EP1572891A2 - Composes agissant contre la resorption osseuse - Google Patents

Composes agissant contre la resorption osseuse

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Publication number
EP1572891A2
EP1572891A2 EP02782164A EP02782164A EP1572891A2 EP 1572891 A2 EP1572891 A2 EP 1572891A2 EP 02782164 A EP02782164 A EP 02782164A EP 02782164 A EP02782164 A EP 02782164A EP 1572891 A2 EP1572891 A2 EP 1572891A2
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EP
European Patent Office
Prior art keywords
polypeptide
seq
amino acid
rankl
rank
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP02782164A
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German (de)
English (en)
Inventor
Jonathan c/o Barnes-Jewish Hospital LAM
F. Patrick c/o Barnes-Jewish Hospital ROSS
Steven L. Barnes-Jewish Hospital TEITELBAUM
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Barnes Hospital
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Barnes Hospital
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Publication of EP1572891A2 publication Critical patent/EP1572891A2/fr
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • 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
    • 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
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to polypeptides that bind to RANK and whose amino acid sequences comprise one or more external surface loops AA" , CD, EF, and DE of RANKL. Further provided are fragments, analogs, and derivatives of said polypeptides .
  • the invention also relates to pharmaceutical compositions comprising the polypeptides provided herein, and/or fragments, analogs, and derivatives thereof.
  • the invention further provides methods for inhibiting osteoclast differentiation and methods for competitively inhibiting RANKL comprising administering an effective amount of said compositions. Also provided are methods for inhibiting bone resorption and methods for treating diseases or conditions which are at least partially characterized by loss of bone mass.
  • bone loss Other conditions known to involve bone loss include juvenile osteoporosis, osteogenesis imperfecta, hypercalcemia, hyperparathyroidism, osteomalacia, osteohalisteresis, osteolytic bone disease, osteonecrosis, Paget's disease of bone, bone loss due to rheumatoid arthritis, inflammatory arthritis, osteomyelitis, corticosteroid treatment, metastatic bone diseases, periodontal bone loss, bone loss due to cancer, age-related loss of bone mass, and other forms of osteopenia. Additionally, new bone formation is needed in many situations, e.g., to facilitate bone repair or replacement for bone fractures, bone defects, plastic surgery, dental and other implantations and in other such contexts.
  • Bone is a dense, specialized form of connective tissue. Bone matrix is formed by osteoblast cells located at or near the surface of existing bone matrix. Bone is resorbed
  • osteoclast a type of macrophage
  • RANK ligand also known as osteoprotegerin ligand (OPGL) , TNF-related activation induced cytokine (TRANCE) , and osteoclast differentiation factor (ODF)
  • OPGL osteoprotegerin ligand
  • TRANCE TNF-related activation induced cytokine
  • ODF osteoclast differentiation factor
  • RANKL The cell surface receptor for RANKL is RANK, Receptor Activator of Necrosis Factor (NF) -kappa B.
  • RANKL is a type-2 transmembrane protein with an intracellular domain of less than about 50 amino acids, a transmembrane domain of about 21 amino acids, and an extracellular domain of about 240 to 250 amino acids. RANKL exists naturally in transmembrane and soluble forms .
  • the deduced amino acid sequence for at least the murine, rat and human forms of RANKL and variants thereof are known. See e.g., Anderson, et al . , U.S. Pat. No. 6,017,729, Boyle, U.S. Pat. No. 5,843,678, and Xu J. et al . , J " . Bone Min . Res . (2000/15:2178) which are incorporated herein by reference. Furthermore, we have solved the crystal structure of RANKL ectodomain, as disclosed in application Ser. No. 60/311,163, filed August 9, 2001 and Ser. No. 10/215,446 filed August 9, 2002.
  • RANKL (OPGL) has been identified as a potent inducer of bone resorption and as a positive regulator of osteoclast development. Lacey et al . , supra . In addition to its role as a factor in osteoclast differentiation and activation, RANKL has been reported to induce human dendritic cell (DC) cluster formation. Anderson et al., supra and mammary epithelium development J.Fata et al., "The osteoclast differentiation factor osteoprotegerin ligand is essential for mammary gland development," Cell , 103:41-50 (2000).
  • RANKL plays a role in anabolic bone formation processes and can be utilized in methods for stimulation of osteoblast proliferation or bone nodule mineralization, as disclosed in applications Ser. No. 60/277,855, filed March 22, 2001 and Ser. No. 10,105,057 filed March 22, 2002.
