# Patent

## Title
Site-specific pegylated linear salmon calcitonin analogues

## Abstract

The present invention relates to site-specific PEGylated linear salmon calcitonin analogs, or pharmaceutically acceptable salts thereof, process for their preparation, pharmaceutical compositions comprising them, and their use for the preparation of a medicament for the treatment or prevention of diseases associated with bone metabolism, e.g., osteoporosis.

## Background
This application incorporates by reference the contents of a 5.3 kb text file created on Jul. 16, 2015 and named “20150716_12664326substitutesequencelisting.txt” which is the sequence listing for this application.
TECHNICAL FIELD
The present invention relates to site-specific PEGylated linear salmon calcitonin analogues, process for their preparation, pharmaceutical compositions comprising them, and their use for the treatment of diseases associated with bone metabolism, e.g., osteoporosis and bone pain.
BACKGROUND ART
Calcitonin is secreted by thyroid parafollicular cells (C cells) of mammals or by post-branchial body of vertebrates such as fishes and birds. It is a type of single-strand polypeptide compound containing 32 amino acid residues, and is one of important hormones to maintain calcium-phosphorus metabolism in vivo. It mainly takes the action of inhibiting bone absorption, and reducing blood calcium level. Although natural calcitonins isolated from different genera are somewhat different in terms of components of amino acid residues, all of them include in their molecules the following structural features: a dithio ring structure formed by cysteines at 1,7-positions of N-terminal and a proline amide group at C-terminal. Natural calcitonin as secreted by vertebrates such as fishes exhibits the highest activity, while that as secreted by mammals exhibits a relatively low activity, for example, the activity of salmon calcitonin (sCT, which first order sequence is:
H-cyclo-(Cys1-Ser-Asn-Leu-Ser5-Thr-Cys7)-Val-Leu-Gly-Lys11-Leu-Ser-Gln-Glu-Leu16-His-Lys-Leu-Gln-Thr21-Tyr-Pro-Arg-Thr-Asn26-Thr-Gly-Ser-Gly30-Thr31-Pro-NH2) is 30 times greater than that of human calcitonin (hCT). Calcitonin can effectively prevent osteoporosis, and can simultaneously alleviate the symptoms such as bone pain and anergy of the patients suffering from osteoporosis. At present, the synthetic analogues of sCT, hCT and eel calcitonin (eCT) are mainly used in clinic for the treatment of senile osteoporosis, postmenopausal osteoporosis, Paget's disease, hypercalcemia and bone pain caused by osteoporosis or bone tumor.
sCT, as developed by Sandoz Co., was mainly used for the treatment of osteoporosis of postmenopausal women and etc., and was also suitable for patients with estrogen contra-indications and male patients suffering from osteoporosis, and its injection had already been marketed in USA in 1986, with trade name Miacalcic, and a conventional dose of 10-20 μg/day. sCT could activate adenylate cyclase (cAMP). It was demonstrated by study that cAMP, as one important second messenger in osteoclasts, participated in the inhibitory action on osteoclasts. sCT could also act on human osteoblasts, to simulate the proliferation and differentiation of osteoblasts. The action of sCT of reducing calcium and phosphorus levels in blood was mainly effectuated by inhibiting the transformation of bone calcium to blood calcium. At the same time, sCT could also promote the excretion of calcium and phosphorus in urine and bile, and inhibit the absorption of calcium and phosphorus ions in digestive tract. In addition, sCT could also specifically bind with calcitonin receptor in cerebrum and hypothalamus, and mediate central analgesic effect.
However, natural sCT can be easily inactivated by enzymolysis in vivo, and has a relatively short action time, which therefore shall be administered parenterally. Natural sCT also has a relatively poor stability in solution as well as with enzyme, and this, someone thinks, may be relevant to its dithio ring. Thus, in clinic, natural sCT shall be administered by injection frequently for a long period of time in order to achieve the treatment effectiveness, which thereby results in a poor compliance of the patients with medical treatment, and a reduction in treatment quality. Moreover, due to the presence of dithio ring, the synthesis of calcitonin is relatively difficult, and the cost thereof is increased, so that the drug obtained is too expensive to accept by the patients.
It was proved by experiment that dithio ring took different actions with respect to the activity of different calcitonins Dithio ring in sCT was not an essential group for its activity, and the linear analogue, i.e., sCT analogue free of 1,7-dithio ring, could still retain a preferable bioactivity. The synthesis of linear salmon calcitonin analogue (hereinafter referred to as sCT(L)) would become less difficult, and the cost thereof would be reduced. In addition, it was demonstrated by study that polypeptide drugs, after PEGylated, could still retain a good bioactivity, and could have a notably prolonged half life in organism. At present, the PEGylation of sCT has already been reported. It was discovered, by studying the metabolism in kidney homogenate, that the metabolic half life of the products PEGylated at three sites was far higher than that of sCT (4.8 min), i.e., the metabolic half life of the product PEGylated at N-terminal was 125.5 min, that of the product PEGylated at Lys11 was 157.3 min, and that of the product PEGylated at Lys18 was 281.5 min (K C Lee, et al, Pharm. Res., 1999, 16:813-818). In addition, the site-specific PEGylation of sCT at 8-amino of Lys18 was also reported (Y S Youn, et al, Pharm. Dev. Technol., 2005, 10(3): 389-396; J Controlled Release, 2006, 114(3): 334-342; 2007, 117(3): 371-379).
Then, by virtue of the characteristics of PEG capable of prolonging the action time, increasing the bioavailability and etc. of peptide drugs, it is possible to carry out an exact site-specific mono-PEGylation of linear sCT analogues using a reaction-specific chemical modification process. Therefore, linear sCT analogues shall include, or to which shall be re-introduced, an amino acid residue comprising a reaction-specific functional group, to increase the specificity of the reaction. The preparation of the linear sCT analogues may be implemented by using any conventional technique in the art, preferably a chemical synthetic process.
The object of the present invention is to provide a type of PEGylated sCT(L) analogues, which is in favor of developing long-acting medicaments and preparations for the treatment of bone diseases, e.g., osteoporosis.
CONTENTS OF THE INVENTION
The abbreviations used in the present invention have the following meanings:

    
    
        sCT—salmon calcitonin
        PEG—polyethylene glycol
        Ala—alanine
        Arg—arginine
        Asn—asparagine
        Cys—cysteine
        Gln—glutamine
        Glu—glutamic acid
        Gly—glycine
        His—histidine
        Leu—leucine
        Lys—lysine
        Pro—proline
        Ser—serine
        Thr—threonine
        Tyr—tyrosine
        Val—valine
        Fmoc—fluorenylmethyloxycarbonyl
        DMF—dimethylformamide
        DCC—Dicyclohexylcarbodiimide
        HBTU—2-(1H-1-hydroxybenzotriazole)-1,1,3,3-tetramethyl-uronium-hexafluorophosphate
        HOBt—1-hydroxybenzotriazole
        TFA—trifluoroacetic acid
        EDT—mercaptoethanol
        RP-HPLC—reversed phase-high performance liquid chromatography
    
    


One object of the present invention is to provide site-specific PEGylated linear salmon calcitonin analogues, which is obtained by changing certain amino acid residues in the amino acid sequence of the linear salmon calcitonin analogues to remain only one amino, carboxyl or mercapto group on the side chain, and carrying out a site-specific PEGylation on said amino, carboxyl or mercapto group. The site-specific PEGylated linear salmon calcitonin analogues of the invention have the structures of the formula (I), formula (II) or formula (III) as given and defined below.
Another object of the present invention is to provide a process for preparing the site-specific PEGylated linear salmon calcitonin analogues.
The present invention further relates to a pharmaceutical composition comprising at least one type of the site-specific PEGylated linear salmon calcitonin analogues, or a stereoisomer or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable vehicles or excipients.
The present invention still further relates to use of the site-specific PEGylated linear salmon calcitonin analogues for the preparation of a medicament for the treatment and prevention of diseases or symptoms associated with bone metabolism, e.g., osteoporosis and bone pain.
The site-specific PEGylated linear salmon calcitonin analogues of the invention have the structure of formula (I):

PEG-M-Cys-sCT(L)  (I)

    
    
        wherein,
        PEG is RO(CH2CH2O)n—CH2CH2—, R═H or CH3, n=25-2500;
    
    






Cys is cysteine, which may lie in any site of the linear salmon calcitonin analogues, including N-terminal, C-terminal and any site in the sequence;
sCT(L) is linear salmon calcitonin peptide analogue, having the following structural features:







(SEQ ID NO: 1)


Aa1-Ser-Asn-Leu-Ser-Thr-Aa2-Aa3-Leu-Gly-Aa4-Leu-


 


Ser-Gln-Glu-Aa5-Aa6-Aa7-Aa8-Pro-Aa9-Thr-Asn-Thr-


 


Gly-Ser-Aa10-Thr-Pro-NH2 






    
    
        wherein,
        Aa1 is a non-polar amino acid selected from Cys, AcmCys, N-α-propinol-Cys, Ala, D-Ala, Val, Leu, Ile, Gly, Ser, Thr, Phe, Met, Trp;
        Aa2 is a non-polar amino acid selected from Cys, AcmCys, Ala, D-Ala, Val, Leu, Ile, Gly, Ser, Thr, Phe, Met, Tip, and Aa1 and Aa2 are not both Cys;
        Aa3 is a non-polar amino acid selected from Val, Gly, Met, Leu, Ile, Cys, Ala, D-Ala;
        Aa4 is an amino acid selected from Lys, Cys, Arg, Gln;
        Aa5 is an amino acid selected from Leu, Ala, D-Ala, Ile, Cys, Val, Gly;
        Aa6 is an amino acid selected from His, Cys, Lys, Arg;
        Aa7 is an amino acid selected from Lys, Arg, Cys, His, Gln;
        Aa8 is selected from -Leu-Gln-Thr-Tyr- (SEQ ID NO:13), -Gln-Thr-Tyr-, -Thr-Tyr-, -Leu-Gln-Thr-, -Leu-Gln-, -Cys-Tyr-, Leu, Tyr, Ala, D-Ala, Cys;
        Aa9 is an amino acid selected from Arg, Lys, His, Cys;
        Aa10 is an amino acid selected from Gly, Ala, D-Ala, Cys, Pro, D-Pro.
        Unless especially specified, all of the amino acids are L-amino acids.
    
    


According to one preferred embodiment of the present invention, the sCT(L) in the formula (I) has the following structural features:

    
    
        Aa1 is Cys, Ala, D-Ala, Val;
        Aa2 is Cys, Ala, D-Ala, and Aa1 and Aa2 are not both Cys;
        Aa3 is Val, Gly, Ala, D-Ala;
        Aa4 is Lys, Arg;
        Aa5 is Leu, Ala, D-Ala, Cys;
        Aa6 is His, Arg;
        Aa7 is Lys, Arg;
        Aa8 is -Leu-Gln-Thr-Tyr-, -Ala-Gln-Thr-Tyr-;
        Aa9 is Arg, His;
        Aa10 is Gly, Ala, D-Ala.
        Unless especially specified, all of the amino acids are L-amino acids.
    
    


According to one further preferred embodiment of the present invention, the sCT(L) in the formula (I) has the following structural features:

    
    
        Aa1 is D-Ala, Val;
        Aa2 is Cys;
        Aa3 is Val, Gly;
        Aa4 is Lys;
        Aa5 is Leu, Ala;
        Aa6 is His;
        Aa7 is Lys;
        Aa8 is -Leu-Gln-Thr-Tyr-;
        Aa9 is Arg;
        Aa10 is Ala, D-Ala.
        Unless especially specified, all of the amino acids are L-amino acids.
    
