WO2001008699A1 - Methodes d'inhibition de l'osteoclastogenese - Google Patents

Methodes d'inhibition de l'osteoclastogenese Download PDF

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WO2001008699A1
WO2001008699A1 PCT/US2000/020510 US0020510W WO0108699A1 WO 2001008699 A1 WO2001008699 A1 WO 2001008699A1 US 0020510 W US0020510 W US 0020510W WO 0108699 A1 WO0108699 A1 WO 0108699A1
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amino acid
linkage
absent
amide
covalent linkage
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PCT/US2000/020510
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English (en)
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Kazuhiro Aoki
William Carle Horne
Roland Baron
Mark I. Greene
Ramachandran Murali
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The Trustees Of The University Of Pennsylvania
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Priority to CA002380009A priority Critical patent/CA2380009A1/fr
Priority to JP2001513429A priority patent/JP2003505514A/ja
Priority to EP00953710A priority patent/EP1221963A4/fr
Priority to AU66111/00A priority patent/AU777634B2/en
Publication of WO2001008699A1 publication Critical patent/WO2001008699A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to the methods of down-modulating osteoclastogenesis activity, thereby inhibiting bone matrix erosion and thus preventing bone loss and treating bone diseases.
  • the present invention also relates to peptides and peptide analogues which inhibit TN binding to its cellular receptors, methods of designing similar peptides and peptide analogues, and methods of using such compounds to inhibit the biological activities of TNF particularly those activities relating to bone resorption, thereby antagonizing its undesirable clinical effects.
  • Osteoclasts are large multinuclear cells which function to erode bone matrix. They are related to macrophage and other cells that develop from monocyte cells. Like macrophage, osteoclasts are derived from haematopoietic progenitor cells.
  • Bone matrix erosion is a normal process which occurs in coordination with bone matrix formation, a process in which osteoblasts are involved. Essentially, osteoblasts erode bone matrix and tunnel into bone while osteoblasts follow, line the walls of the tunnel and form new bone matrix. Typically, in a normal adult, about 5-10% of bone is replaced by these processes annually.
  • Bone diseases such as osteoporosis and Paget's disease are characterized by a loss of bone.
  • metastatic bone disease, rheumatoid arthritis, and periodontal bone disease are also characterized by bone loss.
  • bone loss leads to fractures in patients.
  • patients become physically impaired which often leads to complications having negative consequences on patient health and quality of life.
  • the economic costs attributable to these diseases are tremendous.
  • Receptors and ligands of the Tumor Necrosis Factor (TNF) family have recently been shown to play an essential part in the differentiation and activity of osteoclasts and therefore play a role in bone resorption.
  • TNF Tumor Necrosis Factor
  • TNF- ⁇ is known to promote osteoclastogenesis, the generation of osteoclasts.
  • a TNF- like molecule present on and/or secreted by osteoclasts and stromal cells referred to interchangeably in the field and herein as "Receptor activator of NF- ⁇ B ligand"
  • RTKL "Osteoclast differentiation factor” (ODF), “Osteoprotegerin ligand” (OPGL), and “TNF -related activation-induced cytokine” (TRANCE)
  • ODF Osteoclast differentiation factor
  • OPF Osteoprotegerin ligand
  • TRANCE TNF -related activation-induced cytokine
  • TNF- ⁇ antagonists such as a monoclonal antibodies
  • Suda, et al. (Endocrine Reviews 20(3):345-357, 1999), which is inco ⁇ orated herein by reference, describe osteoclast differentiation and function.
  • Filvaroff, E and R. Derynck (Curr. Biol. 8:R679- R682, 1998) which is incorporated herein by reference, refer to bone remodeling and a signaling system for osteoclast regulation.
  • the present invention relates to methods of inhibiting osteoclastogenesis and the resorbing activity of mature osteoclasts. According to the present invention, an amount of a TRANCE/RANK inhibitor effective to inhibit osteoclast bone erosion activity is administered to a patient.
  • the present invention relates to methods of treating patients who have diseases characterized by bone loss. According to the present invention, an amount of a TRANCE/RANK inhibitor effective to inhibit osteoclastogenesis is administered to a patient.
  • the present invention relates to pharmaceutical compositions which comprise a TRANCE/RANK inhibitor in an amount effective to inhibit osteoclastogenesis.
  • the present invention relates to methods of modulating dendritic cell maturation, T cell proliferation, and/or CD40 receptor systems in an individual comprising the step of administering to the individual an amount of a TRANCE/RANK inhibitor effective to modulate dendritic cell maturation, T cell proliferation, and/or CD40 receptor systems.
  • the present invention relates to the use of peptides and peptide analogues designed from a binding loop of a TNF-R superfamily member. In particular, it relates to the use of peptides and peptide analogues designed from three binding loops of TNF-R.
  • the invention relates to peptides and peptide analogues which inhibit activities relating to bone reso ⁇ tion.
  • compounds used in the present invention are cyclic peptides or peptide analogues which are modified at their termini with hydrophobic moieties.
  • the peptide corresponds in primary sequence to a binding loop of a member of the TNF-R superfamily or a portion thereof.
  • the peptide used in the invention corresponds in primary sequence to a binding loop of TNF-R p55 or a portion thereof.
  • one or more amino acid residues within the peptide are substituted with other amino acid residues.
  • the amino acid substitutions are conservative, t.e.,the amino acid residues are replaced with other amino acid residues having similar physical and/or chemical properties.
  • the analogues is obtained by replacing at least one amide linkage in the peptide with a substituted amide or an isostere of amide.
