CN1635849A - Bone anti-resorptive compounds - Google Patents

Abstract

This invention relates to peptides that bind to RANK and contain an amino acid sequence of an outer ring around RANKL. The invention also relates to fragments, analogs, and derivatives of such peptides. Furthermore, the invention relates to compositions containing said peptides. The invention further includes methods for inhibiting osteoclast differentiation, methods for inhibiting bone resorption, and methods for competitively inhibiting RANKL activity. The invention also provides methods for treating diseases or symptoms characterized at least in part by bone loss.

Description

Bone Antiresorptive Compounds

This application is related to and claims the benefit of the following U.S. applications, which are hereby incorporated by reference:

U.S. Patent 60/277,855, filed March 22, 2001; U.S. Patent 10/105,057, filed March 22, 2002; U.S. Patent 60/311,163, filed August 9, 2001; US Patent 10/215,446, US Patent 60/329,231 filed October 12, 2001; US Patent 60/329,393 filed October 15, 2001, US Patent 60/329,360 filed October 15, 2001; 2001 U.S. Patent 60/328,876, filed Oct. 12; U.S. nonprovisional application titled "RANKL Mimetics and Uses Thereof," Lam et al., filed Oct. 15, 2002; U.S. Nonprovisional Application, Lam et al., filed October 15, 2002.

This invention was made in part with government support under grants AR32788, AR46123, and DE05413 from the National Institutes of Health (NIH). The government has certain rights in this invention.

field of invention

The present invention relates to a polypeptide that binds to RANK, and the polypeptide contains one or more amino acid sequences of RANKL outer surface loops AA", CD, EF and DE. In addition, the present invention also includes fragments, analogs and derivatives of the polypeptide.

The present invention also relates to pharmaceutical compositions containing the above polypeptides and/or fragments, analogs and derivatives thereof. The present invention also provides a method of inhibiting osteoclast differentiation and a method of competitively inhibiting RANKL by administering an effective amount of the composition. The invention also provides methods of inhibiting bone resorption and methods of treating diseases characterized at least in part by loss of bone mass.

technical background

A number of diseases manifested by bone loss or bone thinning are on the rise and have become serious health problems. It is estimated that the disease of osteoporosis alone threatens 30 million Americans and 1 billion people worldwide. Mundy et al., Science 286: 1946-1949 (1999). Other disorders associated with bone loss include juvenile osteoporosis, osteogenesis imperfecta, hypercalcemia, hyperparathyroidism, osteomalacia, osteopenia, osteolytic disease, osteonecrosis, Paget's of bone disease, bone loss from rheumatoid arthritis, inflammatory arthritis, osteomyelitis, corticosteroid therapy, metastatic bone disease, periodontal bone loss, bone loss from cancer, age-related bone loss, and other forms of Osteopenia. Additionally, new bone formation is required in many situations, such as: to facilitate bone repair or replacement of fractures, bone defects, orthopedic surgery, dental and other implants, and other such situations.

Bone is a specialized form of dense connective tissue. The bone matrix is formed by osteoblasts located on or near the surface of existing bone matrix. Bone is resorbed (eroded) by other types of cells known as osteoclasts, a type of macrophage. These cells secrete acids that dissolve the inorganic matter of bone and hydrolytic enzymes that digest its organic components. Thus, bone formation and remodeling is a kinetic process involving the ongoing interaction of the generative and erosive activities of osteoblasts and osteoclasts. Alberts et al., Molecular Biology of the Cell, Garland Press, N.Y. (Third Edition, 1994), pp.1182-1186.

Currently approved clinical drugs for the treatment of bone loss are mainly antiresorptive in nature because they inhibit the bone resorption process. Drugs used or recommended for the treatment of osteoporosis due to their ability to inhibit bone resorption include: estrogens, selective estrogen receptor modulators (SERMs), calcium, calcitriol, calcitonin (Sambrook, P et al., N. Engl. J. Med. 328: 1747-1753), alendronate (Saag, K et al., N. Engl. J. Med. 339: 292-299) and other diphosphates. Luckman et al., J. Bone Min. Res. 13, 581 (1998). However, current antiresorptive drugs have undesired effects in inhibiting bone resorption/remodeling or other undesired side effects.

Therefore, other compounds capable of treating bone loss diseases are desired. Recently, a key advance in the field of bone cell biology was the discovery of RANK ligand (RANKL, also known as osteoprotegerin ligand (OPGL), TNF-related activation-induced cytokine (TRANCE), and osteoclast differentiation ODF (ODF), a ligand expressed in stromal cells, osteoblasts, activated T lymphocytes, and mammary epithelium, is a unique molecule required for macrophage differentiation into osteoclasts. Lacey et al., Cell (Cell) 93: 165-176 (1998) (osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation), the cell surface receptor of RANKL is RANK (Receptor Activatorof N ecrosis Factor (NF) -K appa B). RANKL is a type 2 transmembrane protein containing an intracellular domain of less than 50 amino acids, a transmembrane domain of approximately 21 amino acids, and an extracellular domain of approximately 240 to 250 amino acids. Native RANKL exists as a transmembrane and soluble form of the protein. The deduced amino acid sequence of RANKL is known to exist in at least mouse, rat and human, as well as various other variants. See: Anderson et al., US Patent 6,017,729, Boyle, US Patent 5,843,678, and Xu J. et al., J. Bone Min. Res. (2000/15:2178), incorporated herein by reference. Additionally, we have solved the crystal structure of the RANKL exodomain, as published in US Patent Application 60/311,163, filed August 9, 2001, and US Patent Application 10/215,446, filed August 9, 2002.

RANKL (OPGL) has been identified as a strong inducer of bone resorption and at the same time it is a positive regulator of osteoclast development. Lacey et al., supra. In addition to its role as an osteoclast differentiation and activator, RANKL has been reported to induce human dendritic cell (DC) cluster formation. Anderson et al, supra and mammalian epithelium development J. Fata et al, "The osteoclast differentiation factor osteoprotegerin ligand is essential for mammalian gland development" Cell, 103:41-50 (2000). We have recently established that RANKL plays an important role in the anabolism of bone formation and can be used to stimulate osteoblast proliferation or mineralization of bone nodules. The results are published in US Patent Application 60/277,855, filed March 22, 2001, and US Patent Application 10,105,057, filed March 22, 2002. We also recently confirmed the precise three-dimensional structure of the RANKL external domain and mapped the amino acid sequence of the unique external surface loop of RANKL that interacts with RANK. The results are published in US Patent Application 60/311,163, filed August 9, 2001, and US Patent Application 10/215,446, filed August 9, 2002.

