se or in the form of a pharmaceutical composition. Pharmaceutical 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.

[0117] For topical administration the compounds of the invention may be formulated as solutions, gels, ointments, creams, suspensions, etc., as are well-known in the art.

[0118] 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.

[0119] For injection, 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. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[0120] Alternatively, the compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0121] For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0122] For oral administration, 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. For oral solid formulations such as, for example, powders, capsules and tablets, 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. If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0123] If desired, solid dosage forms may be sugar-coated or enteric-coated using standard techniques.

[0124] For oral liquid preparations such as, for example, suspensions, elixirs and solutions, suitable carriers, excipients or diluents include water, glycols, oils, alcohols, etc. Additionally, flavoring agents, preservatives, coloring agents and the like may be added.

[0125] For buccal administration, the compounds may take the form of tablets, lozenges, etc., formulated in conventional manner.

[0126] For administration by inhalation, 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. In the case of a pressurized aerosol 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.

[0127] 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.

[0128] In addition to the formulations described previously, 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. Thus, for example, 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.

[0129] Alternatively, other pharmaceutical delivery systems may be employed. 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. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the therapeutic agent. Various of 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.

[0130] As 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.

Effective Dosage:

[0131] The compounds of the invention will generally be used in an amount effective to achieve the intended purpose. For use to treat or prevent FAS-related pathologies, the compounds of the invention, or pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount. By 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.

[0132] For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays. For example, 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.

[0133] 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.

[0134] 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.

[0135] In cases of local administration or selective uptake, 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.

[0136] 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.

[0137] 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. In the case of Fas-related pathologies, 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).

[0138] 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.

Toxicity:

[0139] In some embodiments, a therapeutically effective dose of the compounds described herein will provide therapeutic benefit without causing substantial toxicity.

[0140] 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_1Oo (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. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et ah, 1975, In: The Pharmacological Basis of Therapeutics, Ch.1, p.l). EXAMPLES

[0141] The present invention is also described by means of the following examples. However, the use of these or other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described herein. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification and can be made without departing from its spirit and scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which the claims are entitled.

Experimental Protocols

[0142] The following experimental protocols and materials were used at least for the experiments in Examples 1 through 3, except where otherwise noted:

[0143] Materials: Human recombinant TNFα was obtained from Roche Diagnostics (Indianapolis, Ind.). Flag-tagged soluble human Fas ligand (FasL-Flag) and human Fas extra cellular domain-IgGFc fusion protein (Fas-Fc) were purchased from Kamiya Biomedical (Seattle, Wash.). Human recombinant TNF-receptor (I) extracellular domain-IgGFc fusion protein (TNFRI-Fc) was obtained from R & D systems (Minneapolis, Minn.). Anti-Flag-HRP antibody, hydrogen peroxide solution and 3,3',5,5'-tetramethylbenzidine (TMBZ) were from Sigma Biochemical Co. (St. Louis, Mo.).

[0144] 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.

[0145] Molecular Modeling: Computer modeling and the structural analysis were performed using both QUANTA and INSIGHT (Molecular Simulation, San Diego, Calif). The model peptides designed were constructed from their sequences and folding using CHARMM. The side chain of amino acid residues were first positioned to permitted conformation using the Ponders rotamer (Ponder, J. W. et al, J MoI Biol, 193:775-791 (1987)) database provided in QUANTA. Then the folded peptides were minimized to convergence with a dielectric constant set to 80. The molecular model of the human Fas/FasL complex was built by using Modeller 3.0 (SaIi, A. et al, J MoI Biol, 212:403-428 (1990); SaIi, A. et al, Trends in Biochem Set, 15:235- 240 (1990)) using the crystal structure of TNF receptor and molecular model of Fas (Bajorath supra) from the Brookhaven database. (Bernstein, F. C. et al, JMoI Biol, 112:535-542 (1977)). The conformation of loops were constructed using both loop search algorithm and CONGEN. (Bruccoleri, R. E. et al., Nature, 335:564-568 (1988)). The quality of the model were assessed using Ramachandran plot (for phi, psi violations) and profile analysis. (Zhang, K. Y. et al, Protein Sci, 3:687-695 (1994)). The 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.

[0146] 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.

[0147] 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.

[0148] The 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 μg/ml in distilled water adjusted to pH 8.0 by (NH₄)₂CO₃ 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.

[0149] Peptides containing internal lactam bridges may be prepared using appropriate protecting groups and cyclizing on the resin. For example, 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%.