  • a compound used in the invention has the following formula:
  • AC is a peptide of 3-18 amino acid residues which corresponds in primary sequence to a binding loop of a TNF-R superfamily member, and which may optionally contain one or more conservative amino acid substitutions, or an analogue thereof wherein at least one amide linkage is replaced with a substituted amide or an isostere of amide;
  • AB is a moiety having a first functional group capable of forming a covalent linkage with one terminus of AC, a second functional group capable of forming a covalent linkage with AB 2 and a third functional group capable of forming a covalent linkage with AA , ;
  • AB 2 is a moiety having a first functional group capable of forming a covalent linkage with the second terminus of AC, a second functional group capable of forming a covalent linkage with AB, and a third functional group capable of forming a covalent linkage with AA 2 ;
  • AA is a moiety having hydrophobic properties and a functional group capable of forming a covalent linkage with the third functional group of AB
  • AA 2 is a moiety having hydrophobic properties and a functional group capable of forming a covalent linkage with the third functional group of AB 2
  • AC is a peptide which corresponds in primary sequence to a binding loop of TNF-R p55 and which optionally may contain one or more conservative amino acid substitutions, or an analogue thereof.
  • the peptides and peptide analogues specifically inhibit osteoclastogenesis.
  • TNF-R p55 SEQ ID NO:l
  • TNF-R p 75 SEQ ID NO:2
  • TNF-R- ⁇ SEQ ID NO: 3
  • NGF-R p75 SEQ ID NO: 4
  • CD27 SEQ ID NO:5
  • CD30 proximal
  • CD30 distal
  • CD40 SEQ ID NO:10
  • 4-IBB SEQ ID NO:l l
  • the invention provides methods for treating individuals that have diseases characterized by bone loss.
  • TRANCE RANK inhibitors are administered to the individual in an amount effective to inhibit osteoclastogenesis and/or osteoclast function and thereby reduce bone loss, i.e. a therapeutically effective amount.
  • the invention also provides novel therapeutic pharmaceutical compositions for treating diseases characterized by bone loss.
  • the pharmaceutical compositions comprise a therapeutically effective amount of TRANCE/RANK inhibitors and a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical compositions are injectable pharmaceutical compositions, i.e. they are sterile, pyrogen-free, free of particulate matter, essentially isotonic, and are otherwise suitable for injection into humans.
  • TRANCE/RANK inhibitors refers to peptides and peptide analogues which inhibit osteoclastogenesis and/or osteoclast function. TRANCE/RANK inhibitors can function as an antagonist of the cellular receptor RANK by inhibiting TRANCE/RANK.
  • diseases characterized by bone loss is meant to refer to diseases, conditions, disorders and syndromes which have as a symptom or pathology a decrease in bone mass or density.
  • diseases characterized by bone loss include, but are not limited to, osteoporosis, Paget' s disease, metastatic bone disease, rheumatoid arthritis and periodontal bone disease.
  • bone reso ⁇ tion refers to the undesired loss of bone caused at least in part by osteoclast activity.
  • therapeutically effective amount is meant to refer to an amount of a compound which produces a medicinal effect observed as reduction in the rate of bone loss in an individual when a therapeutically effective amount of a compound is administered to an individual who is susceptible to or suffering from a disease characterized by bone loss.
  • Therapeutically effective amounts are typically determined by the effect they have compared to the effect observed when a composition which includes no active ingredient (i.e. a control) is administered to a similarly situated individual.
  • the term “inhibit” means to decrease the amount, quality, or effect of a particular activity and is used interchangeably with the terms “reduce”, “minimize”, and “lessen” and refers to, for example, the reduction of osteoclast bone erosion activity caused by the administration of a therapeutically effective amount of the compounds of the present invention to a patient.
  • alkyl refers to a saturated branched, straight chain or cyclic hydrocarbon group. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl, and the like. In preferred embodiments, the alkyl groups are (C ⁇ ._C 5 ) alkyl, with (C r C 3 ) being particularly preferred. As used herein, the term “substituted alkyl” refers to an alkyl group wherein one or more hydrogen atoms are each independently replaced with other substituents.
  • alkenyl refers to an unsaturated branched, straight chain or cyclic hydrocarbon group having at least one carbon-carbon double bond. The group may be in either the cis or trans conformation about the double bond(s).
  • Typical alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, tert-butenyl, pentenyl, hexenyl and the like.
  • the alkenyl group is (C,-C 6 ) alkenyl, with (C,-C 3 ) being particularly preferred.
  • alkynyl refers to an unsaturated branched, straight chain or cyclic hydrocarbon group having at least one carbon-carbon triple bond.
  • Typical alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, isobutynyl, pentynyl, hexynyl and the like.
  • the alkynyl group is (C r C 6 ) alkynyl, with (C,-C 3 ) being particularly preferred.
  • substituted alkynyl refers to an alkynyl group wherein one or more hydrogen atoms are each independently replaced with other substituents.
  • alkoxy refers to an -OR group, where R is alkyl, alenyl or alkynyl, as defined above.
  • typical aromatic moieties include, but are not limited to, benzene, naphthalene, anthracene, azulene, indacene, and the like.
  • the aromatic moiety contains 5-20 carbons in the ring system, with 5-10 carbon atoms being particularly preferred.
  • substituted aromatic moiety refers to an aromatic moiety wherein one or more hydrogen atoms are each independently replaced with other substituents.
  • heteromatic moiety refers to an aromatic moiety wherein one or more of the ring carbon atoms is replaced with another atom such as N, O or S.
  • heteroaromatic moieties include, but are not limited to, pyran, pyrazole, pyridine, pyrrolke, pyrazine, pyridazine, pyrimidine, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, selenophene, thiophere, tellurophene, xanthene and the like.
  • substituted heteroaromatic moiety refers to a heteroaromatic moiety wherein one or more hydrogen atoms are each independently replaced with other substituents.
  • Applicants have discovered that the peptides described infra are useful to inhibit osteoclastogenesis and/or osteoclast function.
  • osteoclastogenesis and/or osteoclast function By inhibiting osteoclastogenesis and/or osteoclast function, bone erosion can be minimized or even prevented, and bone loss can be reduced.
  • Patients suffering from diseases characterized by bone loss can be treated by administering an amount of compound effective to inhibit osteoclastogenesis and/or osteoclast function.
  • patients identified as being susceptible to diseases characterized by bone loss can be prophylactically treated by administering an amount of compound effective to inhibit osteoclastogenesis and/or osteoclast function.