  • polypeptides comprising one or more of the external surface loops AA" , CD, DE, or EF of RANKL and having the ability to bind to RANK.
  • the RANKL loops correspond to the portions of RANKL molecule (SEQ ID NO 6) described below: AA" contains amino acid residues 170-193 (SEQ ID NO 2) , CD contains amino acid residues 224-233 (SEQ ID NO 3) , DE contains amino acid residues 245-251 (SEQ ID NO 4) , and
  • EF contains amino acid residues 261-269 (SEQ ID NO 5) .
  • a polypeptide containing a portion of AA" loop sequence is also included in the invention, and it includes amino acid residues 175-185 (SEQ ID NO 1) .
  • Polypeptides of SEQ ID NO: 7 and SEQ ID NO: 11 are natural occurring variants of the AA" loop of human RANKL. Both are natural variants of human RANKL.
  • SEQ ID NO : 8, SEQ ID NO : 9 and SEQ ID NO: 10 are surface loop polypeptide sequences of human RANKL loops CD, DE and EF respectively.
  • the invention encompasses polypeptides that bind to RANK and consist • of sequences selected from the group consisting of SEQ ID NO 1 - SEQ ID NO 5 and SEQ ID NO: 7 - SEQ ID NO: 11.
  • the present invention provides pharmaceutical compositions comprising the polypeptides.
  • the compositions may further include pharmaceutically acceptable carriers, adjuvants, solubilizers, stabilizers, and/or anti- oxidants .
  • the invention also encompasses methods for inhibiting osteoclast differentiation, methods for competitively inhibiting RANKL, and methods for inhibiting bone resorption. Such methods include administration of compositions of the invention.
  • Such methods are used to treat osteoporosis, osteolytic bone disease, rheumatoid arthritis, and skeletal metastasis.
  • FIG. 1 is a graph depicting the effect of increasing concentration of PSGSHKVTLSS peptide (SEQ ID NO 1) on osteoclast generation as measured by TRAP activity.
  • amino acid notations used herein for the twenty genetically encoded L-amino acids are conventional and are abbreviated as follows :
  • Arg designates D-arginine and L-arginine
  • R designates D-arginine and L-arginine
  • amino acids can be conveniently classified into two main categories -- hydrophilic and hydrophobic--depending primarily on the physical-chemical characteristics of the amino acid side chain. These two main categories can be further classified into subcategories that more distinctly define the characteristics of the amino acid side chains.
  • hydrophilic amino acids can be further subdivided into acidic, basic and polar amino acids.
  • hydrophobic amino acids can be further subdivided into non-polar and aromatic amino acids.
  • the definitions of the various categories of amino acids are as follows: "Hydrophilic amino acid” refers to an amino acid exhibiting a hydrophobicity of less than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al . , 1984, J. Mol. Biol. 179:125-142.
  • hydrophilic amino acids include Thr (T) , Ser (S) , His (H) , Glu (E) , Asn (N) , Gin (Q) , Asp (D) , Lys (K) and Arg (R) .
  • 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.
  • Genetically encoded acidic amino acids include Glu (E) and Asp (D) .
  • Baseic 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 His (H) , Arg (R) and Lys (K) .
  • “Polar amino acid” refers to a hydrophilic amino acid having a side chain that is uncharged at physiological pH, but which has at least one 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 Asn (N) , Gin (Q) Ser (S) and Thr (T) .
  • Hydrophobic amino acid refers to an amino acid exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg, 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophobic amino acids include Pro (P) , Lie (I) , Phe (F) , Val (V) , Leu (L) , Trp (W) , Met (M) , Ala (A) , Gly (G) and Tyr (Y) .
  • Amatic amino acid refers to a hydrophobic amino acid with a side chain having at least one aromatic or heteroaromatic ring.
  • the aromatic or heteroaromatic ring may contain one or more substituents such as -OH, -SH, -CN, -F, -CI, -Br, -I, -N0 2 , -NO, -NH 2 , -NHR,
  • each R is independently (C ⁇ -C ⁇ ) alkyl, substituted (C ⁇ -C 3 ) alkyl, (C 2 -C 6 ) alkenyl, substituted (C 2 -C ⁇ ) alkenyl, (C 2 -C e ) alkynyl, substituted (C 2 C 6 ) alkynyl, (C 5 -C 20 ) aryl, substituted (C 5 -C 20 ) aryl, (C 6 -C 2e ) arylalkyl, substituted (C 3 -C 26 ) arylalkyl, 5-20 membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 membered heteroarylalkyl or substituted 6-26 membered heteroarylalkyl.