[0150] Solid phase ligand binding assay: 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. After 2 h incubation at room temperature, 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 μg/ml of TMBZ and 0.005% (v/v) H₂O₂) and stopped with 2N H₂SO₄. The absorbance at 450 nm was measured with an ELISA reader.

[0151] 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.

[0152] Cytotoxicity assay. Twenty microliters of Jurkat cells at 1 x 10⁵ 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 μl of mixture was added to each well. After an incubation period of 24 hours, each culture was pulsed with 1 μCi 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. Survival (%) by several doses of peptides was plotted. Incorporation of the radioactive label was measured by liquid scintillation counting (Wallac, Finland) and expressed as the arithmetic mean counts per minute (cpm) of triplicate cultures.

[0153] Statistical analysis: 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.

Example 1: Molecular Model of a Fas Receptor Complex

[0154] 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 β-turns in proteins are considered to mediate roles in molecular recognition and binding. (Leszczynski, J. F. et al, Science, 234:849-855 (1986)). To develop a cystine -knot peptide mimetic, sites of protein-protein interaction that might be disrupted or influenced by small molecules were identified.

[0155] 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. (Watanabe-Fukunaga et al, supra; Naismith, J. H. et al., J Biol Chem, 270:13303-13307 (1995); Banner, D. W. et al, Cell, 73: 431-445 (1993); Bajorath, l. J Comput Aided MoI Des, 13:409-418 (1999)). 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. However, the ectodomain of the Fas receptor appears to be rotated about 10° compared to the transmembrane domain. 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)).

[0156] 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)).

[0157] 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 amino acid positions are obtained from the amino acid sequence of given in SEQ ID NO 15

E

Example 2: Inhibition of FasL Binding to Fas Receptor

[0158] 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 μM of peptide and 20 nM of soluble Fas receptor (FIG. 2A). The results indicated that Kp7 loop is a preferred surface for the design of mimetics as a template. Additional generations of exocyclic peptide mimetics derived from the Kp7 loop surface were also engineered. By the analysis of the interaction site between FasL and Fas, and biological activity of different mimetics, the aspartic and glutamic acids in Kp7 loop appear to represent the most relevant residues involved in the interaction. However, the particular acidic amino acid at each position is not critical. Hence, for example, 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).

[0159] To compare the inhibitory activities of the Kp7 peptide series, dose-response studies were performed. The best activity was found with Kp7-6 (i.e., cyclic peptide comprising the sequence YCDEHFCY (SEQ ID NO: 5)), which inhibited 50% of the FasL-Flag molecule binding to immobilized Fas-Fc at 150 μM (FIG. 2B). Example 3: Binding Affinity and Specificity of Kp7 Mimetics

[0160] The kinetics of binding of Kp7-6, which mediated the best inhibitory activity to FasL, was performed using surface plasmon resonance (BIAcore.TM.) analysis. FasL-Flag was immobilized onto a sensor chip and various solutions containing different concentrations of KpI- 6 were passed over the surface. 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 10¹ 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_off 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. et al, JExp Med, 185:1487-1492 (1997)), the k_off rate was similar with usual antigen-antibody interaction, which suggests that Kp7-6 forms a stable receptor complex and may be useful practically. (Berezov, A. et al, J Med Chem, 44:2565-2574 (2001)).

[0161] To assess the specificity of Kp7-6 binding interaction, Fas, FasL and TNFα were immobilized on a sensor chip. Kp7-6 bound to FasL but not to TNFα, which indicates Kp7- 6 bound to FasL specifically (FIG. 3B). Kp7-6 also bound to Fas (FIG. 3B). This observation is reminiscent of features of the soluble TNF receptor I (p55) which has been shown to form anti- parallel homodimeric complexes in the absence of ligand. (Naismith, J. H. et al, Structure, 4:1251-1262 (1996)). These results suggest that Fas may also form such antiparallel dimers and the Kp7 loop may contribute to antiparallel dimer formation. No significant binding to TNF-R was detected.

Example 4: Kp7-6 Can Inhibit Contact Hypersensitivity (CHS) in Mice

[0162] Measuring contact hypersensitivity (CHS) in mice to an epicutaneously applied hapten(s) is a standard mouse model for allergic contact dermatitis in humans. Interaction of FAS with its ligand, FASL, appears to be a central pathway in the development of CHS. As shown in Figure 4, topical application of Kp7-6 interferes with this interaction and processes associated with the receptor and the ligand and lessens CHS responses in treated mice.