  • TRANCE/RANK inhibitors useful in the invention are compounds described infra such as peptides and peptide analogues designed from a binding loop of a TNF-R superfamily member which function to inhibit osteoclastogenesis and/or osteoclast function. Such compounds may be produced by the methods described infra or by other techniques well known to those skilled in the art.
  • TRANCE/RANK inhibitors useful in the invention to treat diseases characterized by bone loss may be formulated and administered in the manner taught infra, or by other techniques well known to those skilled in the art.
  • TRANCE/RANK inhibitors useful in the invention to treat diseases characterized by bone loss may be formulated and administered in the manner taught infra, or by other techniques well known to those skilled in the art.
  • Some preferred dosages range from InM to 500mM.
  • Some preferred dosages range from ImM to 500mM.
  • Some preferred dosages range from lmg to 500 mg.
  • Some preferred dosages range from lOOOmg to 3000 mg.
  • Some preferred dosages range from 1500 mg to 2500 mg.
  • TRANCE/RANK inhibitors are administered one to four times per day.
  • compositions according to the present invention comprise TRANCE/RANK inhibitors formulated in therapeutically effective doses.
  • the pharmaceutical composition is sterile and pyrogen free.
  • TRANCE/RANK- inhibitors in methods involving other cell types in which TRANCE/RANK-mediated signaling is involved in cell development and/or activity.
  • cell types include antigen presenting cells such as dendritic cells and lymphocytes. Anderson et al. (Nature
  • TRANCE/RANK inhibitors formulated in effective doses as described supra can be used to modulate dendritic cell maturation and function, T cell proliferation and CD40 receptor systems.
  • TNF exerts its biological activities by binding to two TNF-R: p55 and p75.
  • a comparison of these receptors with several other cell surface receptors revealed certain shared structural features that led to their classification as a superfamily (Beutler et al, Science 264:667 ', 1994).
  • the TNF-R superfamily members possess characteristic extracellular Cys-rich domains, yet share only about 25% sequence homology. There are at least ten members in this superfamily, including: TNF-R p55 and p75, TNF-R related protein ( ⁇ ), CD40, Fas antigen (CD95), low-affinity nerve growth factor receptor ( ⁇ 74), CD27, CD30, 4-1BB and OX40 (Beutler et al., Ann. NY Acad. Sci. pp. 118-133, 1994; Gruss and Dower, Cytokines and Mol. Ther. 1:75-105, 1995).
  • Loops and turns in many proteins have been shown to play functionally important roles in protein-protein interactions.
  • cyclic peptides were designed from three binding loops of TNF-R p55 which inhibited the binding of TNF to its cellular receptors.
  • peptides designed from loop 1 of domain 3 exhibited the strongest inhibitory activities.
  • no further increase in inhibitory effects were observed, indicating that loop 1 of domain 3 is a dominate ligand binding site in TNF-R.
  • TNF-R superfamily members from which inhibitory peptides and peptide analogues can be designed are readily identified by amino acid sequence alignment with the three specific binding sites of TNF- R p55 (Fig. 1). Since the dominate binding site of TNF-R p55 falls within amino acid residues #119 to 136, which sequence starts and ends with Cys, the same region in each TNF-R superfamily member may be used to design peptides and peptide analogues that are within the scope of the present invention. In cases where the regions do not start or end with Cys, the region may extend to then next Cys. For example, the corresponding region in Fas is deleted, and thus this region in starts at residue #97 and ends with #143.
  • NGF-R the region ends at the Cys at position at 135.
  • residues 74-81 and 97-110 may also be used to design additional peptides and peptide analogues within the scope of the present invention. Such compounds are then cyclized and modified at their termini with hydrophobic moieties as described in greater detail below.
  • a compound used in the present invention is a cyclic peptide or peptide analogue, such as those disclosed in U.S. Application Serial No. 60/146,090, filed July 28, 1999, which is hereby inco ⁇ orated by reference in its entirety.
  • the peptide or peptide analogue is modified at its termini with hydrophobic moieties.
  • the peptide corresponds in primary sequence to a binding loop of a member of the TNF-R superfamily of a portion thereof.
  • the peptide corresponds in primary sequence to a binding loop of TNF-R p55 or a portion thereof.
  • one or more amino acid residues within the peptide are substituted with other amino acid residues.
  • amino acid substitutions are conservative, i.e., the amino acid residues are replaced with other amino acid residues having physical and/or chemical properties similar to the residues they are replacing.
  • conservative amino acid substitutions are those wherein an amino acid is replaced with another amino acid encompassed within the same designated class, as will be described more thoroughly below.
  • the analogue is obtained by replacing at least one amide linkage in the peptide with a substituted amide or an isostere of amide.
  • a compound used in the invention has the following formula:
  • AC is a peptide of 3-18 amino acid residues, preferably 5-8 amino acid residues, which corresponds in primary sequence to a binding loop of a TNF-R and which may optionally contain conservative amino acid substitutions, or an analogue thereof wherein at least one amide linkage is replaced with a substituted amide or an isostere of amide;
  • AB is a moiety having a first functional group capable of forming a covalent linkage with one terminus of AC, a second functional group capable of forming a covalent linkage with AB 2 and a third functional group capable of forming a covalent linkage with AA, ;
  • AB 2 is a moiety having a first functional group capable of forming a covalent linkage with the second terminus of AC, a second functional group capable of forming a covalent linkage with AB, and a third functional group capable of forming a covalent linkage with AA 2 ;
  • AA is a moiety having hydrophobic properties and a functional group capable of forming a covalent linkage with the third functional group of AB 2 .
  • AA 2 is a moiety having hydrophobic properties and a functional group capable of forming a covalent linkage with the third functional group of AB 2 .
  • the designation X n in each case represents an amino acid at the specified position in the compound.
  • the designation Z n represents an amino acid or other moiety which is capable of forming covalent linkages with other Z n , such as disulfide bridges.
  • the amino acid residues denoted by X n or Z n may be the genetically encoded L-amino acids, naturally occurring non-genetically encoded L-amino acids, synthetic L-amino acids or D-enantiomers of all of the above.