  • Apolar amino acid refers to a hydrophobic amino acid having a side chain that is uncharged at physiological pH and which has bonds in which the pair of electrons shared in common by two atoms is generally held equally by each of the two atoms (i.e., the side chain is not polar) .
  • Genetically encoded apolar amino acids include Leu (L) , Val (V) , He (I) , Met (M) , Gly (G) and Ala (A) .
  • “Aliphatic amino acid” refers to a hydrophobic amino acid having an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include Ala (A) , Val (V) , Leu (L) and He (I) .
  • Hydroxyl-substituted aliphatic amino acid refers to a hydrophilic polar amino acid having a hydroxyl-substituted side chain. Genetically-encoded hydroxyl-substituted aliphatic amino acids include Ser (S) and Thr (T) .
  • Cys (C) is unusual in that it can form disulfide bridges with other Cys (C) residues or other sulfanyl-containing amino acids.
  • the ability of Cys (C) residues (and other amino acids with -SH containing side chains) to exist in a peptide in either the reduced free -SH or oxidized disulfide-bridged form affects whether Cys (C) residues contribute net hydrophobic or hydrophilic character to a peptide.
  • Cys (C) exhibits a hydrophobicity of 0.29 according to the normalized consensus scale of Eisenberg (Eisenberg, 1984, supra)
  • Cys (C) is categorized as a polar hydrophilic amino acid, notwithstanding the general classifications defined above.
  • the above-defined categories are not mutually exclusive.
  • amino acids having side chains exhibiting two or more physical-chemical properties can be included in multiple categories.
  • amino acid side chains having aromatic moieties that are further substituted with polar substituents, such as Tyr (Y) may exhibit both aromatic hydrophobic properties and polar or hydrophilic properties, and can therefore be included in both the aromatic and polar categories.
  • His (H) has a side chain that falls within the aromatic and basic categories . The appropriate categorization of any amino acid will be apparent to those of skill in the art, especially in light of the detailed disclosure provided herein.
  • amino acid substitutions need not be, and in certain embodiments preferably are not, restricted to the genetically encoded amino acids. Indeed, since many of the compounds described herein may be produced synthetically, they may comprise one or more genetically non-encoded amino acids. Thus, in addition to the naturally occurring genetically encoded amino acids, amino acid residues in the core peptides of structure (1) may be substituted with naturally occurring non-encoded amino acids and synthetic amino acids.
  • Certain commonly encountered amino acids of which the compounds of the invention may be comprised include, but are not limited to, ⁇ -alanine ( ⁇ -Ala) and other omega-amino acids such as 3-aminopropionic acid, 2 , 3-diaminopropionic acid (Dpr) , 4-aminobutyric acid and so forth; ⁇ -aminoisobutyric acid (Aib) ; ⁇ -aminohexanoic 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) ; nor
  • Table 3 The classifications of the genetically encoded and common non-encoded amino acids according to the categories defined above are summarized in Table 3, below. It is to be understood that Table 3 is for illustrative purposes only and does not purport to be an exhaustive list of amino acid residues that can be used in the invention. Additional amino acids may be found in Fasman, 1989, Practical Handbook of
  • a "recombinant nucleic acid” is defined either by its method of production or its structure. In reference to its method of production, e.g., a product made by a process, the process is use of recombinant nucleic acid techniques, e.g., involving human intervention in the nucleotide sequence, typically selection or production. Alternatively, it can be a nucleic acid made by generating a sequence comprising fusion of two fragments which are not naturally contiguous to each other, but is meant to exclude products of nature, e.g., naturally occurring mutants.
  • products made by transforming cells with any unnaturally occurring vector is encompassed, as are nucleic acids comprising sequences derived using any synthetic oligonucleotide process. Such is often done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a single genetic entity comprising a desired combination of functions not found in the commonly available natural forms. Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design.
  • site specific targets e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design.
  • polynucleotide and “oligonucleotide” are used interchangeably and mean a polymer of at least 2 nucleotides joined together by phosphodiester bonds and may consist of either ribonucleotides or deoxyribonucleotides .
  • sequence means the linear order in which monomers occur in a polymer, for example, the order of amino acids in a polypeptide or the order of nucleotides in a polynucleotide .