    


The site-specific PEGylated linear salmon calcitonin analogues of the invention may also have the structure of formula (II):

[PEG-X—(CH2)mCO—NH]z-Cys-sCT(L)  (II)

    
    
        wherein,
        PEG is RO(CH2CH2O)n—CH2CH2—, R═H or CH3, n=25-2500;
        X=O, NH or NHCO;
        m=0-6;
        z=1;
    
    


Cys is cysteine, which may lie in any site of the linear salmon calcitonin analogues, including N-terminal, C-terminal and any site in the sequence;
sCT(L) is linear salmon calcitonin peptide analogue, the definition and preferred embodiments of which are the same as those described with formula (I).
The site-specific PEGylated sCT(L) linear salmon calcitonin analogues of formula (II) are obtained by covalently binding reaction of a PEGylating agent having carboxyl active group, aldehyde group, isocyano group, isothiocyano group and the like with terminal amino group, lysine amino group, histidine amino group or other amino group as introduced in sCT(L).
The site-specific PEGylated linear salmon calcitonin analogues of the present invention may also have the structure of formula (III):

Cys-sCT(L)-[CO—X-PEG]z  (III)

    
    
        wherein,
        PEG is RO(CH2CH2O)n—CH2CH2—, R═H or CH3, n=25-2500;
        X=O, NH or NHCO;
        z=1;
    
    


Cys is cysteine, which may lie in any site of the linear salmon calcitonin analogues, including N-terminal, C-terminal and any site in the sequence;
sCT(L) is linear salmon calcitonin peptide analogue, the definition and preferred embodiments of which are the same as those described with formula (I).
The site-specific PEGylated linear salmon calcitonin analogues of formula (III) are obtained by covalently binding reaction of a PEGylating agent having carboxyl active group, amino group and the like with terminal carboxyl group, aspartic acid carboxyl group, glutamic acid carboxyl group or other carboxyl group as introduced in sCT(L).
The site-specific PEGylated linear salmon calcitonin analogues further include the compounds obtained by replacing any amino acid residue in the linear amino acid sequence with cysteine and then modifying with mPEG-MAL, PEG-VS or PEG-IODO.
According to the present invention, the following linear salmon calcitonin analogues capable of site-specific PEGylation are preferred:










 (1)
[D-Ala1, Cys7, D-Ala30]sCT;
D-Ala-Ser-Asn-Leu-Ser-Thr-Cys-





Val-Leu-Gly-Lys-Leu-Ser-Gln-




Glu-Leu-His-Lys-Leu-Gln-Thr-




Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-




D-Ala-Thr-Pro (SEQ ID NO: 2)


 


 (2)
[Val1, Cys7, Des-19,
Val-Ser-Asn-Leu-Ser-Thr-Cys-Val-



Ala30]sCT;
Leu-Gly-Lys-Leu-Ser-Gln-Glu-




Leu-His-Lys-Gln-Thr-Tyr-Pro-Arg-




Thr-Asn-Thr-Gly-Ser-Ala-Thr-Pro




(SEQ ID NO: 3)


 


 (3)
[Val1, Ala7, Cys16, Ala30, Des-
Val-Ser-Asn-Leu-Ser-Thr-Ala-Val-



19-22]sCT;
Leu-Gly-Lys-Leu-Ser-Gln-Glu-




Cys-His-Lys-Pro-Arg-Thr-Asn-




Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID




NO: 4)


 


 (4)
[Val1, Ala7, Gln11,18, Cys16,
Val-Ser-Asn-Leu-Ser-Thr-Ala-Val-



Ala30, Des-19-22]sCT;
Leu-Gly-Gln-Leu-Ser-Gln-Glu-




Cys-His-Gln-Pro-Arg-Thr-Asn-




Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID




NO: 5)


 


 (5)
[Val1, Cys7, Des-19-22,
Val-Ser-Asn-Leu-Ser-Thr-Cys-Val-



Ala30]sCT;
Leu-Gly-Lys-Leu-Ser-Gln-Glu-




Leu-His-Lys-Pro-Arg-Thr-Asn-




Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID




NO: 6)


 


 (6)
[Val1, Cys7, Ala30, Des-
Val-Ser-Asn-Leu-Ser-Thr-Cys-Val-



19]sCT;
Leu-Gly-Lys-Leu-Ser-Gln-Glu-




Leu-His-Lys-Gln-Thr-Tyr-Pro-Arg-




Thr-Asn-Thr-Gly-Ser-Ala-Thr-Pro




(SEQ ID NO: 7)


 


 (7)
[Val1, Cys7, Gln18, Ala30, Des-
Val-Ser-Asn-Leu-Ser-Thr-Cys-Val-



19-22]sCT;
Leu-Gly-Lys-Leu-Ser-Gln-Glu-




Leu-His-Gln-Pro-Arg-Thr-Asn-




Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID




NO: 8)


 


 (8)
[Val1, Cys7, Gln11, Ala30, Des-
Val-Ser-Asn-Leu-Ser-Thr-Cys-Val-



19-22]sCT;
Leu-Gly-Gln-Leu-Ser-Gln-Glu-




Leu-His-Lys-Pro-Arg-Thr-Asn-




Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID




NO: 9)


 


 (9)
[Val1, Cys7, Gln11,18, Ala30,
Val-Ser-Asn-Leu-Ser-Thr-Cys-Val-



Des-19-22]sCT;
Leu-Gly-Gln-Leu-Ser-Gln-Glu-




Leu-His-Gln-Pro-Arg-Thr-Asn-




Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID




NO: 10)


 


(10)
[Val1, Ala7, Cys19, Ala30, Des-
Val-Ser-Asn-Leu-Ser-Thr-Ala-Val-



20-22]sCT;
Leu-Gly-Lys-Leu-Ser-Gln-Glu-




Leu-His-Lys-Cys-Pro-Arg-Thr-




Asn-Thr-Gly-Ser-Ala-Thr-Pro




(SEQ ID NO: 11)


 