  • the amino acid notations used herein for the twenty genetically encoded L-amino acids and common non-encoded amino acids are conventional and are as follows:
  • the compounds used in 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 amino classes may be further divided into subclasses.
  • Hydrophilic amino acids include amino acids having acidic, basic or polar side chains and hydrophobic amino acids including amino acids having aromatic or apolar side chains.
  • Apolar amino acids ma be further subdivided to include, among others, aliphatic amino acids.
  • the definitions of the classes of amino acids as used herein are as follows:
  • “Hydrophobic Amino Acid” refers to an amino acid having a side chain that is uncharged at physiological pH and that is repelled by aqueous solution.
  • Examples of genetically encoded hydrophobic amino acids include Ile, Leu and Val.
  • Examples of non- genetically encoded hydrophobic amino acids include t-BuA.
  • “Aromatic Amino Acid” refers to a hydrophobic amino acid having a side chain containing at least one ring having a conjugated ⁇ -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, ⁇ -2-thienylalanine, l,2,3,4-tetrahydroisoquinoline-3- carboxylic acid, 4-chloro-phenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine and 4-fluorophyenylalanine.
  • Apolar Amino Acid refers to a hydrophobic amino acid having a side chain that is generlaly uncharged at physiological pH and that is not polar.
  • Examples of genetically encoded apolar amino acids include glycine, pro line and methionine.
  • Examples of non- encoded apolar amino acids include Cha.
  • Aliphatic Amino Acid refers to an apolar amino acid having a saturated or unsaturated straight chain, branched or cyclic hydrocarbon side chain.
  • genetically encoded aliphatic amino acids include Ala, Leu, Nal and Ile.
  • non- encoded aliphatic amino acids include ⁇ le.
  • Hydrophilic Amino Acid refers to an amino acid having a side chain that is attracted by aqueous solution.
  • examples of genetically encoded hydrophilic amino acids include Ser and Lys.
  • examples of non-encoded hydrophilic amino acids include Cit and hCys.
  • Acidic Amino Acid refers to a hydrophilic amino acid having a side chain pK value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Examples of genetically encoded acidic amino acids include aspartic acid (aspartate) and glutamic acid (glutamate).
  • Basic Amino Acid refers to a hydrophilic amino acid having a side chain pK value of greater than 7.
  • Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion.
  • genetically encoded basic amino acids include arginine, lysine and histidine.
  • non-genetically encoded basic amino acids include the non-cyclic amino acids ornithine, 2, 3- diaminopropionic acid, 2.4-diaminobutyric acid and homoarginine.
  • Poly Amino Acid refers to a hydrophilic amino acid having a side chain that is uncharged at physiological pH, but which has a bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms.
  • genetically encoded polar amino acids include asparagine and glutamine.
  • non-genetically encoded polar amino acids include citrulline, ⁇ -acetyl lysine and methionine sulfoxide.
  • cyste-Like Amino Acid refers to an amino acid having a side chain capable of forming a covalent linkage with a side chain of another amino acid residue, such as a disulfide linkage.
  • cysteine-like amino acids generally have a side chain containing at least one thiol (SH) group.
  • examples of genetically encoded cysteine-like amino acids include cysteine.
  • examples of non-genetically encoded cysteine-like amino acids include homocysteine and penicillamine.
  • the above classifications are not absolute — several amino acids exhibit more than one characteristic property, and can therefore be included in more than one category.
  • tyrosine has both an aromatic ring and a polar hydroxyl group.
  • tyrosine has dual properties and can be included in both the aromatic and polar categories.
  • cysteine in addition to being able to form disulfide linkages, cysteine also has apolar character.
  • cysteine can be used to confer hydrophobicity to a peptide.
  • Certain commonly encountered amino acids which are not genetically encoded of which the peptides and peptide analogues of the invention may be composed include, but are not limited to, ⁇ -alanine (B-Ala) and other omega-amino acids such as 3- aminopropionic acid (Dap), 2.3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth; ⁇ -aminoisobutyric acid (Aib); e-aminohyxanoic acid (Aha); ⁇ -aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly); ornithine (Orn); citrulline (Cit); t- butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (Melle); phenylglycine (phg); cyclohexylalanine (Cha); norleucine (Nle
  • Table 1 is for illustrative pu ⁇ oses only and does not pu ⁇ ort to be an exhaustive list of amino acid residues which may comprise the peptides and peptide analogues described herein.
  • Other amino acid residues which are useful for making the peptides and peptide analogues described herein can be found, e.g., in Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc., and the references cited therein.
  • Amino acids not specifically mentioned herein can be conveniently classified into the above-described categories on the basis of known behavior and/or their characteristic chemical and/or physical properties as compared with amino acids specifically identified.
  • Z n in each case represents an amino acid or other moiety capable of forming covalent linkages with other Z n so as to allow cyclization of the peptide.
  • amino acid residues which are capable of forming covalent linkages with one another include cysteine-like amino acids such as Cys, hCys, ⁇ -methyl Cys and Pen, which are capable of forming disulfide bridges with one another.
  • Preferred cysteine-like amino acid residues include Cys and Pen.
  • Amino acids used to cyclize a peptide need not be cysteine-like amino acids. Pairs of amino acids that have side chain functional groups capable of forming covalent linkages with one another can also be used. Such pairs of functional groups are known to those of skill in the art and include, inter alia, -COOH and -OH, -COOH and -NH 2 , and -COOH and -SH. Thus, pairs of amino acids that can be used to cyclize a peptide include, inter alia, Asp and Lys; Glu and Lys; Asp and Arg; Glu and Arg; Asp and Ser; Glu and Ser; Asp and Thr; Glu and Thr; Asp and Cus; and Glu8 and Cys.
  • Z n groups used to cyclize a peptide need not be amino acids.
  • Z n may be any molecule having three functional groups — one functional group capable of forming a covalent linkage with a terminus of the peptide, a second functional group capable of forming a covalent linkage with the second functional group of another Z n , and a third functional group capable of forming a covalent linkage with hydrophobic moieties B n .