Therefore, in view of the limited efficacy of current drugs in the treatment of bone loss, it is urgent to develop new drugs for the treatment of such diseases.

Brief description of the invention

Therefore, the object of the present invention is to provide a polypeptide containing one or more RANKL outer surface rings AA", CD, DE or EF, and the polypeptide has RANK binding ability simultaneously. The RANKL ring corresponds to the RANKL molecule (SEQ ID No:: 6) The sections are 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);

Polypeptides containing part of the AA" loop sequence are also included in the present invention, and it also includes amino acid residues 175-185 (SEQ ID No: 1). The polypeptides of SEQ ID No: 7 and SEQ ID No: 11 are natural Variants of the AA" loop of human RANKL that exist. Both are natural variants of human RANKL. SEQ ID No: 8, SEQ ID No: 9 and SEQ ID No: 10 are the surface loop polypeptide sequences of the CD, DE and EF loops of human RANKL, respectively.

In one aspect, the present invention includes a RANK binding polypeptide and it consists of a sequence selected from SEQ ID No: 1-SEQ ID No: 5 and SEQ ID No: 7-SEQ ID No: 11.

Another object of the present invention is to provide a polypeptide containing the outer surface loop of RANKL and capable of competitively inhibiting the activity of RANKL.

A further object of the present invention is to provide fragments, analogues and derivatives of such polypeptides and to describe methods for obtaining said fragments, analogues and derivatives.

In another aspect, the present invention provides a pharmaceutical composition containing the polypeptide. The composition may further contain pharmaceutically acceptable carriers, adjuvants, solubilizers, stabilizers and/or antioxidants.

The invention also includes methods of inhibiting osteoclast differentiation, methods of competitively inhibiting RANKL, and methods of inhibiting bone resorption. These methods include methods of administering compositions of the invention. Also provided are methods of treating diseases characterized at least in part by loss of bone mass. In preferred embodiments, these methods are used to treat osteoporosis, osteolytic disease, rheumatoid arthritis, and skeletal metastasis.

Other purposes and features will be introduced separately in the following sections.

Brief description of the drawings

Figure 1 is a graph depicting the effect of elevated concentrations of PSGSHKVTLSS peptide on osteoclastogenesis by measuring the activity of TRAP.

Abbreviations and Definitions

In order to facilitate understanding of the present invention, some terms are defined as follows:

The amino acids mentioned here are 20 genetically coded L-amino acids, conventionally abbreviated as follows: amino acid single letter symbols three letter symbol Alanine A Ala Arginine R Arg Asparagine N Asn aspartic acid D. Asp cysteine C Cys Glutamine Q Gln glutamic acid E. Glu Glycine G Gly Histidine h His Isoleucine I Ile Leucine L Leu Lysine K Lys Methionine m met Phenylalanine f Phe proline P Pro serine S Ser threonine T Thr Tryptophan W Trp Tyrosine Y Tyr Valine V Val

Unless otherwise specified herein, three-letter symbols and one-letter abbreviations refer to amino acids in either the D-configuration or the L-configuration. For example, Arg refers to D-arginine and L-arginine, and R refers to D-arginine and L-arginine.

Unless otherwise indicated, when a series of one-letter and/or three-letter abbreviations are used to represent a polypeptide sequence, by convention the sequence is shown in the N→C direction. Here "C" refers to the alpha carbon of an amino acid residue. In order to determine the conservative amino acid substitutions of the above-mentioned different peptides and peptide analogs and to describe the different peptides and peptide analogs, amino acids are conventionally divided into 2 main categories based on the physicochemical properties of the amino acid side chains: Hydrophilic and hydrophobic amino acids. These 2 broad categories can be further divided into subcategories that define the properties of amino acid side chains in more detail. For example: hydrophilic amino acids can be further subdivided into acidic, basic and polar amino acids. Hydrophobic amino acids can be further subdivided into nonpolar and aromatic amino acids. The different classes of amino acids are defined as follows:

"Hydrophilic amino acid" refers to an amino acid having an amino acid hydrophobicity of less than 0 according to the standardized hydrophobicity assay of Eisenberg et al., 1984, J. Mol. Biol. 179: 125-142. Typically hydrophilic encoded amino acids include: Thr(T), Ser(S), His(H), Glu(E), Asn(N), Gln(Q), Asp(D), Lys(K), and Arg (R).

"Acidic amino acid" refers to a hydrophilic amino acid with a side chain pK value of less than 7. Acidic amino acids generally have negatively charged side chains at physiological pH due to the lack of hydrogen ions. Commonly acidic coded amino acids include Glu(E) and Asp(D).

"Basic amino acid" refers to a hydrophilic amino acid with a side chain pK value greater than 7. Basic amino acids generally have positively charged side chains under physiological pH conditions because of hydrogen ions. Common basic coded amino acids include His (H) and Arg (A) and Lys (L).

"Polar amino acid" refers to a hydrophilic amino acid whose side chain is uncharged at physiological pH, but which contains at least one bond where two atoms share a pair of electrons and the closer atom retains the electron pair. Common polar coded amino acids include Asn (N), Gln (Q), Ser (S) and Thr (T).

"Hydrophobic amino acid" refers to an amino acid having an amino acid hydrophobicity greater than 0 according to the standardized hydrophobicity assay of Eisenberg et al., 1984, J. Mol. Biol. 179: 125-142. Common hydrophobically encoded amino acids include Pro(P), Ile(I), Phe(F), Val(V), Leu(L), Trp(W), Met(M), Ala(A), Gly(G) and Tyr(Y).

"Aromatic amino acid" refers to a hydrophobic amino acid having at least one aromatic or heteroaromatic ring in its side chain. The aromatic ring or heteroaromatic ring may contain one or more substituents, such as: -OH, -SH, -CN, -F, -Cl, -Br, -I, -NO ₂ , -NO, -NH ₂ , -NHR, -NHR, -C(O)R, -C(O)OH, -C(O)OR, -C(O)NH ₂ , -C(O)NHR, -C(O)NRR, etc., R here can be (C ₁ -C ₆ ) alkyl, substituted (C ₁ -C ₆ ) alkyl, (C ₂ -C ₆ ) alkenyl, substituted (C ₂ -C ₆ ) alkenyl, respectively group, (C ₂ -C ₆ ) alkynyl group, substituted (C ₂ -C ₆ ) alkynyl group, (C ₅ -C ₂₀ ) aromatic group, substituted (C ₅ -C ₂₀ ) aromatic group, (C ₆ -C ₂₆ )Aralkyl, substituted (C ₆ -C ₂₆ )Aralkyl, 5-20 atom heteroaryl, substituted 5-20 atom heteroaryl, 6-26 atom Heteroaralkyls and substituted heteroarylalkyls of 6-26 atoms. Typically, aromatic encoded amino acids include His (H), Phe (F), Tyr (Y) and Trp (W).