  • peptide and “polypeptide” and “protein” are used interchangeably and mean a compound that consists of two or more amino acids that are linked by means of peptide bonds .
  • recombinant protein means that the protein, whether comprising a native or mutant primary amino acid sequence, is obtained by expression of a gene carried by a recombinant DNA molecule in a cell other than the cell in which that gene and/or protein is naturally found. In other words, the gene is heterologous to the host in which it is expressed. It should be noted that any alteration of a gene, including the addition of a polynucleotide encoding an affinity purification moiety to the gene, makes that gene unnatural for the purposes of this definition, and thus that gene cannot be ⁇ naturally' found in any cell.
  • mutant includes fragments, derivatives, and analogs of polypeptides .
  • RANK refers to RANK protein, recombinant RANK proteins, RANK fusion proteins, analogs, derivatives and mimics thereof.
  • the term "animal” includes human beings.
  • the phrase "preventing or inhibiting” is being affected either by being inhibited, expressed in another manner, or reduced to such an extent that the observed property is measurably lower than is the case when the treatment is not employed. Measurement of the degree of inhibition can be determined in vi tro by methods known to the person skilled in the art .
  • an effective amount is meant an amount of the substance in question which produces a statistically significant effect.
  • an “effective amount” for therapeutic uses is the amount of the composition comprising an active compound herein required to provide a clinically significant increase in healing rates in fracture repair; reversal or inhibition of bone loss in osteoporosis; prevention or delay of onset of osteoporosis; repair or prevention of dental defects; or treatment or inhibition of other bone loss conditions, diseases or defects, including but not limited to those discussed herein above.
  • Such effective amounts will be determined using routine optimization techniques and are dependent on the particular condition to be treated, the condition of the patient, the route of administration, the formulation, and the judgment of the practitioner and other factors evident to those skilled in the art .
  • the dosage required for the compounds of the invention is manifested as that which induces a statistically significant difference in bone mass between treatment and control groups.
  • This difference in bone mass may be seen, for example, as at least 1-2%, or any clinically significant enhancement in bone mass for the treatment group.
  • Other measurements of clinically significant increases in healing may include, for example, tests for breaking strength and tension, breaking strength and torsion, 4-point bending, and other biomechanical tests well known to those skilled in the art .
  • General guidance for treatment regimens is obtained from the experiments carried out in animal models of the disease of interest.
  • treatment includes both prophylaxis and therapy.
  • the compounds of the invention may be administered to a subject already suffering from loss of bone mass or to prevent or inhibit the occurrence of such condition.
  • a polypeptide whose sequence represents a subset of the AA" loop of RANKL acts as a competitive antagonist of RANKL by preventing RANKL from inducing cellular differentiation of osteoclast precursors. Furthermore, the observed inhibition of osteoclast differentiation was dose- dependent .
  • AA" loop (residues 170-193 of RANKL protein) bridges strands A and A' , the CD loop (residues 224-233) connects strands C and D, the EF loop (residues 261-269) links strands E and F, and the loop DE (residues 245-251) connects strands D and E.
  • RANKL possesses a longer AA" loop and a shorter EF loop than the typical TNF family member.
  • the AA" loop, together with the displacement of the CD loop confers a unique surface to the upper third of the RANKL molecule, whereas a subtle shift of the DE loop shapes the receptor binding groove at the base of RANKL molecule.
  • polypeptides containing RANKL external surface loop sequences and muteins thereof for inhibiting osteoclast differentiation. Consequently, such polypeptides may be used to treat diseases or conditions manifested at least in part by loss of bone mass.
  • the present invention provides polypeptides comprising one or more of external surface loops AA" , CD, DE, or EF of RANKL and having the ability to bind to RANK.
  • the RANKL loops correspond to the portions of RANKL molecule (SEQ ID NO 6) described below:
  • AA contains amino acid residues 170-193 (SEQ ID NO 2)
  • CD contains amino acid residues 224-233 (SEQ ID NO 3) ,
  • DE contains amino acid residues 245-251 (SEQ ID NO 4) .
  • EF contains amino acid residues 261-269 (SEQ ID NO 5) .
  • the invention is illustrated by a polypeptide containing a portion of the AA" loop sequence which includes amino acid residues 175-185 (SEQ ID NO 1) .
  • the RANKL loops correspond to the portions of human RANKL including polypeptides of SEQ ID NO: 7 and SEQ ID NO: 11 which are natural occurring variants of the AA" loop of human RANKL. Both are natural variants of human RANKL.