[0163] 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 μl 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 lOμl 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).

[0164] To determine whether Kp7-6 could limit CHS when applied during the sensitization phase of CHS, 0.2% Kp7-6 in 10% propylene glycol or vehicle alone was applied to the abdomen of groups of mice 10 minutes after a sensitizing application of DNFB. Five days later both groups were challenged with DNFB and ear swelling measured (Figure 4, left). Both vehicle and Kp7-6 treated mice developed robust CHS responses indicating that Kp7-6 in propylene glycol does not affect CHS sensitization. In contrast, when Kp7-6 was applied following challenge with DNFB, a small decrease in CHS was observed on days 1 and 2

[0165] Next, to test whether the small decrease in CHS observed when Kp7-6 was applied after challenge could be enhanced with use of a more compatible vehicle, the same experiment was repeated with two different vehicles: Kp7-6 dissolved in propylene glycol (Figure 4, left) was compared with Kp7-6 dissolved in ethanoholive oil (Figure 4, right). As in the experiment in the immediate paragraph above, Kp7-6 produced a statistically insignificant reduction in CHS. However, Kp7-6 in ethanoholive oil dramatically reduced CHS (p=0.0056) compared with vehicle treated animals. 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.

Example 5: Kp7-6 and Chemotherapy-induced Hair Loss in a Murine Model

[0166] 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.

[0167] The 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.

[0168] To perform the experiments, forty-five C57B1/6J female 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 ³A inch across the back. None of the mice experienced lesions or irritations from the waxing. All exposed skin was pink in color indicating dorsal skin hair follicles were in the telogen phase, or resting phase of the hair cycle. Depilation synchronizes the hair cycle in this mouse strain when they are 7 weeks old. One week later, all hairs in the depilated region are in anagen phase, the most susceptible phase of the hair cycle to chemotherapy-induced hair loss (Figure 5).

[0169] After depilation, mice were returned to cages for one week.

[0170] 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 3 : Post chemotherapy treatment with Kp7-6

Group 4: Post chemotherapy treatment with Vehicle

Group 5: Wax only (Control-no chemotherapy, no Kp7-6, no Vehicle)

[0171] On day 7 and day 8 post depilation, mice in Group 1 received two IP injections per day (one AM and one PM injection) of lOOμl of Kp7-6 in 10% DMSO/PBS at 6mg/kg mouse, and mice in Group 2 received the same volume of Vehicle only. On day 9, all mice (except the negative control, Group 5) received cyclophosphamide (CYP) treatment at 150 mg/kg mouse via IP injection. On days 10-12, 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.

[0172] On day 14, after a total of 6 days of Kp7-6 or vehicle treatments (pre- and post- chemotherapy treatment with CYP), mice in groups 1 and 2 were euthanized via CO₂, photographed and the dorsal skin harvested into Zamboni's fixative.

[0173] On days 14 and 15, 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 Canfϊeld 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.

[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:

Treatment Groups % Hair in Depilated Area at Experimental Endpoint

1 : Pre and Post w/ Kp7-6 47%

2 : Pre and Post w/Vehicle 22%

3: Post w/ Kp7-6 55%

4: Post w/Vehicle 22%

5 : Wax only (Control) 100%

[0175] The following observations were noted: CYP appeared to inhibit the hair from re-growing in the depilated area, but CYP did not appear to affect the remaining coat and there was no additional hair loss; mice in Group 1 showed hair re-growth in patches of shiny short hair; mice in Group 3 showed hair re-growth; unlike Group 1 , Group 3 hair came in thinly across the waxed area; Group 3 hair was longer than Group 1 hair; and Control (Group 5) mouse hair in depilated area was luxuriant and completely filled in the depilated area. Importantly, no adverse events were observed in the Kp7-6 treated mice. The skin of Kp7-6 appeared normal and unaffected by the treatment with Kp7-6.

[0176] 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

[0177] An animal model of skin contact sensitivity was used to test the ability of Kp7-6 to prevent such sensitivity, and whether ideal Kp7-6 administration was external application or IP injection, as follows:

1. Preparation: Balb/c 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 μl 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.