"Non-polar amino acid" refers to a hydrophobic amino acid whose side chain is uncharged at physiological pH, but which contains a bond in which two atoms share a pair of electrons and each atom is equally capable of retaining that electron ( That is: the side chain has no polarity). Commonly non-polar encoded amino acids include Leu (L), Val (V), Ile (I), Met (M), Gly (G) and Ala (A).

"Aliphatic amino acid" refers to a hydrophobic amino acid containing an aliphatic carbon side chain, typically aliphatic encoded amino acids include Ala (A), Val (V), Leu (L) and Ile (I).

"Hydroxy-substituted aliphatic amino acid" refers to an aliphatic hydrophilic polar amino acid having a hydroxy-substituted side chain. Commonly hydroxy-substituted aliphatic encoded amino acids include Ser (S) and Thr (T).

The amino acid residue Cys(C) is uncommon, and it can form disulfide bridges with other Cys(C) residues or amino acids containing sulfhydryl groups. Cys(C) residues in peptides (and other amino acids with -SH side chains) exist either in the form of free -SH or in the form of oxidized disulfide bridges, whether Cys(C) residues are bonded or not Directly affects the net hydrophobic or hydrophilic properties of the peptide. According to the standardized hydrophobicity test of Eisenberg et al. (1984, supra), Cys(C) has a hydrophobic value of 0.29. It can therefore be understood that Cys(C) is classified as a polar hydrophilic amino acid in the present invention, although the conventional classification is as described above.

Those skilled in the art will appreciate that the categories defined above are not mutually exclusive. Thus, amino acids with side chains of 2 or more physicochemical properties can be included in various classifications. For example, an amino acid with an aromatic side chain can have a polar substituent, such as Tyr(Y), and the amino acid has both aromatic hydrophobic properties and polar or hydrophilic properties. They can therefore be classified as aromatic and polar amino acids. Other examples are: the side chain of His(H) belongs to aromatic and basic amino acids. The exact classification of any amino acid is well known to those skilled in the art. No further details here.

Although the above categories are illustrated in terms of genetically encoded amino acids, substituting amino acids is preferably limited to genetically encoded amino acids only in certain embodiments. In fact, many of the compounds described can be produced synthetically, and they may contain one or more common non-coded amino acids. Thus, in addition to the naturally occurring coded amino acids, naturally occurring non-coded amino acids and synthetic amino acids can be substituted for amino acid residues on the core peptide of structure (1).

Some common amino acids in compounds of the invention include, but are not limited to, β-alanine (β-Ala) and other omega-amino acids such as 3-aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4 -Aminobutyric acid, etc.; α-aminoisobutyric acid (Aib), ε-aminocaproic acid (Aha), δ-aminovaleric acid (Ava), N-methylglycine or sarcosine (MeGly), ornithine (Orn), Guanidine (Cit), t-Butylalanine (t-BuA), t-Butylglycine (t-BuG), N-methylisoleucine (MeIle), Phenylglycine (Phg), cyclohexylalanine (Cha), norleucine (Nle), naphthylalanine (Nal), 4-chlorophenylalanine (Phe(4-C1)), 2- Fluorophenylalanine (Phe(2-F)), 3-fluorophenylalanine (Phe(3-F)), 4-fluorophenylalanine (Phe(4-F)), cyanine Mycamine (Pen), 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), β-2-thienylalanine (Thi), methionine sulfoxide (MSO), Homoarginine (hArg), N-acetyllysine (AcLys), 2,4-diaminopimelic acid (Dbu), 2,3-diaminobutyric acid (Dab), p-aminophenylalanine (Phe(pNH ₂ )), N-methylvaline (MeVal), homocysteine (hCys), homophenylalanine (hPhe) and homoserine (hSer), hydroxyproline (Hyp) , homoproline (hPro), N-methylated amino acid and peptoid (N-substituted glycine).

Genetically encoded and conventionally non-encoded amino acids were classified according to the classification definitions above, summarized in Table 3 below. In the present invention, Table 3 is only for the purpose of illustration, not a list of all amino acid residues. Other amino acids can be found in Fasman, 1989, Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc, pp. 3-70, and is incorporated herein by reference.

A "recombinant nucleic acid" is defined herein by the method of preparation of the nucleic acid or by the structure of the nucleic acid. Reference to a preparation method such as: a product prepared by a method using recombinant nucleic acid technology, for example: involving human intervention on nucleic acid sequences, generally selection or preparation. Alternatively, two fragments that are naturally discrete from each other are fused to form a nucleic acid sequence. However, natural products such as naturally occurring mutants are not excluded. Thus includes, for example, the product obtained by transforming cells with any non-naturally occurring vector containing nucleic acid obtained by any method of oligonucleotide synthesis. Codons are usually replaced with redundant codons encoding identical or conservative amino acids, and sequence recognition sites are generally introduced or removed. Alternatively, functional nucleotide fragments of interest are joined together to produce a single genetic entity recombined with a functional of interest that does not normally occur in nature. Usually the restriction enzyme recognition site is the target of this artificial operation, and it will also be designed together to include other special targets such as: promoters, DNA replication sites, regulatory sequences, control sequences or other useful sequences.

Here, "polynucleotide" and "oligonucleotide" are interchangeable, and refer to a polymer composed of at least 2 nucleotides or deoxynucleic acid linked together by phosphate bonds .

The "sequence" here refers to the linear sequence of monomer polymers, for example: the amino acid sequence of a polypeptide or the nucleotide sequence of a polynucleic acid.

Here, "peptide", "polypeptide" and "protein" are interchangeable and refer to a compound consisting of two or more amino acids linked by peptide bonds.