  • SEQ ID NO: 8 SEQ ID NO : 9 and SEQ ID NO: 10 are surface loop polypeptide sequences of human RANKL loops CD, DE and EF respectively .
  • the invention encompasses polypeptides that bind to RANK and consist of sequences selected from the group consisting of SEQ ID NO 1 - SEQ ID NO 5 and SEQ ID NO : 7 - SEQ ID NO: 11.
  • the invention encompasses concatemers of one or more polypeptides. selected from SEQ ID NO 1 - SEQ ID NO 5 and SEQ ID NO : 7 - SEQ ID NO: 11.
  • the invention encompasses therapeutic combinations of said polypeptides .
  • the invention also provides polypeptides comprising RANKL external surface loops and having the ability to competitively inhibit RANKL.
  • polypeptides can be synthesized using conventional synthesis procedures commonly used by one skilled in the art.
  • the polypeptides can be chemically synthesized using an automated peptide synthesizer (such as one manufactured by Pharmacia LKB Biotechnology Co., LKB Biolynk 4170 or Milligen, Model 9050 (Milligen, Millford, MA)) following the method of Sheppard, et al . , Journal of
  • the resin is washed with dimethylformamide containing piperidine, and the protecting group of the amino functional group of the C-terminal acid is removed.
  • the next amino acid corresponding to the desired peptide is coupled to the C-terminal amino acid.
  • the deprotecting process is then repeated.
  • Successive desired amino acids are fixed in the same manner until the peptide chain of the desired sequence is formed.
  • the protective groups other than the acetoamidomethyl are then removed and the peptide is released with solvent .
  • the polypeptides can be synthesized by using nucleic acid molecules which encode the polypeptides of this invention in an appropriate expression vector which include the encoding nucleotide sequences.
  • DNA molecules may be prepared using an automated DNA sequencer and the well-known codon-amino acid relationship of the genetic code. Such a DNA molecule also may be obtained as genomic DNA or as cDNA using oligonucleotide probes and conventional hybridization methodologies. Such DNA molecules may be incorporated into expression vectors, including plasmids, which are adapted for the expression of the DNA and production of the polypeptide in a suitable host such as bacterium, e . g. , Escherichia coli , yeast cell, mammalian cell, or insect cell. Mammalian expression systems may facilitate glycosylation that may improve pharmaceutical and/or immunologic properties of the compound.
  • fragments refer to compounds modified in such manner as to retain the ability to bind RANK.
  • a fragment may be any suitable portion of the peptide of the present invention so long as the RANK binding functionality is retained by the fragment.
  • Modifications may be achieved by any of the techniques known in the art for derivatization of polypeptides into fragments, analogs, or derivatives thereof.
  • analog also specifically include peptide, non-peptide, small molecules and other compounds that function as RANKL mimics that bind RANKL.
  • modifications in the amino acid sequence of a peptide, polypeptide, or protein can result in equivalent, or possibly improved, second generation peptides, etc., that display equivalent or superior functional characteristics when compared to the original amino acid sequence .
  • the present invention accordingly encompasses such modified amino acid sequences. Alterations can include but are not limited to amino acid insertions, deletions, substitutions, truncations, fusions, cyclization, disulfide bridging, shuffling of subunit sequences, and the like, provided that the peptide sequences produced by such modifications retain the ability to bind RANK. Such modifications may be undertaken to improve compound half-life, biological activity, absorption, distribution, metabolism, excretion, toxicity or the like.
  • hydropathic index of amino acids One factor that can be considered in making such changes is the hydropathic index of amino acids .
  • the importance of the hydropathic amino acid index in conferring interactive biological function on a protein has been discussed by Kyte and Doolittle (J " . Mol . Biol . , 157: 105-132, 1982). It is accepted that the relative hydropathic character of amino acids contributes to the secondary structure of the resultant protein. This, in turn, affects the interaction of the protein with molecules such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.
  • each amino acid has been assigned a hydropathic index as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (- 1.6); histidine (-3.2); glutamate/glutamine/aspartate/asparagine (-3.5); lysine (- 3.9) ; and arginine (-4.5) .
  • amino acids in a peptide or protein can be substituted for other amino acids having a similar hydropathic index or score and produce a resultant peptide or protein having similar biological activity, i.e., which still retains biological functionality.