(11)
[Val1, Ala7, Cys19, Ala30, Des-
Val-Ser-Asn-Leu-Ser-Thr-Ala-Val-



20-21]sCT.
Leu-Gly-Lys-Leu-Ser-Gln-Glu-




Leu-His-Lys-Cys-Tyr-Pro-Arg-




Thr-Asn-Thr-Gly-Ser-Ala-Thr-Pro




(SEQ ID NO: 12)






According to the present invention, the following site-specific PEGylated linear salmon calcitonin analogues are preferred:










 1
[D-Ala1, Cys7(mPEG5000-
D-Ala-Ser-Asn-Leu-Ser-Thr-Cys(mPEG5000-MAL)-




MAL), D-Ala30]sCT;
Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-




Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-D-Ala-




Thr-Pro (SEQ ID NO: 2)


 


 2
[Val1, Cys7(mPEG5000-
Val-Ser-Asn-Leu-Ser-Thr-Cys(mPEG5000-MAL)-Val-



MAL), Des-19,
Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Gln-Thr-



Ala30]sCT;
Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Ala-Thr-Pro (SEQ




ID NO: 3)


 


 3
[Val1, Ala7,
Val-Ser-Asn-Leu-Ser-Thr-Ala-Val-Leu-Gly-Lys-Leu-



Cys16(mPEG5000-MAL),
Ser-Gln-Glu-Cys(mPEG5000-MAL)-His-Lys-Pro-Arg-



Des-19-22, Ala30]sCT;
Thr-Asn-Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID NO: 4)


 


 4
[Val1, Ala7, Gln11,18,
Val-Ser-Asn-Leu-Ser-Thr-Ala-Val-Leu-Gly-Gln-Leu-



Cys16(mPEG5000-MAL),
Ser-Gln-Glu-Cys(mPEG5000-MAL)-His-Gln-Pro-Arg-



Des-19-22, Ala30]sCT;
Thr-Asn-Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID NO: 5)


 


 5
[Val1, Cys7(mPEG5000-
Val-Ser-Asn-Leu-Ser-Thr-Cys(mPEG5000-MAL)-Val-



MAL), Des-19-22,
Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Pro-Arg-



Ala30]sCT;
Thr-Asn-Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID NO: 6)


 


 6
[Val1, Cys7(mPEG5000-
Val-Ser-Asn-Leu-Ser-Thr-Cys(mPEG5000-MAL)-Val-



MAL), Des-19,
Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Gln-Thr-



Ala30]sCT;
Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Ala-Thr-Pro (SEQ




ID NO: 7)


 


 7
[Val1, Cys7(mPEG5000-
Val-Ser-Asn-Leu-Ser-Thr-Cys(mPEG5000-MAL)-Val-



MAL), Gln18, Des-19-22,
Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Gln-Pro-Arg-



Ala30]sCT;
Thr-Asn-Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID NO: 8)


 


 8
[Val1, Cys7(mPEG5000-
Val-Ser-Asn-Leu-Ser-Thr-Cys(mPEG5000-MAL)-Val-



MAL), Gln11, Des-19-22,
Leu-Gly-Gln-Leu-Ser-Gln-Glu-Leu-His-Lys-Pro-Arg-



Ala30]sCT;
Thr-Asn-Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID NO: 9)


 


 9
[Val1, Cys7(mPEG5000-
Val-Ser-Asn-Leu-Ser-Thr-Cys(mPEG5000-MAL)-Val-



MAL), Gln11,18, Des-19-
Leu-Gly-Gln-Leu-Ser-Gln-Glu-Leu-His-Gln-Pro-Arg-



22, Ala30]sCT;
Thr-Asn-Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID NO: 10)


 


10
[Val1, Ala7,
Val-Ser-Asn-Leu-Ser-Thr-Ala-Val-Leu-Gly-Lys-Leu-



Cys19(mPEG5000-MAL),
Ser-Gln-Glu-Leu-His-Lys-Cys(mPEG5000-MAL)-Pro-



Des-20-22, Ala30]sCT;
Arg-Thr-Asn-Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID NO: 11)



and



 


11
[Val1, Ala7,
Val-Ser-Asn-Leu-Ser-Thr-Ala-Val-Leu-Gly-Lys-Leu-



Cys19(mPEG5000-MAL),
Ser-Gln-Glu-Leu-His-Lys-Cys(mPEG5000-MAL)-Tyr-



Des-20-21, Ala30]sCT.
Pro-Arg-Thr-Asn-Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID NO: 12)