A "recombinant protein" herein refers to a protein that contains a native or mutated original amino acid sequence. The protein is obtained by gene expression of a cell carrying a recombinant DNA molecule rather than from the cell in which the gene and/or protein is naturally found. In other words, the gene is heterologous to the expression host. It is worth mentioning that the replacement of any gene, including the addition of a polynucleotide encoding an affinity-purifying moiety to a gene, is by definition non-native, and these genes cannot naturally exist in any cell.

"Mute proteins" herein include fragments, derivatives and analogs of polypeptides.

Here, "RANK" refers to RANK protein, recombinant RANK protein, RANK fusion protein, analogs, derivatives and mimetics of the above proteins, etc.

The term "animal" herein includes humans.

The phrase "preventing or inhibiting" means inhibiting or attenuating the effect, manifested otherwise, of reducing an observable indicator of a property as compared to untreated. The degree of inhibition in vitro can be detected by methods well known to those skilled in the art.

The term "effective amount" refers to the amount of a substance that produces a statistically significant effect. For example: "effective amount" for therapeutic use refers to the amount of the composition containing the active compound in fracture repair, reversal or inhibition of bone loss in osteoporosis, prevention or delay of the onset of osteoporosis, repair or prevention of tooth loss, Or when treating or inhibiting other bone loss symptoms, diseases or deficient diseases, etc., to achieve a clinically significant increase in the cure rate required. The diseases include the above-mentioned diseases, but are not limited thereto. Effective amounts can be determined using routine optimization techniques and will depend largely on the particular condition to be treated, the condition of the patient, the route of administration, the dosage form, the physician's diagnosis, and other factors well known to those skilled in the art. The amount that induces a statistically significant difference between the treatment group and the control group dictates the required dose of the compound of the invention (eg, to treat osteoporosis). Differences in bone mass can be seen, eg, at least 1-2%, or any clinically significant improvement in bone mass in the treatment group. Other indicators for assessing the clinically significant increase in cure include: breaking strength and tensile tests, breaking strength and torsional force tests, 4-point bending tests, and other biochemical tests well known in the art. Usually the guiding scheme of drug treatment is obtained from the animal model experiment of the target disease.

"Treatment" herein includes prophylaxis and treatment. That is, when treating an individual, the compound of the present invention is administered to an individual afflicted with a bone loss disorder to prevent or inhibit the occurrence of such symptoms.

Detailed description of the invention

In accordance with the present invention, applicants have discovered that a polypeptide representing the AA" loop portion of RANKL acts as a competitive antagonist of RANKL by preventing RANKL from inducing differentiation of osteoclast precursor cells. Furthermore, it has been found that the inhibition of osteoclast differentiation exhibits a dosage dependency.

U.S. Patent Applications 60/311,163 and 10/215,446, filed August 9, 2001 and 2002, respectively, identify the RANKL surface responsible for binding RANK, which includes the outer surface rings AA", CD, DE, and EF. The outer surface of RANKL The (solvent-exposed) surface loops are unique in the TNF family in that they exhibit distinctive lengths and configurations: the AA" loop (residues 170-193 of the RANKL protein) connects the A and A' strands, the CD loop (224-233 residues) connects the C chain and the D chain, the EF loop (261-269 residues) connects the E chain and the F chain, and the DE loop (245-251 residues) connects the D chain and the E chain. Compared with general TNF family members, RANKL has a longer AA" loop and a shorter EF loop. The arrangement of the AA" loop and the CD loop forms a unique surface in the upper third of the RANKL molecule, whereas the DE Subtle changes in the loop create a receptor-binding groove at the base of the RANKL molecule. A detailed description of the RANKL loop and the binding specificity of the RANK/RANKL interaction can be found in US Patent Applications 60/311,163 and 10/215,446, filed August 9, 2001 and 2002, respectively.

Therefore, the applicant considers the application of the polypeptide containing the outer surface loop sequence of RSNKL and its mutant protein that inhibit osteoclast differentiation. Also, such polypeptides can be used in the treatment of diseases and conditions manifesting at least partial loss of bone mass.

The polypeptide provided by the present invention contains one or more outer surface AA", CD, DE or EF rings of RANKL, and has RANK binding ability. The RANKL molecule (SEQ ID No: 6) part corresponding to the RANKL ring is introduced as follows:

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),

The present invention describes polypeptides containing a portion of the AA" loop sequence, which includes amino acid residues 175-185 (SEQ ID No: 1).

In one aspect, the RANKL loop corresponds to a portion of human RANKL, comprising the peptides SEQ ID No: 7 and SEQ ID No: 11, which are variants of the AA" loop of naturally occurring human RANKL. Both of the above sequences are variants of human RANKL .SEQ ID No: 8, SEQ ID No: 9 and SEQ ID No: 10 are the peptide sequences of the surface loops of CD loop, DE loop and EF loop of human RANKL, respectively.

In another aspect, the present invention encompasses RANK binding peptides consisting of peptides selected from the group consisting of SEQ ID No: 1-SEQ ID No: 5 and SEQ ID No: 7-SEQ ID No: 11. In addition, the present invention includes the concatenation of one or more polypeptides selected from Sequence 1-Sequence 5 and Sequence 7-Sequence 11. Furthermore, the invention encompasses therapeutic combinations of said polypeptides. The present invention also provides a peptide containing the outer surface loop of RANKL and having the activity of competitively inhibiting RANKL.

Apparently, according to the present invention, the above-mentioned polypeptides can be synthesized by adopting conventional synthesis techniques routinely used by those skilled in the art. For example: according to the method of Sheppard et al., Journal of Chemical Society Perkin I, p.538 (1981), an automatic peptide synthesizer (such as: Pharmacia LKB Biotechnology Company, LKB Biolynk 4170 or Milligen, Model 9050 (Millien, Millford, MA) ) to chemically synthesize the above peptides. The operation process step is to add N,N'-dicyclohexylcarbodiimide to the amino acid whose amino functional group is protected by 9-fluorenylmethoxycarbonyl (Fmoc) and the synthesized target amino acid anhydride. Fmoc amino acid anhydrides are used in peptide synthesis. Using dimethylaminopyridine as a catalyst, the anhydride carboxyl group of Fmoc amino acid corresponding to the C-terminal amino acid residue was immobilized on Ulrtosyn A resin. Then, the resin was washed with piperidine-containing dimethylformamide, and the protecting group of the C-terminal amino acid functional group was removed. Then, the next amino acid corresponding to the synthetic peptide of interest is coupled to the C-terminal amino acid. The deprotection step is then repeated. Follow the same steps to continuously immobilize the target amino acid until the peptide chain of the target sequence is completed. The protective group is removed with acetamidomethyl, and the peptide is released with solvent.