  • amino acids having hydropathic indices within ⁇ 2 are substituted for one another. More preferred substitutions are those wherein the amino acids have hydropathic indices within +1. Most preferred substitutions are those wherein the amino acids have hydropathic indices within ⁇ 0.5.
  • hydrophilicity values have been assigned to amino acids: arginine/lysine (+3.0); aspartate/glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine/glutamine (+0.2); glycine (0) ; threonine (-0.4); proline (-0.5 ⁇ 1); alanine/histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine/isoleucine (-1.8); tyrosine (-2.3); phenylalanine (- 2.5) ; and tryptophan (-3.4) .
  • amino acids having hydropathic indices within +2 are preferably substituted for one another, those within ⁇ 1 are more preferred, and those within ⁇ 0.5 are most preferred.
  • amino acid substitutions in the polypeptides of the present invention can be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, etc.
  • Exemplary substitutions that take various of the foregoing characteristics into consideration in order to produce conservative amino acid changes resulting in silent changes within the present polypeptides, etc. can be selected from other members of the class to which the naturally occurring amino acid belongs .
  • Amino acids can be divided into the following four groups: (1) acidic amino acids; (2) basic amino acids; (3) neutral polar amino acids; and (4) neutral non-polar amino acids.
  • amino acids within these various groups include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, and glutamine; and (4) neutral non-polar amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. It should be noted that changes which are not expected to be advantageous can also be useful if these result in the production of functional sequences.
  • the fragment, derivative or analog of the polypeptides of the present invention may be, for example and without limitation, (i) one in which one or more amino acid residues are substituted with a conserved or non-conserved amino acid residue, and such substituted amino acid residue may or may not be one encoded by the genetic code; (ii) one in which one or more of the amino acid residues includes a substituent group; (iii) one in which the mature protein is fused to another compound such as a compound to increase the half-life of the protein; (iv) one in which additional amino acids are fused to the protein to aid in purification or in detection and identification; or (v) one in which additional amino acid residues are fused to the protein to aid in modifying tissue distribution or localization of the protein to certain locations such as the cell membrane or extracellular compartments; or (vi) one in which another molecule, possibly a small, non-peptide molecule, mimics the RANK-binding functionality of the polypeptide.
  • the polypeptide sequence may be flanked at either of both of its N- and/or C-termini by residues.
  • flanking residues should not significantly alter the ability of the core sequence to bind to RANK and inhibit RANK/RANKL interaction.
  • Flanking residues may include Cysteines to facilitate disulfide bridging.
  • the polypeptides of the invention may include flanking residues at each terminus that may be fewer than 5 residues each. In a preferred embodiment, fewer than 3 flanking residues at each terminus, and most preferably no flanking residues.
  • substituted amide linkages may generally include, but are not limited to, groups of the formula -C(0)N(R)-, where R is (C ⁇ -C 6 ) alkyl, substituted (C ⁇ -C 6 ) alkyl, (C 2 -C 6 ) alkenyl, substituted (C 2 -C 6 ) alkenyl, (C 2 -C 6 ) alkynyl, substituted (C 2 -C 3 ) alkynyl, (C 5 -C 2 o) aryl, substituted (C 5 -C 20 ) aryl, (C 6 -C 2 6) arylalkyl, substituted (C 6 -C 26 ) arylalkyl, 5-20 membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 membered heteroarylalkyl and substituted 6-26 membered heteroarylalkyl.
  • Compounds having such non-amide linkages and methods for preparing such compounds are well-known in the art (see, e.g., Spatola, March 1983, Vega Data Vol. 1, Issue 3; Spatola, 1983, "Peptide Backbone Modifications" In -. Chemistry and Biochemistry of Amino Acids Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p.
  • amide linkages can be replaced with peptidomimetic or amide mimetic moieties which do not significantly interfere with the structure or activity of the peptides. Alternatively, all of amide linkages may be replaced with peptidomimetic moieties.
  • Suitable amide mimetic moieties are described, for example, in Olson et al . , 1993, J. Med. Chem. 36:3039-3049.
  • the peptides and peptide analogs may optionally include a peptide or peptide analog at either or both termini that may be 1 to 5 residues or more in length.
  • Peptide analogs typically contain at least one modified interlinkage, such as a substituted amide or an isostere of an amide, as described above.
  • Such additional peptides or peptide analogs may have an amino acid sequence derived from another portion of the RANKL amino acid sequence or, alternatively, their sequences may be completely random. Peptides including such random sequences may be tested for biological activity, i.e. their ability to bind to RANK.