The compounds of the present invention are prepared by using conventional polypeptide synthetic processes, including solid-phase polypeptide synthetic process, liquid-phase polypeptide synthetic process and solid-liquid phase polypeptide synthetic process, wherein amino acid is protected by Fmoc-/tBu- or Boc-/Bzl-, and the linking mode includes linking N-terminal to C-terminal in sequence, or firstly synthesizing fragments and then linking the fragments. In the solid-phase synthetic process, various resins (e.g., MBHA, PAL, Rink amide resin, and etc.) capable of forming amide terminal are used as the support, various common condensing agents (e.g., DCC/HOBT, BOP/DIEA, HBTU/HOBt, TBTU, and etc.) are used for carrying out the condensation reaction, then, after completion of the reaction, trifluoroacetic acid or anhydrous HF is used for splitting the polypeptide obtained from the resin. The linking of the polypeptide with a PEGylating agent is carried out in an aqueous solution or a phosphate buffer solution, while appropriately controlling pH of the reaction solution, and monitoring the PEGylated product by RP-HPLC, and then the final product is separated and purified, and determined by MALDI-TOF-MS.
A part of preferred compounds according to the present invention are relatively effective for reducing blood calcium level of animals, and simultaneously exhibit a long-acting performance in preliminary activity study in vivo in animals.
The present invention further relates to a pharmaceutical composition comprising as active ingredient an effective amount of at least one PEGylated polypeptide and/or its stereoisomer or its physiologically nontoxic salt, and pharmaceutically acceptable vehicles or excipients. The term “pharmaceutically acceptable vehicles or excipients” used herein includes any one or all solvents, dispersing mediums, coatings, antibacterial agents or antifungal agents, isotonizing and sustained release agents, and similar physiologically compatible preparations, which are preferably suitable for administration by intravenous injection, intramscular injection, subcutaneous injection, or other parenteral routes. According to the administration route, the active compound may be coated to protect it from inactivation under the influence of acid or other natural conditions.
The term “physiologically nontoxic salt” used herein refers to salts capable of retaining the expected physiological activity of the parent compound while producing no unexpected toxic side-effect, or compositions comprising them, for example, hydrochlorides, hydrobromides, sulfates, phosphates, nitrates, and acetates, oxalates, tartrates, succinates, malates, benzoates, pamoates, alginates, mesylates, napsylates, and etc. According to the cations contained, the salts may also be divided into: inorganic salts such as potassium salts, lithium salts, zinc salts, copper salts, barium salts, bismuth salts, calcium salts, and organic salts such as trialkylammonium salts.
The PEGylated polypeptide compound and its stereoisomer or the pharmaceutical composition comprising it of the invention may be administered in any known route, e.g., orally, intramuscularly, subcutaneously, nasally, and etc. The administration dosage form includes tablets, capsules, buccal tablets, chewable tablets, elixirs, suspensions, transdermal agents, microencapsulated agents, implants, syrups, and etc. It may be common preparations, sustained release preparations, controlled release preparations and various particulate delivery systems. In order to prepare a unit dosage form into a tablet, various biodegradable or biocompatible vehicles as well-known in the art can be widely used. The examples of the vehicle include saline and various buffer aqueous solutions, ethanol or other polyols, liposomes, polylactic acid, vinyl acetate, polyanhdyrides, polyglycolic acid, collagen, poly(ortho ester), and etc.
The administration dose of the PEGylated polypeptide compound of the invention depends on various factors, such as nature and degree of severity of the disease to be prevented or treated; gender, age, body weight, sensitivity and individual reaction of the patient or animal; specific compound used; administration route; administration frequency and treatment effectiveness as expected to be achieved, and etc. The above dose may be administered in the form of a single dose or in the form of several doses, e.g., two, three, four doses.
MODE OF CARRYING OUT THE INVENTION
The following examples and biological active experiments are used to further illustrate the present invention, but shall not be understood to limit the present invention in any manner.
MBHA resin and PAM resin used as support for solid-phase synthesis in the examples are manufactured by Tianjin Nankai Synthesis Co., Ltd.; DCC, HOBT, BOP, DIEA and Fmoc-protected natural amino acids are manufactured by GL Biochem (Shanghai) Ltd. and Suzhou Tianma New Technology Co., Ltd.