Alternatively, the polypeptide can be synthesized using an appropriate expression vector containing a nucleic acid encoding the polypeptide of the invention. Such DNA molecules can be prepared using an automatic DNA sequencer using the codon-amino acid relationship of the genetic code. Such DNA molecules can also be obtained as genomic DNA or cDNA using oligonucleotide probes and traditional hybridization methods, and integrated into expression vectors, including: plasmids, making them suitable for use in suitable hosts The DNA and synthetic polypeptides are expressed. The host includes bacteria, such as Escherichia coli, yeast cells, mammalian cells or insect cells. Mammalian expression systems facilitate glycosylation, which can improve the pharmaceutical and/or immunological properties of compounds.

Another aspect of the invention provides fragments, analogs and derivatives of the polypeptides of the invention. As used herein, the terms "fragment", "derivative" and "analogue" refer to a compound modified in some form that retains RANK binding activity. As long as the fragment retains the RANK binding function, the fragment may be any part of the polypeptide of the present invention. Modifications may be made using any technique known in the art for derivatizing peptides into fragments, analogs or derivatives. These terms, especially "analogue" also specifically include peptides, non-peptides, small molecules and other RANK-binding compounds that act as RANKL mimetics.

Those skilled in the art understand that amino acid sequence modifications of peptides, polypeptides or proteins can be carried out to obtain equivalent, possibly improved, second-generation peptides, etc., which exhibit equivalent or superior functional characteristics compared with the original peptide amino acid sequence . The invention accordingly includes such modified amino acid sequences. Modifications may include, but are not limited to, insertions, deletions, substitutions, truncations, fusions, cyclizations, disulfide bridging, rearrangements of subunit sequences, and others, provided that the peptide sequence so modified still retains RANK binding capacity. Such modifications can improve the half-life, biological activity, absorption, distribution, metabolism, elimination, toxicity, and others of the compound. One factor to consider when making these changes is the hydrophobic index of the amino acid. The importance of the amino acid hydrophobicity index in the biological function of protein interactions was discussed by Kyte and Doolittle (J. Mol. Biol., 157:105-132, 1982). It is well understood that the relative hydrophobic character of amino acids has an impact on the secondary structure of the final protein. In turn, it affects the interaction between proteins and enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

Based on its hydrophobic and charge properties, each amino acid is assigned a Hydrophobic Index with the following values: Isoleucine (+4.5), Valine (+4.2), Leucine (+3.8), Phenylalanine acid (+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), Glutamine/Glutamic Acid/Aspartic Acid Acids/Aspartate (-3.5), Lysine (-3.9), Arginine (-4.5). As known in the art, certain amino acids of peptides or proteins can be replaced by other amino acids with similar hydrophobicity index or hydrophobic value, and the final peptide or protein obtained has similar biological activity, that is, always maintains its biological function . When making such changes, it is preferred that amino acids with a difference in hydrophobicity index of ±2 are substituted for each other, more preferably amino acids with a difference in hydrophobicity index of ±1 are substituted, and most preferably amino acids with a difference in hydrophobicity index of ±0.5 are substituted.

Amino acids can also be substituted according to their hydrophilicity. US Patent No. 4,554,101 announced the average value of the maximum local hydrophilicity of a protein, which is mainly affected by the hydrophilicity of adjacent amino acids and is related to the biological characteristics of the protein. Hydrophilic values are assigned to each amino acid as follows: arginine/lysine (+3.0), aspartic acid/glutamic acid (+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). An amino acid on a peptide, polypeptide or protein can be replaced by another amino acid with similar hydrophilic value and still obtain the final protein with similar biological activity, that is, always retain the correct biological function. When making such changes, it is preferred that amino acids with a difference in hydrophilic index between ±2 are substituted for each other, more preferably amino acids with a difference in hydrophilic index between ±1 are substituted, and most preferably amino acids with a difference in hydrophilic index between ±0.5 are substituted. amino acid substitutions.

As outlined above, amino acid substitutions on polypeptides of the invention may be made based on the similarity of amino acid side chain substituent groups. For example: their hydrophobicity, hydrophilicity, charge, size, etc. In order to obtain conservative amino acid changes, consider the above-mentioned multiple characteristics to finally obtain an example of silent changes in the polypeptide of the present invention, and other members of the natural amino acid category can be selected. Amino acids can be divided into the following four classes: (1) acidic amino acids; (2) basic amino acids; (3) neutral polar amino acids and (4) neutral nonpolar amino acids. Representative amino acids of these categories include, but are not limited to: (1) acidic (negatively charged) amino acids, such as: aspartic acid and glutamic acid; (2) basic amino acids (positively charged), such as: arginine, Histidine and lysine; (3) neutral polar amino acids such as: glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine and glutamine; (4 ) neutral nonpolar amino acids such as: alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. A caveat is that changes that are not necessarily beneficial may also be useful if a functional sequence is ultimately produced.

Therefore, fragments, derivatives or analogs of the polypeptides of the present invention may be, but not limited to, the following examples: (i) one or more amino acid residues are substituted with conservative or non-conservative amino acid residues, and these substituted amino acid residues The residue may or may not be an amino acid encoded by the genetic code; (ii) one or more amino acid residues contain a substituent; (iii) the fusion product of a mature protein and other compounds such as those that increase the half-life of the protein; (iv) the protein Fused with other amino acids to facilitate purification, detection or identification, or (v) the protein is fused to other amino acids to improve tissue distribution or localize the protein to certain locations such as the cell membrane or extracellular matrix; or (vi) other A molecule, possibly a small, non-peptidic molecule, mimics the RANK binding function of the polypeptide.

It is standard practice in the art to modify polypeptides to improve their pharmaceutical efficacy. The polypeptide can be modified without completely affecting its binding to RANK and/or its activity to inhibit osteoclast differentiation. For example: RANKL oligomerization has emerged as a useful technique for delaying protein internalization. Additionally, cyclization can also stabilize the mimetics of the invention. Similarly, existing treatments to increase stability or other beneficial properties of polypeptides, such as substitution of D-amino acids for L-amino acids or PEG-winging, can be used to achieve RANKL mimetics of the present invention.