  • One method of testing compound binding to RANK is determined by performing an assay such as, e.g., a binding assay between a desired compound and RANK. In one aspect, this is done by contacting said compound to RANK and determining its dissociation rate. Numerous possibilities for performing binding assays are well known in the art. The indication of a compound's ability to bind to RANK is determined, e.g., by a dissociation rate, and the correlation of binding activity and dissociation rates is well established in the art .
  • blocked forms of the peptides and peptide analogs in which the N- and/or C-terminus is blocked with a moiety capable of reacting with the N-terminal -NH 2 or C-terminal -C(0)OH.
  • Such blocked compounds are typically N-terminal acylated and/or C-terminal amidated or esterified.
  • Typical N-terminal blocking groups include R 1 C(0)-, where R 1 is hydrogen, (C ⁇ -C 6 ) alkyl, (C 2 -C 3 ) alkenyl, (C 2 -C 6 ) alkynyl, (C 5 -C 0 ) aryl, (C 6 -C 2 6) arylalkyl, 5-20 membered heteroaryl or 6-26 -membered heteroarylalkyl.
  • Preferred N-terminal blocking groups include acetyl, formyl and dansyl .
  • Typical C-terminal blocking groups include -C(0)NR 1 R 1 and -C(0)0R 1 , where each R 1 is independently as defined above.
  • Preferred C-terminal blocking groups include those in which each R 1 is independently (C ⁇ -C 3 ) alkyl, preferably methyl, ethyl, propyl or isopropyl.
  • a method of preventing or inhibiting bone loss, a method of inhibiting osteoclast differentiation, and a method of competitively inhibiting RANKL activity are provided by administering compositions comprising compounds identified by the screening methods described herein.
  • the bone forming compositions of the present invention may be utilized by providing an effective amount of such compositions to a subject in need thereof .
  • the methods and compositions may be used to treat many diseases or conditions characterized by bone loss or thinning.
  • Such diseases and conditions include osteoporosis, juvenile osteoporosis, osteogenesis imperfecta, hypercalcemia, hyperparathyroidism, osteomalacia, osteohalisteresis, osteolytic bone disease, osteonecrosis, Paget's disease of bone, bone loss due to rheumatoid arthritis, inflammatory arthritis, osteomyelitis, corticosteroid treatment, metastatic bone diseases, periodontal bone loss, bone loss due to cancer, age-related loss of bone mass, and other forms of osteopenia.
  • the methods and compositions of the invention are used to treat osteoporosis, osteolytic bone disease, bone loss due to rheumatoid arthritis, and skeletal metastasis .
  • compositions of the invention can be formulated as pharmaceutical or veterinary compositions.
  • a summary of such techniques is found in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, PA.
  • the administration of the compositions of the present invention may be pharmacokinetically and pharmacodynamically controlled by calibrating various parameters of administration, including the frequency, dosage, duration mode and route of administration. Variations in the dosage, duration and mode of administration may also be manipulated to produce the activity required.
  • the dosage of the compounds of the invention is typically 0.01- lOOmg/kg.
  • dosage levels are highly dependent on the nature of the disease or situation, the condition of the patient, the judgment of the practitioner, and the frequency and mode of administration. If the oral route is employed, the absorption of the substance will be a factor effecting bioavailability. A low absorption will have the effect that in the gastro-intestinal tract higher concentrations, and thus higher dosages, will be necessary.
  • the appropriate dosage of the substance should suitably be assessed by performing animal model tests, wherein the effective dose level (e.g. ED 50 ) and the toxic dose level [ e . g. TD 50 ) as well as the lethal dose level (e.g. LD 50 or LD X0 ) are established in suitable and acceptable animal models. Further, if a substance has proven efficient in such animal tests, controlled clinical trials should be performed.
  • the compounds of the invention may be used alone or in combination with other compositions for the treatment of bone loss .
  • Such compositions include anti-resorptives such as a bisphosphonate, a calcitonin, a calcitriol, an estrogen, SERM' s and a calcium source, or a supplemental bone formation agent like parathyroid hormone or its derivative, a bone orphogenetic protein, osteogenin, NaF, or a statin.
  • anti-resorptives such as a bisphosphonate, a calcitonin, a calcitriol, an estrogen, SERM' s and a calcium source, or a supplemental bone formation agent like parathyroid hormone or its derivative, a bone orphogenetic protein, osteogenin, NaF, or a statin.