EXAMPLE 1
Synthesis of [D-Ala1,Cys7(mPEG5000-MAL),D-Ala30]sCT (Compound 12)
Step 1: Solid-phase synthesis of [D-Ala1,Cys7,D-Ala30]sCT (Compound 1)
Using 250 mg MBHA resin (0.12 mmol) as solid-phase support, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-D-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-Cys(Trt)-OH as raw material, and DCC-HOBt as a condensing agent, a polypeptide resin was synthesized by a standard Fmoc solid-phase polypeptide synthetic process according to the amino acid sequence of linear sCT. The polypeptide was split from the resin by reacting at 0° C. for 1 h with 10 ml anhydrous HF as a splitting solution. The crude polypeptide obtained was dissolved in water, and lyophilized, to obtain 400 mg of a white dry powder. The crude polypeptide obtained was purified by RP-HPLC, and determined by Bio Mass Spectrometry to have a molecular weight of 3415.52, and a retention time of 11.9 min.
Step 2: Reaction of Polypeptide with a PEGylating Agent
[D-Ala1,Cys7,D-Ala30]sCT, as purified by RP-HPLC, was dissolved in water, and adjusted with a phosphate buffer to pH=7-8, to which a suitable amount of mPEG5000-MAL was added. Then, the system was reacted at room temperature, while monitoring the reaction process and the separated product by RP-HPLC. As analyzed by MALDI-TOF-MS, the product exhibited Mn=8550, a difference in molecular weights of two neighbouring peaks of 44, a typical structural feature of polyethylene glycol, and a retention time in RP-HPLC of 13.65 min.
EXAMPLE 2
Synthesis of [Val1,Cys7(mPEG5000-MAL),Des-19,Ala30]sCT (Compound 13)
The synthetic process was identical with that described in Example 1.
The peptide analogue obtained had a molecular weight of 3287.38, and a retention time of 8.13 min. As analyzed by MALDI-TOF-MS, the PEGylated product obtained exhibited Mn=8465, a difference in molecular weights of two neighbouring peaks of 44, a typical structural feature of polyethylene glycol, and a retention time in RP-HPLC of 13.16 min.
EXAMPLE 3
Synthesis of [Val1, Ala7, Cys16(mPEG5000-MAL), Des-19-22, Ala30]sCT (Compound 14)
The synthetic process was identical with that described in Example 1.
The peptide analogue obtained had a molecular weight of 2896.12, and a retention time of 7.87 min. As analyzed by MALDI-TOF-MS, the PEGylated product obtained exhibited Mn=8292, a difference in molecular weights of two neighbouring peaks of 44, a typical structural feature of polyethylene glycol, and a retention time in RP-HPLC of 13.01 min.
EXAMPLE 4
Synthesis of [Val1,Ala7,Gln11,18,Cys16(mPEG5000-MAL),Des-19-22,Ala30]sCT (Compound 15)
The synthetic process was identical with that described in Example 1.
The peptide analogue obtained had a molecular weight of 2896.30, and a retention time of 8.56 min. As analyzed by MALDI-TOF-MS, the PEGylated product obtained exhibited Mn=8295, a difference in molecular weights of two neighbouring peaks of 44, a typical structural feature of polyethylene glycol, and a retention time in RP-HPLC of 13.26 min.
EXAMPLE 5
Synthesis of [Val1, Cys7(mPEG5000-MAL),Des-19-22, Ala30]sCT (Compound 16)
The synthetic process was identical with that described in Example 1.
The peptide analogue obtained had a molecular weight of 2937.33, and a retention time of 8.87 min. As analyzed by MALDI-TOF-MS, the PEGylated product obtained exhibited Mn=7907, a difference in molecular weights of two neighbouring peaks of 44, a typical structural feature of polyethylene glycol, and a retention time in RP-HPLC of 11.82 min.
EXAMPLE 6
Synthesis of [Val1,Cys7(mPEG5000-MAL),Des-19,Ala30]sCT (Compound 17)
The synthetic process was identical with that described in Example 1.
The peptide analogue obtained had a molecular weight of 3330.8, and a retention time of 10.66 min. As analyzed by MALDI-TOF-MS, the PEGylated product obtained exhibited Mn=8476, a difference in molecular weights of two neighbouring peaks of 44, a typical structural feature of polyethylene glycol, and a retention time in RP-HPLC of 13.38 min.
EXAMPLE 7
Synthesis of [Val1,Cys7(mPEG5000-MAL), Gln18,Des-19-22, Ala30]sCT (Compound 18)
The synthetic process was identical with that described in Example 1.
The polypeptide compound obtained had a molecular weight of 2938.30, and a retention time of 9.54 min. As analyzed by MALDI-TOF-MS, the PEGylated product obtained exhibited Mn=8033, a difference in molecular weights of two neighbouring peaks of 44, a typical structural feature of polyethylene glycol, and a retention time in RP-HPLC of 11.10 min.
EXAMPLE 8
Synthesis of [Val1,Cys7(mPEG5000-MAL), Gln11,Des-19-22, Ala30]sCT (Compound 19)
The synthetic process was identical with that described in Example 1.
The peptide analogue obtained had a molecular weight of 2938.23, and a retention time of 8.95 min. As analyzed by MALDI-TOF-MS, the PEGylated product obtained exhibited Mn=7899, a difference in molecular weights of two neighbouring peaks of 44, a typical structural feature of polyethylene glycol, and a retention time in RP-HPLC of 13.43 min.
EXAMPLE 9
Synthesis of [Val1,Cys7(mPEG5000-MAL), Gln11,18,Des-19-22,Ala30]sCT (Compound 20)
The synthetic process was identical with that described in Example 1.
The peptide analogue obtained had a molecular weight of 2937.44, and a retention time of 9.47 min. As analyzed by MALDI-TOF-MS, the PEGylated product obtained exhibited Mn=8336, a difference in molecular weights of two neighbouring peaks of 44, a typical structural feature of polyethylene glycol, and a retention time in RP-HPLC of 13.69 min.
EXAMPLE 10
Synthesis of [Val1, Ala7, Cys19(mPEG5000-MAL),Des-20-22, Ala30]sCT (Compound 21)
The synthetic process was identical with that described in Example 1.
The peptide analogue obtained had a molecular weight of 3008.48, and a retention time of 8.77 min. As analyzed by MALDI-TOF-MS, the PEGylated product obtained exhibited Mn=8055, a difference in molecular weights of two neighbouring peaks of 44, a typical structural feature of polyethylene glycol, and a retention time in RP-HPLC of 13.36 min.
EXAMPLE 11
Synthesis of [Val1,Ala7,Cys19(mPEG5000-MAL),Des-20-21, Ala30]sCT (Compound 22)
The synthetic process was identical with that described in Example 1.
The peptide analogue obtained had a molecular weight of 3171.52, and a retention time of 9.46 min. As analyzed by MALDI-TOF-MS, the PEGylated product obtained exhibited Mn=8296, a difference in molecular weights of two neighbouring peaks of 44, a typical structural feature of polyethylene glycol, and a retention time in RP-HPLC of 13.53 min.
EXAMPLE 12
Evaluation on Calcium-Reducing Activity
The calcium-reducing activity of sCT(L)s and their PEGylated analogues was determined by administering them to abdomens of rats via subcutaneous injection, according to the method described in the literature (cf. Qian Deming, Shen Genquan, Ke Ruolun; Biological Assay of Calcitonin by Blood Calcium Determination in Rats, CHINESE JOURNAL OF PHARMACEUTICAL ANALYSIS, 1994, 14(3): 30-34).
The activity results as determined were listed as follows, while the reference activity of salmon calcitonin being 4500 IU/mg.







TABLE 1







The calcium-reducing activity results of linear


sCT analogues and their PEGylated analogues









Compound

Activity


No.
Sequences
(IU/mg)