In one aspect, flanking residues may be added to either or both of the N- and/or C-termini of the polypeptide sequence. When included, these flanking residues should not significantly alter the ability of the core peptide to bind RANK, inhibiting RANK/RANKL interactions. Flanking residues may contain cysteines to facilitate disulfide bond formation. Thus in embodiments, polypeptides of the invention may contain flanking residues of less than 5 amino acids each at each terminus. In preferred embodiments, there are less than 3 flanking residues at each terminus, more preferably no flanking residues.

Such molecules may contain a number of modifications known to those skilled in the art. Such as: Substitutable amide linkages generally include but are not limited to groups of the following formula: -C(O)NR-, where R is (C ₁ -C ₆ ) alkyl, substituted (C ₁ -C ₆ ) alkane group, (C ₂ -C ₆ )alkenyl, substituted (C ₂ -C ₆ )alkenyl, (C ₂ -C ₆ )alkynyl, substituted (C ₂ -C ₆ )alkynyl, (C ₅ - C ₂₀ ) aryl group, substituted (C ₅ -C ₂₀ ) aryl group, (C ₆ -C ₂₆ ) arylalkyl group, substituted (C ₆ -C ₂₆ ) arylalkyl group, 5-20 atom Heteroaryl, substituted heteroaryl of 5-20 atoms, heteroaralkane of 6-26 atoms, and substituted heteroarylalkyl of 6-26 atoms.

Amide-linked isoelectronic arrangements generally include but are not limited to: -CH ₂ NH-, -CH ₂ S-, -CH ₂ CH ₂ -, -CH=CH- (cis and trans), -C( O) _CH2- , -CH(OH) _CH2- and _-CH2SO- . Compounds containing non-amide linkages and methods for preparing such compounds are well known to those skilled 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, de., Marcel Dekker, New York, p.267 (general review); Morley, 1980, Trends Pharm.Sci.1:463-468; Hudson et al., 1979, Int.J .Prot.Res.14: 177-185 (-CH ₂ NH-, -CH ₂ CH ₂ -); Spatola et al., 1986, Life Sci.38: 1243-1249 (-CH ₂ -S); Hann, 1982, J.Chen.Soc.PerkinTrans.I.1:307-314 (-CH=CH-, cis and trans); Almquist et al., 1980, J.Med.Chem.23:1392-1398 (-COCH ₂ -); Jennings-White et al., Tetrahedron. Lett. 23:2533 (-COCH ₂ -); European Patent Application EP45665 (1982) CA 97:39405 (-CH(OH)CH ₂ -); Holladay et al., 1983, Tetrahedron Lett. 24:4401-4404 (-C(OH) _CH2- ), and Hruby, 1982, Life Sci 31:189-199 ( _-CH2- S-).

Additionally, one or more amido linkages may be replaced with a peptidomimetic or amido mimetic moiety that does not significantly interfere with the activity or structure of the peptide. Alternatively, all amide linkages can be replaced with peptidomimetic moieties. See, eg, Olson et al., 1993, J. Med. Chem. 36:3039-3049 for suitable amidomimetics.

Peptides and peptide analogs are optionally peptides or peptide analogs containing 1 to 5 amino acid residues or longer at one or both ends. Peptide analogs generally contain at least one modified intermediate linkage, such as a substituted amido or an isoelectronic form of an amido as described above. These additional peptides or peptide analogs may have amino acid sequences from other parts of RANKL or completely random sequences. These random sequences in the peptides can be used to detect biological activity, ie their ability to bind RANK.

RANK-binding compounds are detected by assaying the compound of interest and RANK binding, among other methods. In one aspect, the compound is contacted with RANK to determine its dissociation rate. Various methods for performing binding assays are known in the art. As an indicator of the dissociation rate by confirming the binding ability of the compound and RANK and using the correlation between binding activity and dissociation activity that has been established in the art.

For example: a reference compound, peptide or protein labeled with ¹²⁵ I radiation, such as RANKL or its isolated outer surface loop, etc., and co-incubated with RANK in a 1.5ml tube. The compound to be tested is added to these reactants and its concentration is gradually increased. After appropriate incubation, the RANK/compound complex is separated, eg, using a chromatographic column, and bound ¹²⁵ I-labeled peptide is measured using a gamma counter. Determine the amount of test compound required to inhibit 50% of the binding of the reference peptide. The determined values were calculated to the concentration of unlabeled reference peptide bound (relative inhibitory concentration (RIC) ^-1 = concentration _detection /concentration _reference ). Small RIC ^-1 values indicate relatively strong binding. Conversely, large RIC ^-1 values indicate relatively weak binding. See, eg: Latek et al., Proc. Natl. Acad. Sci/USA, Vol. 97, No. 21, pp. 11460-11465, 2000. Of course, high-throughput binding assays such as commercial services provided by PerkinElmer, Actelion, and others are also suitable for testing compound binding.

Blocking of peptides and peptides with substances reactive with N-terminal -NH2 or C-terminal -C(O)OH, such N- and/or C-terminal "blocked" forms of peptides and peptides also include within the scope of the present invention. Such blocking compounds are generally acetylated at the N-terminus and/or amidated or esterified at the C-terminus. Common N-terminal blocking groups include: R ¹ C(O)-, where R ¹ is a hydrogen atom, (C ₁ -C ₆ ) alkyl, (C ₂ -C ₆ ) alkenyl, (C ₂ -C ₆ ) alkynyl group, (C ₅ -C ₂₀ ) aromatic group, (C ₆ -C ₂₆ ) aralkyl group, heteroaromatic group with 5-20 atoms, or heteroarylalkyl group with 6-26 atoms . Preferred N-terminal blocking groups include: acetyl, formyl and dansyl. Typical C-terminal blocking groups include: -C(O)NR ¹ R ¹ and -C(O)OR ¹ , wherein each R ¹ is as defined above, respectively. Preferred C-terminal blocking groups include those whose R ¹ is each (C ₁ -C ₆ )alkyl, preferably methyl, ethyl, propyl or isopropyl.