  • Molecules having a suitable functional groups will be apparent to those skilled in the art.
  • Examples of functional groups capable of forming a covalent linkage with the amino terminus of a peptide include carboxylic acids and esters.
  • Examples of functional groups capable of forming a covalent linkage with the carboxyl terminus of a peptide include -OH, -SH, -NH 2 and -NHR where R is (C, -C 6 ) alkyl, (C, - C 6 ) alkenyl and (C, -C 6 ) alkynyl.
  • interlinkages useful to cyclize a peptide can be generated by reaction between two Z n Z n with functional groups suitable for forming such interlinkages, as well as reaction conditions suitable for forming such interlinkages, will be apparent to those of skill in the art.
  • the reaction conditions used to cyclize the peptides are sufficiently mild so as not to degrade or otherwise damage the peptide.
  • Suitable groups for protecting the various functionalities as necessary are well know in the art (see, e.g., Green & Wuts, 1991, 2 nd ed., John Wiley & Sons NY), as are various reaction schemes for preparing such protected molecules.
  • the destination B n in each case represents a hydrophobic moiety.
  • B n designates a peptide of 1-6 amino acids, at least one of which is an aromatic amino acid or an aromatic or heteroaromatic moiety.
  • B n may be illustrated as X 32 -X 33 -X 34 -X 35 -X 36 - ⁇ 37 ⁇ wherein X n is an amino acid at least one of which is an aromatic amino acid. More preferably, X 32 -X 33 -X 34 -X 35 -X 36 are absent and X 37 is an aromatic amino acid.
  • Suitable aromatic amino acids include Tyr, Phe and T ⁇ , with Tyr and Phe being preferred.
  • Suitable aromatic or heteroaromatic moieties include phenyl, naphthyl, purine, pyrimidine, and the like.
  • amino acid refers to other bifunctional moieties having side- chain groups similar to the side chains of the amino acids.
  • acid refers to other bifunctional moieties having side- chain groups similar to the side chains of the amino acids.
  • acidic amino acid refers to a bifunctional molecule capable of forming the desired backbone interlinkages and which has a side chain group similar to the side chain of an acidic amino acid.
  • Substituted amides generally include groups of the formula -C(O)-NR, where R is (C, -C 6 ) alkyl, (C, -C 6 ) alkenyl, (C, - C 6 ) alkynyl, substituted (C, -C 6 ) alkyl, substituted (C, -C 6 ) alkenyl or substituted (C, -C 0 ) alkynyl.
  • a linker is a bifunctional molecule that spaces one moiety from another.
  • Such linkers which may be flexible, semi-rigid or rigid, are well-known in the art and include polypeptides such as poly-Gly and poly-Pro, bifunctional hydrocarbons such as aminocaproic acid, ⁇ - aminovaleric acid and ⁇ -alanine, carbohydrates, nucleic acids, and the like.
  • B, and B, 0 are each independently a peptide of 1-6 amino acids, at least one of which is a hydrophobic amino acid, an aromatic moiety or a heteroaromatic moiety;
  • Z 2 is a moiety that is capable of forming a covalent linkage with B,, X 3 and Z 9 ;
  • Z 9 is a moiety that is capable of forming a covalent linkage with B, 0 , X 8 and Z 2 ;
  • X 3 is absent or a hydrophilic amino acid
  • X 4 is a hydrophobic amino acid
  • X 5 is a hydrophilic amino acid
  • X 6 is a hydrophilic amino acid
  • X 7 is a hydrophobic or hydrophilic amino acid
  • X 8 is a hydrophobic or hydrophilic amino acid
  • "-" is an amide, substituted amide or an isostere of amide thereof
  • the compounds are those of formula (II) wherein:
  • B, and B, 0 are each independently a peptide of 1-3 amino acids, at least one of which is an aromatic amino acid;
  • Z 2 and Z 9 are each independently a Cys-like amino acid
  • X 3 is absent or an acidic amino acid
  • X 4 is an aromatic or apolar amino acid
  • X 5 is a polar amino acid
  • X 6 is a polar amino acid
  • X 7 is an aromatic or polar amino acid
  • X g is an aromatic, apolar or polar amino acid
  • the compounds used in the invention are those of formula (II) wherein:
  • B, and B 10 are each independently Tyr or Phe; Z 2 and Z 9 are each Cys; X 3 is absent or Glu;
  • X g is Tyr or Leu
  • Particularly preferred peptides used in the invention include the following:
  • B n and B 22 are each independently a peptide of 1-6 amino acids, at least one of which is a hydrophobic amino acid, an aromatic moiety or a heteroaromatic moiety;
  • Z, 2 is a moiety that is capable of forming a covalent linkage with B,,, X, 3 and Z 21 ;
  • Z 2 is a moiety that is capable of forming a covalent linkage with B 22 , X 20 and Z, 2 ;
  • X 13 is absent or hydrophobic amino acid
  • X 14 is absent or a hydrophilic amino acid
  • X, 5 is a hydrophilic or hydrophobic amino acid
  • X 16 is hydrophilic amino acid
  • X 17 is absent or a hydrophobic amino acid
  • X 18 is a hydrophilic amino acid
  • X, 9 is a hydrophilic amino acid
  • X 20 is a hydrophilic amino acid
  • the compounds are those of formula (III) wherein: B,, and B 22 are each independently a peptide of 1-3 amino acids, at least one of which is an aromatic amino acid;
  • Z, 2 and Z 2 are each independently a Cys-like amino acid
  • X, 3 is absent or an aromatic amino acid
  • X, 4 is absent or a polar amino acid
  • X )5 is a basic, polar or apolar amino acid
  • X !6 is a polar amino acid
  • X, 7 is absent or an apolar amino acid
  • X, 8 is an acidic amino acid
  • X, 9 is a polar amino acid
  • X 20 is a basic amino acid
  • the compounds are those of formula (III), wherein:
  • B,, and B 22 are each independently Tyr or Phe;
  • Z, 2 and Z 2 are each Cys;
  • X, 3 is absent or Phe;
  • X, 4 is absent or Thr;
  • X, 5 is Ala, Asn or Arg;
  • X 16 is Ser
  • X, 7 is absent or Val
  • X 19 is Asn
  • X 20 is Arg or His
  • Particularly preferred peptides used in the invention include the following:
  • YC FTASENH CY (SEQ. ID NO: 16) YC FTNSENH CY (SEQ. ID NO: 17) YC FTRSENH CY (SEQ. ID NO: 18) FC ASENH CY (SEQ. ID NO: 19) YC ASENH CY (SEQ. ID NO:20) FC NSEN ⁇ CY (SEQ. ID NO:21) FC NSENR CY (SEQ. ID NO:22) FC NSVENR CY (SEQ. ID NO:23)
  • B 23 an B 33 are each independently a peptide of 1-6 amino acids, at least one of which is a hydrophobic amino acid, an aromatic moiety or a heteroaromatic moiety;
  • Z 24 is a moiety that is capable of forming a covalent linkage with B 23 , X 25 and Z 32 ;
  • Z 32 is a moiety that is capable of forming a covalent linkage with B 33 , X 31 and Z 24 ;
  • X 25 is absent or a hydrophilic amino acid
  • X 26 is a hydrophilic amino acid
  • X 27 is a hydrophilic amino acid
  • X 28 is a hydrophobic amino acid
  • X 29 is a hydrophobic amino acid
  • X 30 is absent or a hydrophilic amino acid
  • X 3 is absent or a hydrophobic amino acid
  • "-" is an amide, a substituted amide or an isostere of amide
  • the compounds are those of formula (IV) wherein:
  • B 23 an B 33 are each independently a peptide of 1-3 amino acids, at least one of which is a an aromatic acid;
  • Z 24 and Z 32 are each independently a Cys-like amino acid;
  • X 25 is absent or a basic amino acid;
  • X 26 is a basic amino acid
  • X 27 is an acidic amino acid
  • X 28 is an apolar amino acid
  • X 29 is an apolar amino acid
  • X 30 is absent or a polar amino acid
  • X 3 is absent or a apolar amino acid
  • the compounds used in the invention or analogues thereof are those of formula (IV), wherein: B 23 an B 33 are each independently Tyr or Phe; Z 24 and Z 32 are each Cys;
  • X 28 is Leu, Pro or Met;
  • X 29 is Gly;
  • X 30 is absent or Gin
  • X 31 is absent or Val
  • Particularly preferred peptides used in the invention include the following:
  • amino acid also refers to bifunctional moieties having amino acid-like side chains, as previously described.
  • active peptides or peptide analogues used in the invention are those that exhibit at least about 15% inhibition of TNF-R:TNF interactions as measured in vitro assays such as those described, infra.
  • active peptides used in the invention or analogues thereof will exhibit at least about 20% to 50% or even 80% or more inhibition of TNF-R:TNF- ⁇ binding interactions.
  • the peptides used in the invention or analogues thereof may be prepared using virtually any art-known technique for the preparation of peptides and peptide analogues.
  • the peptides may be prepared in linear or non-cyclized form using conventional solution or solid phase peptide syntheses and cyclized using standard chemistries.
  • the chemistry used to cyclize the peptide will be sufficiently mild so as to avoid substantially degrading the peptide.
  • Suitable procedures for synthesizing the peptides described herein as well as suitable chemistries for cyclizing the peptides are well known in the art.
  • Formation of disulfide linkages is generally conducted in the presence of mild oxidizing agents.
  • Chemical, enzymatic or photo lytic oxidation agents may be used.
  • Various methods are known in the art, including those described, for example, by Tam, J.P. et al, Synthesis 955-957, 1979; Stewart et al, Solid Phase Peptide Synthesis. 2d Ed., Pierce Chemical Company Rockford, IL , 1984; Ahmed et al, J. Biol. Chem. 250:8477-8482, 1975; and Pennington et al. Peptides 1990 164-166, Giralt and Andreu, Eds., ESCOM, 1991; Leiden, The Netherlands.
  • the peptide is composed entirely of gene-encoded amino acids, or a portion of it is so composed, the peptide or the relevant portion may also be synthesized using conventional recombinant genetic engineering techniques.
  • the isolated peptides, or segments thereof, are then condensed, and oxidized, as previously described, to yield a cyclic peptide.
  • a polynucleotide sequence encoding a linear form of the peptide is inserted into an appropriate expression vehicle, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation.
  • an appropriate expression vehicle i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation.
  • the expression vehicle is then transfected into a suitable target cell which will express the linear form of the cyclic peptide.
  • the expressed peptide is then isolated by procedures well-established in the art.
  • 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.
  • 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
  • the expression elements of the expression systems vary in their strength and specificities.
  • any of a number of suitable transcription and translation elements may be used in the expression vector.
  • inducible promoters such as pL of baceriophage ⁇ , plac, pt ⁇ , ptac (pt ⁇ -lac hybrid promoter) and the like may be used;
  • promoters such as the baculovirus polyhedron promoter may be used;
  • 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
  • plant viruses e.g., the 35S RNA promoter of CaMV; the coat protein promoter of TMV
  • sequences encoding the peptides of the invention may be driven by any of a number of promoters.
  • viral promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al, Nature 310: 511-514, 1984), or the coat protein promoter of TMV (Takamatsu et al, EMBO J., 3:17-311, 1987) may be used; alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al, EMBO J.
  • 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 AcNPN 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).
  • recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed, (e.g., see Smith et al, J. Virol, 46:584, 1983; Smith, U.S. Patent No. 4,215,051). Further examples of this expression system may be found in Current Protocols in Molecular Biology, Vol. 2, Ausubel et al, eds., Greene Publish. Assoc. & Wiley Interscience. In mammalian host cells, a number of viral based expression systems may be utilized.