  • the compounds will be formulated into suitable compositions. Formulations may be prepared in a manner suitable for systemic administration or for topical or local administration.
  • Systemic formulations include, but are not limited to those designed for injection (e.g., intramuscular, intravenous or subcutaneous injection) or may be prepared for transdermal, transmucosal, nasal, or oral administration.
  • the formulation will generally include a diluent as well as, in some cases, adjuvants, buffers, preservatives and the like.
  • compositions can be administered also in liposomal compositions or as microemulsions .
  • suitable forms include syrups, capsules, tablets, as is understood in the art.
  • formulations can be prepared in conventional forms as liquid solutions or suspensions or as solid forms suitable for solution or suspension in liquid prior to injection or as emulsions.
  • Suitable excipients include, for example, water, saline, dextrose, glycerol and the like.
  • Such compositions may also contain amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as, for example, sodium acetate, sorbitan monolaurate, and so forth.
  • compositions of the present invention may also be administered locally to sites in patients, both human and other vertebrates, such as domestic animals, rodents and livestock, where decreased bone loss and/or increased bone mass are desired using a variety of techniques known to those skilled in the art.
  • these may include sprays, lotions, gels or other vehicles such as alcohols, polyglycols, esters, oils and silicones.
  • Such local applications include, for example, at a site of a bone fracture or defect to repair or replace damaged bone.
  • an anti-resorptive agent may be administered e.g., in a suitable carrier, at a junction of an autograft, allograft or prosthesis and native bone to assist in binding of the graft or prosthesis to the native bone.
  • Binding to RANK is determined by performing an assay as described above as a binding assay between a desired compound and RANK. In one aspect, this is done by contacting a test compound to RANK and determining its dissociation rate. Numerous possibilities for performing binding assays are well known in the art. The indication of a compound's ability to bind to RANK is determined, e.g., by a dissociation rate, and the correlation of binding activity and dissociation rates is well established in the art. For example, the assay may be performed by radio-labeling a reference compound, e.g.
  • RANK a polypeptide containing a portion of AA" loop sequence SEQ ID NO 1 with 125 I and incubating it with RANK in 1.5 ml tubes . Test compounds are then added to these reactions in increasing concentrations. After optimal incubation, the RANK/compound complexes are separated, e.g., with chromatography columns, and evaluated for bound
  • RIC relative inhibitory concentration
  • the RANKL loops identified in SEQ ID NO 2 - 5 and fragments thereof are also suitable for use individually or in combination as reference compounds in such assays. Again, high throughput assays are also suitable to perform the binding assays involving one or more of the RANKL loops.
  • TRAP tartrate specific acid phosphatase
  • the osteoclast differentiation inhibition is dose-dependent, i.e. increasing the concentration of the test compound in culture decreases TRAP activity, whereas the negative control peptide has no affect on TRAP activity.

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Abstract

Cette invention concerne des polypeptides qui se lient à RANK et qui comprennent des séquences d'acides aminés de boucles de surface extérieure RANKL. Cette invention concerne également des fragments, des analogues et des dérivés de ces polypeptides. En outre, cette invention concerne des compositions renfermant ces polypeptides, ainsi que des méthodes permettant d'inhiber la différenciation ostéoclastique, des méthodes permettant d'inhiber la résorption osseuse ainsi que des méthodes permettant d'inhiber de manière compétitive l'activité RANKL. Cette invention concerne également des méthodes de traitement de maladies ou d'états se caractérisant au moins en partie par une perte de masse osseuse.
EP02782164A 2001-10-15 2002-10-15 Composes agissant contre la resorption osseuse Withdrawn EP1572891A2 (fr)

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US7381792B2 (en) 2002-01-04 2008-06-03 Xencor, Inc. Variants of RANKL protein
WO2006024096A1 (fr) * 2004-08-31 2006-03-09 Chemgenex Pharmaceuticals Limited Procédé servant à moduler l'ostéoclastogénèse
FR2883873B1 (fr) * 2005-03-31 2009-07-10 Pharmamens Sarl Inhibiteurs d'age
KR102072250B1 (ko) * 2011-05-27 2020-03-02 아블린쓰 엔.브이. Ｒａｎｋｌ 결합 펩티드를 이용한 골 재흡수 억제
CN108064231A (zh) * 2015-05-20 2018-05-22 国立大学法人大阪大学 具有炎性细胞因子分泌抑制活性的寡肽
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