In a preferred embodiment of the present invention, compounds identified according to said screening method provide a method of preventing or inhibiting bone loss, a method of inhibiting osteoclast differentiation, a method of competitively inhibiting RANKL activity by administering a composition containing said compound . The compositions of the present invention are used by providing an effective amount of the bone forming composition to an individual in need thereof. The methods and compositions are useful for treating diseases or conditions characterized by loss or thinning of bone mass. These diseases and conditions include: osteoporosis, juvenile osteoporosis, osteogenesis imperfecta, hypercalcemia, hyperparathyroidism, osteomalacia, osteopenia, osteolytic disease, osteonecrosis, Paget's of bone disease, bone loss from rheumatoid arthritis, inflammatory arthritis, osteomyelitis, corticosteroid therapy, metastatic bone disease, periodontal bone loss, bone loss from cancer, age-related bone loss, and other forms of Osteopenia. In preferred embodiments, the methods and compositions of the present invention may be used to treat osteoporosis, osteolytic disease, bone loss due to rheumatoid arthritis and bone metastases.

For the treatment of individual animals, the composition of the present invention can be made into a pharmaceutical or veterinary composition. Depending on the individual to be treated, the mode of administration and the type of treatment desired eg prophylaxis, prophylaxis, therapy, the composition can be formulated to meet the above mentioned parameters. Its technique may be found in Remington's Pharmaceutical Sciences, Last Edition, Mack Publishing Company, Easton, PA.

When administering the composition of the present invention, the following administration parameters can be adjusted to control its pharmacokinetics and pharmacodynamics. These parameters include: frequency of administration, dosage, interval pattern, route. The desired activity can be achieved by various controls on the dosage, interval and mode of administration.

For administration to animal or human subjects, the dosage of the compound of the present invention is generally 0.01-100 mg/kg. However, dosage levels will depend primarily on the natural state of the disease or condition, the condition of the patient, the judgment of the physician, and the frequency and mode of administration. If oral administration is used, absorption of the substance will be a factor affecting bioavailability. Low absorption will result in high drug concentrations in the gastrointestinal tract and require higher doses.

It is understandable that the appropriate dose of the substance is tested and evaluated by corresponding animal models, and effective dose levels (such as: ED ₅₀ ) and toxic dose levels (such as: TD ₅₀ ) and Lethal dose level (eg LD ₅₀ or LD ₁₀ ). And, if a substance proves effective in animal tests, clinical trials will begin.

When used generally in therapy, the compounds of the present invention may be used alone or in combination with other compositions to treat bone loss. These compositions contain antiresorptive substances such as: bisphosphonates, calcitonin, estrogens, SERM's and sources of calcium or bone forming supplements such as parathyroid hormone or its derivatives, bone morphogenetic proteins, osteogenic Inhibin, NaF or inhibin. See reference US Patent 6,080,779. Depending on the mode of administration, the compounds can be formulated into suitable compositions.

The medicament of the present invention can be prepared into appropriate dosage forms for systemic administration or local or localized administration. Dosage forms for systemic administration include, but are not limited to, dosage forms such as those for injection (eg, intramuscular, intravenous or subcutaneous injection) or which may be prepared for transdermal, transmucosal, nasal or oral administration. Dosage forms usually include diluents, and in some cases, excipients, buffers, preservatives, etc.

For oral administration, the composition can be administered in the form of a liposomal composition or a microemulsion. Corresponding forms include those well known in the art: syrups, capsules, tablets. For injection, dosage forms can be prepared as conventional liquids, suspensions, solids suitable for solution in or resuspended in liquid prior to injection, or as emulsions. Suitable excipients include, for example, water, saline, dextrose, glycerol, and the like. Such compositions may contain certain amounts of non-toxic auxiliary substances, such as: wetting or emulsifying agents, pH buffering agents, etc., for example: sodium acetate, sorbitan monolaurate and others.

The compositions of the present invention can be used for localized administration to patients, including humans and other vertebrates, such as domestic animals, mice and livestock. For sites where reduction of bone loss and/or increase in bone mass is desired, various techniques known to those skilled in the art may be used. For example, sprays, lotions, gels, or other carriers such as alcohols, polyethylene glycols, esters, oils, and silicones. Situations requiring topical application include, for example, fractures, repair defects, or repositioning of damaged bones. In addition, antiresorptive drugs can also be added to appropriate carriers, and administered to the joints of autografts, xenografts, prostheses and natural bone to assist the combination of grafts or prostheses and natural bone.

Other embodiments of the invention involve screening for inhibitors of RANKL using assays for competitive binding of the RANKL loop. Binding assays for RANK were performed using the compound of interest and RANK binding assay described earlier. In one aspect, a test compound is brought into contact with RANK and its dissociation rate is determined. A variety of methods are known in the art for binding assays. To determine the index of the binding ability of the compound and RANK, for example, use the dissociation rate that has been maturely established in the art, and correlate the binding activity and the dissociation rate. For example: detection can be carried out by radiolabeling reference compounds, such as: with the polypeptide of the AA" loop sequence portion of SEQ ID NO: 1 with ¹²⁵ I and RANK incubating in a 1.5 ml tube, adding the detection compound at increasing concentrations In these reactions, after appropriate incubation, the RANK/compound complex is separated, e.g., using a chromatographic column, and the bound ¹²⁵ I-labeled peptide is evaluated with a gamma counter. The amount of test compound that inhibits 50% of the binding of the reference peptide is necessary. The Values are calculated to the binding concentration of the unlabeled reference peptide (relative inhibitory concentration (RIC) ^-1 = concentration _detection /concentration _reference ). Small RIC ^-1 values indicate relatively strong binding. Conversely, large RIC ^-1 values indicate relatively strong binding. Weak binding, for example: Latek et al., Proc.Natl.Acad.Sci.USA, Vol.97, No.21, pp.11460-11465, 2000. In these assays, SEQ ID No: 2 can be used alone or in combination -5 Confirmed RANKL rings or their fragments are used as reference compounds. In addition, when one or more RANKL rings are involved, high-throughput analysis methods can also be used for binding analysis.

Additionally, objects and advantages of the present invention will be apparent in the art. The explanations and illustrations presented herein are intended to acquaint others skilled in the art with the principles and applications of the invention. Those skilled in the art can accept and apply the present invention in various forms. At the same time may be very suitable for some special needs. Therefore, the specific embodiments described herein should not be intended to exhaustively comprise or limit the invention.

All publications and patent applications listed in the examples herein are incorporated as references, and each publication or patent application can be used as references individually.

The following examples illustrate the invention, but do not exhaust all aspects of the invention.