  • a coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g. Region El or E3) will result in a recombinant virus that is viable and capable of expressing peptide in infected hosts, (e.g., See Logan & Shenk, Proc. Natl. Acad. Sci. USA 81 :3655-3659, 1984).
  • the vaccinia 7.5 K promoter may be used, (see, e.g., Mackett et al, Proc. Natl. Acad. Sci. USA, 79:7415-7419, 1982; Mackett et al, J. Virol., 49:857-864, 1984; Panicali et al, Proc. Natl. Acad. Sci. 79:4927- 4931, 1982).
  • the peptides and peptide analogues used in the invention can be purified by art- 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.
  • affinity chromatography purification any antibody which specifically binds the peptides or peptide analogues may be used.
  • various host animals including but not limited to rabbits, mice, rats, etc., may be immunized by injection with a linear or cyclic peptide.
  • the peptide may be attached to a suitable carrier, such as BSA, by means of a side chain functional group or linkers attached to a side chain functional group.
  • a suitable carrier such as BSA
  • 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, sufrace 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-Duerin) and Corynebacterium parvum.
  • 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
  • Antibody fragments which contain deletions of specific binding sites may be generated by known techniques.
  • 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.
  • Fab expression libraries may be constructed (Huse et al, Science 246: 1275-1281, 1989) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for the cyclic peptide of interest.
  • the antibody or antibody fragment specific for the desired cyclic peptide can be attached, for example, to agarose, and the antibody-agarose complex is used in immunochromatography to purify cyclic peptides of the invention. See, Scopes, Protein Purification: Principles and Practice, Spriger-Verlag New York, Inc., NY, 1984; Livingstone, Methods Enzymologv: Immunoaffinitv Chromatography of Proteins 34:723- 731, 1974.
  • 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 physiological acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the active peptides or peptide analogues into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the compounds of the invention may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art.
  • Systemic formulations include those designed for administration by injection, e.g. subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral or pulmonary administration.
  • the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks 's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hanks 's solution, Ringer's solution, or physiological saline buffer.
  • the solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • the compounds can be readily formulated by combining the active peptides or peptide analogues with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • suitable excipients include fillers such as sugars, such as lactose, sucrose, mannitol and sorbitol; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium, carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulating agents; and binding agents.
  • disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, atgar, or alginic acid or a salt thereof such as sodium alginate.
  • solid dosage forms may be sugar-coated or enteric-coated using standard techniques.
  • suitable carriers, excipients or diluents include water, glycols, oils, alcohols, etc. Additionally, flavoring agents, preservatives, coloring agents and the like may be added.
  • the compounds may take the form of tablets, lozenges, etc. formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • Liposomes and emulsions are well known examples of delivery vehicles that may be used to deliver peptides and peptide analogues of the invention.
  • Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
  • the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the therapeutic agent.
  • sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
  • the compounds of the invention may contain charged side chains or termini, they may be included in any of the above-described formulations as the free acids or bases or as pharmaceutically acceptable salts.
  • Pharmaceutically acceptable salts are those salts which substantially retain the antimicrobial activity of the free bases and which are prepared by reaction with inorganic acids. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base forms.
  • the compounds of the invention will generally be used in an amount effective to achieve the intended pu ⁇ ose.
  • the compounds of the invention, or pharmaceutical compositions thereof are administered or applied in a therapeutically effective amount.
  • therapeutically effective amount is meant an amount which is effective to ameliorate, or prevent the symptoms of the disease or disorder, 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, especially in light of the detailed disclosure provided herein.
  • a therapeutically effective dose can be estimated initially from in vitro assays.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC 50 as determined in cell culture (i.e., the concentration of test compound that inhibits 50% of TNF-R: TNF-binding interactions). Such information can be used to more accurately determine useful doses in humans.
  • Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the compounds which are sufficient to maintain therapeutic effect.
  • Usual patient dosages for administration by injection range from about 0.1 to 5 mg/kg/day, preferably from about 0.5 to 1 mg/kg/day.
  • Therapeutically effective serum levels may be achieved by administering multiple doses each day.
  • the effective local concentration of the compounds may not be related to plasma concentration.
  • One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.
  • the amount of compound administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
  • Toxicity Preferably, a therapeutically effective dose of the compounds described herein will provide therapeutic benefit without causing substantial toxicity.
  • Toxicity of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD 50 (the dose lethal to 50% of the population) or the LD 100 (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 formulation 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 not toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • TNF- ⁇ and TNF ⁇ /TNF receptor complex have been developed based on atomic structures deduced from the crystal structures of TNF- ⁇ and the TNF ⁇ /TNF receptor complex (Takasaki et al. Nature Biotechnology, 15:1266-1270, 1997).
  • the most critical TNF- ⁇ recognition site was localized to the first loop of the third domain of TNF receptor (residues 107-114).
  • a peptidomimetic (WP9QY) engineered to mimic this recognition site efficiently antagonized the effects of TNF-binding to the TNF- ⁇ receptor in L929 lymphocytes.
  • WP9QY inhibits TRANCE-induced marrow cultures. There was a reciprocal dose-dependence of WP9QY and TRANCE.
  • WP9QY is capable of interfering not only with the TNF/TNF receptor interaction but also with the RANK Ligand/RANK interaction, thereby decreasing the osteoclastogenic potential of this cytokine.
  • TNF- ⁇ and 125 I-labeled TNF- ⁇ were obtained from Amersham Life Science, Inc. (Arlington Heights, IL).
  • TNF-R(I) or p55 extracellular domain-IgG heavy chain chimeric protein was prepared by expression of a cDNA construct (Peppel et al, J. Exp. Med. 174:1483, 1991; Williams et al, Immunol. 84:433, 1995).
  • Anti-TNF- ⁇ monoclonal antibody was prepared according to Doring et al. (Molecular Immunol. 31 : 1059, 1994) and anti-TNF-R(I) monoclonal antibody (htr-9) was obtained from BMA Biomedicals AG (Augst, Switzerland).
  • the crystal structure of the TNF- ⁇ /TNF-R(I) complex was utilized to determine the binding sites of TNF-R for TNF- ⁇ .