3. Challenge: 5 days after sensitization, each mouse was challenged by the application of 20 μl skin sensitizer (0.2% DNFB, 4:1 acetone-olive oil) to the outer aspect of the external ear over about 20 seconds. 30 minutes to 4 hours later,

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.) For some challenges, 20 μl of a topical semi-sticky solution of F2 cream (Latitude) containing 150 mg of F2 cream with of NL0012Ac dissolved in PBS at 20 mg/ml, to provide about 8 mg/ml final NLOO 12 concentration, was used.

4. Measurement of ear thickness. To monitor contact sensitivity, at baseline and daily after ear challenge, the external ear thickness was measured with a hand-held Micrometer.

[0178] In one experiment, 5 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, whereasNL0012Ac (IP) indicates administration by IP injection. For each group, the same treatment was provided to the animals again twice on day 1. However, as some mice weighed much less than others, some experiments for the HCV and control groups were performed on different ears of the same animal.

[0179] As shown in Figure 7, HCV (Hydrocortisone Valerate, the positive control) demonstrated the best activity to reduce contact sensitivity. 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. During the experiment, application of the NL0012Ac F2 solution was found to slightly increase ear thickness. This interference was subtracted during the analysis of the data.

[0180] A second experiment was performed to confirm the activity of NLOO 12Ac using properly designed controls. The control (3 mice) and NLOO 12Ac (4 mice) treatments were performed on different mice. 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.

[0181] As shown in Figure 8, NL0012Ac clearly reduced the contact sensitivity induced by DNFB. We also observed that the reaction to DNFB was stronger in older mice. In this experiment, mice were about 9-11 weeks, wheras in the previous experiment, mice were 7-9 weeks.

[0182] A third experiment was performed to obtain statistically significant results by repeating the experiment with more mice per group (n=7). In addition, one group was included to check NLOO 12Ac activity in complete F2 cream (n=4). Another groups (n=3) was included to investigate the best timing for NL0012 treatment (starting 10 or 30 minutes after challenge as compared to 2 hr). Separate control groups, which were treated with PBS/F2 solution, were used for the different time points.

[0183] At the time of the experiment, 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.

[0184] The activity of NLOO 12Ac was much clearer when given at 30 minutes after challenge (Figure 10), even in the PBS/F2 solution. At day 1, the difference between the control and NLOO 12Ac treated groups was statistically significant.

[0185] Treating mice at an earlier time point, e.g. 10 min after challenge with either PBS/F2 (control) or NLOO 12Ac, prevents contact sensitivity from developing and invalidates this disease model.

EXAMPLE 7: Inhibition of FasL-Induced Cytoxicity by Kp7-6

[0186] To evaluate the effect of various forms of Kp7-6 mimetics on Fas-mediated cytotoxicity, 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 (data not shown) 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. In addition, 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).

Claims

What is Claimed:

1. 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.

2. The method according to claim 1, 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 aspartic acid and glutamic acid.

3. The method according to claim 2, wherein said mimetic is a cyclic peptide of formula (II):

wherein B₁ and B₂ are each independently a peptide of 1-6 amino acids each independently comprising at least one aromatic amino acid;

Z=Z is a cysteine=cysteine disulfide bridge or an aspartate=diaminopropionic acid lactam bridge;

X₃ and X₄ are each aspartic acid or glutamic acid;

X₅ and X₇ 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.

4. The method according to any one of the preceding claims wherein said FAS-related pathology is vitiligo, psoriasis, alopecia areata, toxic epidermal necrolysis, eczematous dermatitis, macropapular rashes, or rosacea.

5. The method according to any one of claims 1 to 3 wherein said FAS-related pathology is inflammation, Lyell syndrome, eczema, toxic epidermal necrolysis, and drug-induced skin eruptions.

6. The method according to any one of claims 1 to 3 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.

7. The method according to claim 6, wherein said chemotherapy induced hair loss is induced by treatment with cyclophosphamide, chlormethine, mustine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, vplβ, ifosfamide, irinotecan, mitozantrone, mitoxantrone, paclitaxel, topotecan, vinblastine, vincristine, gemcitabin, or carboplatin.

8. The method according to any one of claims 3 to 7 wherein Z=Z is a cysteine=cysteine disulfide bridge.

9. The method according to claim 8 wherein said 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).

10. The method according to any one of claims 3 to 7 wherein Z=Z is an aspartate=diaminopropionic acid lactam bridge.

11. The method according to claim 10 wherein said cyclicized peptide is prepared from a peptide comprising the sequence YDDEHF(diaminopropionic acid)Y (SEQ ID NO: 21).