1
[D-Ala1,Cys7,D-Ala30]sCT
5202


2
[Val1,Cys7,Des-19,Ala30]sCT
1136


3
[Val1,Ala7,Cys16,Ala30,Des-19-22]sCT
3394


4
[Val1,Ala7,Gln11,18,Cys16,Ala30,Des-19-22]sCT
807


5
[Val1,Cys7,Des-19-22,Ala30]sCT
67


6
[Val1,Cys7,Ala30,Des-19]sCT
4347


7
[Val1,Cys7,Gln18,Ala30,Des-19-22]sCT
233


8
[Val1,Cys7,Gln11,Ala30,Des-19-22]sCT
344


9
[Val1,Cys7,Gln11,18,Ala30,Des-19-22]sCT
920


10
[Val1,Ala7,Cys19,Ala30,Des-20-22]sCT
892


11
[Val1,Ala7,Cys19,Ala30,Des-20-21]sCT
379


12
[D-Ala1,Cys7,(mPEG5000-MAL),D-Ala30]sCT
1467


13
[Val1,Cys7(mPEG5000-MAL),Des-19,Ala30]sCT
438


14
[Val1,Ala7,Cys116(mPEG5000-MAL),Des-19-22,Ala30]sCT
987


15
[Val1,Ala7,Gln11,18,Cys16(mPEG5000-MAL),Des-19-22,Ala30]sCT
301


16
[Val1,Cys7(mPEG5000-MAL),Des-19-22,Ala30]sCT
717


17
[Val1,Cys7(mPEG5000-MAL),Des-19,Ala30]sCT
1921


18
[Val1,Cys7(mPEG5000-MAL),Gln18,Des-19-22,Ala30]sCT
763


19
[Val1,Cys7(mPEG5000-MAL),Gln11,Des-19-22,Ala30]sCT
815


20
[Val1,Cys7(mPEG5000-MAL),Gln11,18,Des-19-22,Ala30]sCT
1265


21
[Val1,Ala7,Cys19(mPEG5000-MAL),Des-20-22,Ala30]sCT
1208


22
[Val1,Ala7,Cys19(mPEG5000-MAL),Des-20-21,Ala30]sCT
1707









Compounds 1-11 in the Table were respectively non-PEGylated linear salmon calcitonin analogues as synthesized in Examples 1-11.
As can be seen from Table 1, linear salmon calcitonin analogues and their PEGylated analogues all exhibited an effective calcium-reducing activity.
EXAMPLE 13
Study on Long-Acting Performance of PEGylated sCT(L) Analogues
Compound 12 in Table 1 was taken as an example.
60 female Wistar rats, body weight 250-280 g, 16 weeks aged, were randomly divided into six groups: pseudo-castrated contrast group (10 rats); castrated contrast group (10 rats); calcitonin group (10 rats, twice every week, 0.036 μg/kg body weight/time); a low dose group of compound 12 (10 rats, twice every week, 0.216 μg/kg body weight/time); a high dose group of compound (10 rats, twice every week, 1.08 μg/kg body weight/time); and a high dose group of compound (10 rats, once every week, 1.08 μg/kg body weight/time). In each of the groups, the administration was given subcutaneously. During the experiment, the rats in each of the groups freely fed on standard solid forage and drank water. Except for the high dose group of compound 12 with administration once every week, blood samples were respectively taken from the orbits of the rats in other groups at the 6th week of administration, and subjected to the determination of osteocalcin, serum calcium and alkaline phosphatase (Table 2). At the end of the 12th week of administration, in addition to osteocalcin, serum calcium and alkaline phosphatase (Table 3), the bone densities of lumbar vertebrae, tibia and femur in the rats of each of the groups were determined (Table 4).







TABLE 2







Influence of calcitonin and its homologues on bone metabolism


of rats at the 6th week of administration













Alkaline
Serum




Dose
phosphatase
calcium
Osteocalcin


Group
(μg/kg)
(u/100 ml)
(mg/100 ml)
(ng/100 ml)





Pseudo-surgery
—
160.3 ± 19.0
10.7 ± 0.3  
3.63 ± 0.51 


group


Model group

 206.0 ± 25.2##
10.4 ± 0.2  
4.16 ± 0.25#


Calcitonin
0.036
 179.8 ± 31.4*
9.3 ± 0.2**
3.90 ± 1.1* 


group


Low dose group
0.216
187.0 ± 30.4
9.8 ± 0.3**
3.77 ± 0.25*


of compound 12


High dose group
1.08
 180.0 ± 19.4*
9.2 ± 0.2**
3.81 ± 0.24*


of compound 12





Notes:


the administration was given subcutaneously twice every week; n = 10;


#p < 0.05,


##p < 0.01, as compared with the pseudo-surgery group;


*p < 0.05,


**p < 0.01, as compared with the model group.













TABLE 3







Influence of calcitonin and its homologues on bone metabolism


of rats at the 12th week of administration













Alkaline
Serum




Dose
phosphatase
calcium
Osteocalcin


Group
(μg/kg)
(u/100 ml)
(mg/100 ml)
(ng/100 ml)





Pseudo-surgery
—
161.4 ± 33.4
10.7 ± 0.3 
3.33 ± 0.45


group


Model group

 215.6 ± 28.2##
10.6 ± 0.4 
 4.10 ± 0.43#


Calcitonin
0.036
200.9 ± 40.1
 9.3 ± 0.2**
 3.38 ± 0.17**


group


Low dose group
0.216
202.4 ± 44.8
9.8 ± 0.3*
4.01 ± 0.25


of compound 12


High dose group
1.08
188.2 ± 51.2
 9.4 ± 0.4**
3.95 ± 0.24


of compound 12





Notes:


the administration was given subcutaneously twice every week; n = 10;


#p < 0.05,


##p < 0.01, as compared with the pseudo-surgery group;


*p < 0.05,


**p < 0.01, as compared with the model group.













TABLE 4







Influence of calcitonin and its homologues on bone


density of rats at the 12th week of administration













Lumbar





Dose
vertebrae
Femur
Tibia


Group
(μg/kg)
(g/cm2)
(g/cm2)
(g/cm2)





Pseudo-surgery
—
0.189 ± 0.015 
0.264 ± 0.003
0.256 ± 0.014


group


Model group

 0.168 ± 0.016##
 0.226 ± 0.013##
0.242 ± 0.022


Calcitonin group
0.036
0.181 ± 0.013*
0.242 ± 0.027
0.247 ± 0.127


(twice/week)


101 Low dose group
0.216
0.175 ± 0.021 
0.240 ± 0.028
0.245 ± 0.014


(twice/week)


High dose group
1.08
0.181 ± 0.018*
0.241 ± 0.017
0.256 ± 0.017


of compound 12


(twice/week)


High dose group
1.08
0.182 ± 0.014*
 0.237 ± 0.0.022
0.251 ± 0.015


of compound 12


(once/week)





Notes:


n = 10;


##p < 0.01, as compared with the pseudo-surgery group;


*p < 0.05, as compared with the model group.