  • the first (residues 56-73) and second (residues 76-83) loops of domain 2 and the first loop (residues 107-114) of domain 3 of the TNF-R were explored for use in designing peptides.
  • the essential amino acid sequences of TNF-R for binding interactions with TNF- ⁇ were identified as structural templates by superimposing TNF- ⁇ to TNF- ⁇ complexed with its cognate receptor.
  • Linear peptides were synthesized by solid-phase methods, deprotected, and released from the resin utilizing standard methodology well known to those skilled in the art. Peptides were precipitated and purified by high performance liquid chromatography (HPLC) utilizing a C18 column and then lyophilized. The purity of such peptides was greater than 95% as measured by HPLC analysis.
  • HPLC high performance liquid chromatography
  • the peptides containing internal Cys residues were oxidized by dissolving them at 100 ⁇ g/ml in distilled water adjusted or buffered to pH 8.0 - 8.5, for example, by
  • peptides 100 ⁇ g/ml, 50 ⁇ l
  • DTNB lOmM, 50 ⁇ l
  • the cyclized peptides were lyophilized, purified by HPLC utilizing a C18 preparative column and a size exclusion column Protein-Pak 60 (Waters, Milford, MA). The purity of the peptides was shown to be greater than 95% by HPLC analysis. The concentration of each cyclized peptide was calculated based on UV intensity versus the corresponding linear peptide by HPLC analysis.
  • WP5J FC ASENH CY 19 60 FTASENH 66
  • ** WPl and WPIR were derived from an anti-TNF- ⁇ antibody (Di62, CDRIL) and the template sequence is QSVSNDV.
  • TRAP refers to tartrate resistant acid phosphatase which identifies osteoclast-like cells.
  • Osteoprotegerin OPG
  • OPG Osteoprotegerin
  • Administration of OPG in vivo inhibits osteoclastogenesis and associated bone reso ⁇ tion and blocks the pathological increase in osteoclast numbers and activity seen in animal models that mimic osteopenic disorders in humans.
  • OPG can be used as a positive control in the TRAP assay.
  • Cytochemical staining for TRAP is widely used for identifying osteoclasts in vivo and in vitro. Naphthol AS-MX phosphate 5 mg. Sigma, St.
  • TRAP -positive osteoclasts appear as red cells. The incubation period longer than 10 min. should be avoided since cells other than osteoclasts become weakly positive with time. After staining, cells are washed with distilled water, and TRAP -positive multinucleated cells having three or more nuclei are counted as osteoclasts under a microscope. (G.C. Nicholson, J.M. Mosely, P.M. Sexton, F.A.O. Mendelssohn, and T.J. Martin, J. Clin. Invest. 78, 355, 1986, which is inco ⁇ orated herein by reference).

Abstract

L'invention concerne des méthodes destinées à inhiber l'ostéoclastogenèse et l'activité des ostéoclastes. L'invention concerne également des méthodes de traitement de patients atteints de maladies caractérisées par une perte osseuse. La présente invention concerne en outre des peptides et des analogues de peptides mis au point à partir d'une boucle de fixation d'un élément de la superfamille de récepteurs de facteur de nécrose tumorale (TNF-R). Selon ces méthodes, on administre au patient une quantité d'inhibiteur efficace pour inhiber l'ostéoclastogenèse. L'invention concerne également des méthodes de modulation de la maturation des cellules dendritiques, de la prolifération des lymphocytes T, et/ou des systèmes récepteurs CD40 chez un individu. Ces méthodes consistent à administrer à l'individu une quantité d'inhibiteur efficace pour moduler la maturation des cellules dendritiques, la prolifération des lymphocytes T, et/ou les systèmes récepteurs CD40.
PCT/US2000/020510 1999-07-28 2000-07-28 Methodes d'inhibition de l'osteoclastogenese WO2001008699A1 (fr)

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JP2001513429A JP2003505514A (ja) 1999-07-28 2000-07-28 破骨細胞生成を阻害する方法
EP00953710A EP1221963A4 (fr) 1999-07-28 2000-07-28 Methodes d'inhibition de l'osteoclastogenese
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EP1207873A1 (fr) * 1999-07-28 2002-05-29 The Trustees Of The University Of Pennsylvania Procedes d'inhibition de l'activite des osteoclastes
EP1405645A1 (fr) * 2001-06-22 2004-04-07 Daiichi Suntory Biomedical Research Limited Remedes pour maladies eosinophiliques
US6884598B2 (en) 2000-09-22 2005-04-26 Immunex Corporation Screening assays for agonists and antagonists of receptor activator of NF-κB
US7566741B2 (en) 1999-07-28 2009-07-28 The Trustees Of The University Of Pennsylvania Methods of inhibiting osteoclast activity
WO2012121057A1 (fr) * 2011-03-09 2012-09-13 Jitsubo株式会社 Nouveau peptide réticulé contenant une structure réticulée non peptidique, procédé de synthèse de peptides réticulés et nouveau composé organique utilisé dans le procédé
WO2016075221A1 (fr) * 2014-11-14 2016-05-19 Probiocon Gmbh Agent spécifique de rankl pour le traitement d'une maladie métastatique

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JP2014101274A (ja) * 2011-03-09 2014-06-05 Jitsubo Co Ltd 新規な架橋構造を含むｔｎｆレセプターループペプチドの模倣ペプチドを用いた医薬組成物

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WO2016075221A1 (fr) * 2014-11-14 2016-05-19 Probiocon Gmbh Agent spécifique de rankl pour le traitement d'une maladie métastatique
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US10806786B2 (en) 2014-11-14 2020-10-20 Probiocon Gmbh RANKL-specific agent for treating metastatic disease
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EP1221963A1 (fr) 2002-07-17
AU777634B2 (en) 2004-10-28
AU6611100A (en) 2001-02-19
EP1221963A4 (fr) 2006-03-22
CA2380009A1 (fr) 2001-02-08
JP2003505514A (ja) 2003-02-12

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