Example

Example 1

Effect of partial AA"ring polypeptide of RANKL on TRAP activity. Wild-type C3H/HENJ mice were purchased from Harlan Industries (Indianapolis, IN). In order to establish osteoclast precursor cell culture, from 4 to 6 weeks old mice Bone marrow macrophages (BMM) were isolated from whole bone marrow and cultured at 37°C in a tissue culture incubator containing 5% CO _2. After 24 hours of culture, non-adherent cells were collected and placed on a Ficoll Hypaque gradient, collected Cells on gradient interface. Cells were re-plated at 65000/cm ² in α-minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum in the presence of recombinant mouse M-CSF (10 ng.ml) cultured at 37°C in an incubator with 5% CO _2. The cell culture was either treated with increasing concentrations of the polypeptide of SEQ ID No: 1 (subpart of the AA loop), or with the same molecular weight as the test compound Negative control scrambled peptides were processed.

Figure 1 shows the results of this experiment. As shown in the figure, the addition of test compounds to osteoclast precursors competitively inhibited the ability of RANKL to induce osteoclast differentiation as seen by the tartrate-specific acid phosphatase (TRAP) assay. The above-mentioned phosphatase is an osteoclast-specific enzyme, and its activity corresponds to the differentiation of osteoclasts. The ability of the enzyme to cleave a substrate in a chromogenic reaction is assayed to detect TRAP activity.

It can also be seen from Figure 1 that the inhibition of osteoclast differentiation was dose-dependent. That is, as the concentration of the test compound in the culture increases, the activity of TRAP is reduced. Meanwhile, the negative control peptide had no effect on TRAP activity.

Sequence List

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Ross, F. Patrick

Teitelbaum, Steven

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Claims (25)

1. An isolated polypeptide, containing one or more amino acid sequences selected from SEQ ID No: 1-SEQ ID No: 5 and SEQ ID No: 7-SEQ ID No: 11, the polypeptide has RANK binding ability. 2. The polypeptide according to claim 1, having conservative or non-conservative modifications and still having the ability to bind RANK. 3. A polypeptide comprising a RANKL sequence involved in binding to RANK, wherein said sequence consists essentially of one or more amino acid sequences selected from the group consisting of SEQ ID No: 1-SEQ ID No: 5 and SEQ ID No: 7-SEQ ID No: 11 , and the polypeptide has the ability to bind RANK. 4. The polypeptide of claim 3, which has a conservative or non-conservative modification and has the ability to inhibit RANKL from binding to RANK. 5. A non-RANKL polypeptide containing one or more outer surface loops of RANKL, said polypeptide having the ability to bind RANK. 6. A polypeptide comprising one or more outer surface loops of RANKL, said polypeptide having the activity of competitively inhibiting the binding of RANKL to RANK. 7. The polypeptide of claim 1, wherein it consists essentially of an amino acid sequence selected from SEQ ID No: 1-SEQ ID No: 5 and SEQ ID No: 7-SEQ ID No: 11. 8. A compound comprising a fragment, analog, mimetic or derivative of the polypeptide of claim 1, 2, 3, 4, 5, 6 or 7, said compound having the ability to bind RANK. 9. A pharmaceutical composition comprising an effective amount of the polypeptide according to claims 1, 2, 3, 4, 5, 6, 7 in a pharmaceutically acceptable carrier, adjuvant, co-solvent, stabilizer and/or antioxidant. 10. A pharmaceutical composition comprising an effective amount of the compound of claim 8 in a pharmaceutically acceptable carrier, adjuvant, co-solvent, stabilizer and/or antioxidant. 11. A method for inhibiting osteoclast differentiation, comprising administering an effective amount of the pharmaceutical composition of claim 9. 12. A method of inhibiting bone resorption comprising administering an effective amount of the pharmaceutical composition of claim 9. 13. A method of competitively inhibiting RANKL, said method comprising administering an effective amount of a compound of claims 1-8. 14. A method of competitively inhibiting RANKL, said method comprising administering an effective amount of the pharmaceutical composition of claim 9. 15. The treatment is selected from osteoporosis, juvenile osteoporosis, osteogenesis imperfecta, hypercalcemia, hyperparathyroidism, osteomalacia, osteopenia, osteolytic disease, osteonecrosis, Paget's disease of bone, Rheumatoid arthritis, inflammatory arthritis, osteomyelitis, corticosteroid therapy, bone loss due to metastatic bone disease, periodontal bone loss, bone loss due to cancer, aging-related bone loss, and other forms of bone loss A method for reducing diseases or symptoms, wherein said method comprises administering an effective amount of the pharmaceutical composition of claim 9. 16. The method of claim 15, wherein the disease or condition comprises osteoporosis, osteolytic disease, rheumatoid arthritis, and skeletal metastases. 17. A polypeptide comprising (a) a RANKL sequence related to binding to RANK, wherein said sequence consists essentially of at least one amino acid sequence selected from the group consisting of SEQ ID No: 1-SEQ ID No: 5 and SEQ ID No: 7-SEQ ID No: 11 and said polypeptide has RANK binding ability; and (b) additional amino acid residues which flank said sequence without eliminating the ability of said sequence to bind RANK. 18. The polypeptide of claim 1 having one or more of the following modifications: (i) a modification in which one or more amino acid residues of the polypeptide are substituted with conservative or non-conservative amino acid residues, which may or may not be encoded by the genetic code; (ii) modifications in which one or more amino acid residues contain substituents; (iii) modifications in which the polypeptide is fused to other compounds, such as compounds that increase the half-life of the protein; (iv) modifications wherein additional amino acids are fused to the polypeptide to facilitate purification or detection and identification; or (v) Modifications wherein additional amino acid residues are fused to the polypeptide to assist in altering the tissue distribution of the protein or localization to specific sites such as the cell membrane or extracellular compartments. 19. A method for screening RANKL binding compounds, said method comprising performing a binding analysis between a test compound, a reference compound and RANK, wherein said reference compound is basically selected from the group consisting of SEQ ID No: 1-SEQ ID No: 5 and SEQ ID No: 1-SEQ ID No: 5 ID No: 7-SEQ ID No: at least one sequence composition of 11. 20. A polynucleotide encoding the polypeptide of claim 1; 21. The polynucleotide of claim 20, which exhibits regular degeneracy according to the degeneracy of the genetic code. 22. A polynucleotide encoding the polypeptide of claim 20 exhibiting conservative substitutions to the polypeptide of claim 1. 23. An expression vector comprising the polynucleotide of claim 20, 21 or 22. 24. A host cell comprising the expression vector of claim 23. 25. The method of treating a disease according to claim 15, comprising administering an effective amount of one or more pharmaceutical compositions to enhance bone growth to the individual in need thereof.