As can be seen from Tables 2, 3 and 4, at the 6th and 12th weeks of administration, high dose group of compound 12 was substantially equivalent to the positive contrast sCT group in the experiments relating to the influence on bone metabolism of rats. During a experiment relating to the influence on bone density lasted for 12 weeks, high dose group of compound 12 and the positive contrast sCT group both increased the bone density of lumbar vertebrae in the rats; in addition, the high dose group of PEGylated compound (administered once every week) exerted a substantially equivalent influence on the bone density of lumbar vertebrae as compared with the calcitonin (sCT) group (administered twice every week). The results exhibited the long-acting performance of PEGylated sCT(L).

## Claims

1. A linear salmon calcitonin analogue or the pharmaceutically acceptable salt thereof, which has the following structural features:
Aa1-Ser-Asn-Leu-Ser-Thr-Aa2-Aa3-Leu-Gly-Aa4-Leu-Ser-Gln-Glu-Aa5-Aa6-Aa7-Aa8-Pro-Aa9-Thr-Asn-Thr-Gly-Ser-Aa10-Thr-Pro-NH2 (SEQ ID NO: 1)

wherein,
Aa1 is selected from Cys, AcmCys, N-α-propinol-Cys, Ala, D-Ala, Val, Leu, Ile, Gly, Ser, Thr, Phe, Met, and Trp;
Aa2 is selected from Cys, AcmCys, Ala, D-Ala, Val, Leu, Ile, Gly, Ser, Thr, Phe, Met, and Trp, wherein Aa1 and Aa2 are not both Cys;
Aa3 is selected from Val, Gly, Met, Leu, Ile, Cys, Ala, and D-Ala;
Aa4 is selected from Lys, Cys, Arg, and Gln;
Aa5 is selected from Leu and Cys;
Aa6 is selected from His, Cys, Lys, and Arg;
Aa7 is selected from Lys, Arg, Cys, His, and Gln;
Aa8 is selected from -Cys-Tyr- and Cys or is absent;
Aa9 is selected from Arg, Lys, His, and Cys;
Aa10 is selected from Ala and D-Ala;

unless especially specified, all of the amino acids are L-amino acids, and
wherein at least one of Aa5 and Aa8 is Cys, and the Cys is unattached or is attached via its thiol group to a group selected from the group consisting of:
(a) PEG-M-, wherein
PEG is RO(CH2CH2O)n—CH2CH2—, R=H or CH3, n=25-2500; and





(b) [PEG-X—(CH2)mCO—NH]z—, wherein
PEG is RO(CH2CH2O)n—CH2CH2—, R=H or CH3, n=25-2500;
X=O, NH or NHCO;
m=0-6; and
z=1; and

(c) —[CO—X-PEG]z, wherein
PEG is RO(CH2CH2O)n—CH2CH2—, R=H or CH3, n=25-2500;
X=O, NH or NHCO; and
z=1.

2. The linear salmon calcitonin analogue or the pharmaceutically acceptable salt thereof according to claim 1, wherein
Aa1 is Cys, Ala, D-Ala, or Val;
Aa2 is Cys, Ala, or D-Ala, wherein Aa1 and Aa2 are not both Cys;
Aa3 is Val, Gly, Ala, or D-Ala;
Aa4 is Lys or Arg;
Aa6 is His or Arg;
Aa7 is Lys or Arg; and
Aa9 is Arg or His.

3. The linear salmon calcitonin analogue of the pharmaceutically acceptable salt thereof according to claim 2, wherein
Aa1 is D-Ala or Val;
Aa2 is Cys;
Aa3 is Val or Gly;
Aa4 is Lys;
Aa6 is His;
Aa7 is Lys;
Aa9 is Arg.

4. The linear salmon calcitonin analogue or the pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein the PEG as a molecular weight ranging from 2,000 to 100,000.

5. The linear salmon calcitonin analogue or the pharmaceutically acceptable salt thereof according to claim 4, wherein the PEG has a molecular eight ranging from 5,000 to 60,000.

6. The linear salmon calcitonin analogue or the pharmaceutically acceptable salt thereof according to claim 5, which is selected from the group consisting of:
(1) Val-Ser-Asn-Leu-Ser-Thr-Ala-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Cys-His-Lys-Pro-Arg-Thr-Asn-Thr-Gly-Scr-Ala-Thr-Pro (SEQ ID NO:4);
(2) Val-Ser-Asn-Leu-Ser-Thr-Ala-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Cys(mPEG5000-MAL)-His-Lys-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID NO:4);
(3) Val-Ser-Asn-Leu-Ser-Thr-Ala-Val-Leu-Gly-Gln-Leu-Ser-Gln-Glu-Cys-His-Gln-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID NO:5);
(4) Val-Ser-Asn-Leu-Ser-Thr-Ala-Val-Leu-Gly-Gln-Leu-Ser-Gln-Glu-Cys(mPEG5000-MAL)-His-Gln-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID NO:5);
(5) Val-Ser-Asn-Leu-Ser-T hr-Ala-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Cvs-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID NO: 11);
(6) Val-Ser-Asn-Leu-Ser-Thr-Ala-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Cys(mPEG5000-MAL)-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID NO: 11);
(7) Val-Ser-Asn-Leu-Ser-Thr-Ala-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Cys-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID NO:12); and
(8) Val-Ser-Asn-Leu-Ser-Thr-Ala-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Cys(mPEG5000-MAL)-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Ala-Thr-Pro (SEQ ID NO:12).

7. A pharmaceutical composition, comprising the linear salmon calcitonin analogue or the pharmaceutically acceptable salt thereof according to any of claims 1, 2, 3, 4, 5, or 6, and pharmaceutically acceptable vehicles or excipients.

8. A method for the treatment of a disease associated with bone metabolism which comprises administering to a subject
(i) the linear salmon calcitonin analogue or the pharmaceutically acceptable salt thereof according to any of claims 1, 2, 3, 4, 5, or 6 or
(ii) the pharmaceutical composition according to claim 7, wherein the disease is selected from the group consisting of senile osteoporosis, postmenopausal osteoporosis, Paget's disease, hypercalcemia, bone pain caused by osteoporosis, and bone pain caused by bone tumor.