Calcitonin analogs with C-terminal D-amino acid substituents
FIELD OF THE INVENTION 
     This invention relates to calcitonin analogs having the ability to reduce the serum calcium concentration and to peptides which can be converted to calcitonin analogs having the ability to reduce the serum calcium concentration. 
     BACKGROUND OF THE INVENTION 
     Correlation of calcitonin structure to biological activity has proven difficult. Often the combination of single amino acid substitutions (ability to reduce level of serum calcium) has not resulted in additive effects on biological activity, perhaps due to a lack of information on the metabolic properties of the hormone. There is a wide variation in activity in the naturally occurring calcitonins with an approximate 40-fold range in biopotency. All calcitonins share some common structural features. Each is 32 amino acids long with a C-terminal prolinamide and an N-terminal disulfide linked ring from position 1 through 7. Salmon 1 calcitonin, for example, has the following formula (Niall, H. D. (1969) Proc. Natl. Acad. Sci. USA 64, 771-778): ##STR1## Other natural calcitonins have different degrees of homology with this sequence (Queener, S. F. and Bell, N. H. (1975) Metabolism 24, 555-567; Lasmoles, F. et al. (1985) FEBS Lett. 180, 113-116). The amino acid L-threonine (2S,3R configuration) at position 31 also occurs in eel, ovine, bovine, porcine and chicken calcitonins. The same position 31 is occupied by the amino acid L-alanine in human, murine and L-valine in salmon 2 calcitonin and salmon 3 calcitonin. Eel calcitonin differs from salmon 1 calcitonin by having the amino acids L-Asp at position 26, L-Val at position 27 and L-Ala at position 29. 
     Chicken calcitonin differs from salmon 1 calcitonin by having the amino acid L-Ala at position 2, L-Ser at position 3, L-Asp at position 26, L-Val at position 27 and L-Ala at position 29. Salmon 2 calcitonin differs from salmon 1 calcitonin by having L-Asp at position 15, L-Phe at position 22, L-Ala at position 29 and L-Val at position 31. Salmon 3 calcitonin differs from salmon 1 calcitonin by having L-Met at position 8, L-Asp at position 15, L-Phe at position 22, L-Ala at position 29 and L-Val at position 31. 
     The calcitonins of mammalian origin differ more markedly from salmon 1 calcitonin, as shown by the following comparison. The disulfide linkage between positions 1 and 7 is omitted from the following sequence for clarity. 
     
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Position:                                                                 
       1      2       3    4       5    6     7                           
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Salmon 1                                                                  
       Cys    Ser     Asn  Leu     Ser  Thr   Cys                         
Human  &#34;      Gly     &#34;    &#34;       &#34;    &#34;     &#34;                           
Murine &#34;      &#34;       &#34;    &#34;       &#34;    &#34;     &#34;                           
Bovine &#34;      Ser     &#34;    &#34;       &#34;    &#34;     &#34;                           
Porcine                                                                   
       &#34;      &#34;       &#34;    &#34;       &#34;    &#34;     &#34;                           
Ovine  &#34;      &#34;       &#34;    &#34;       &#34;    &#34;     &#34;                           
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Position:                                                                 
       8      9       10   11      12   13    14                          
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Salmon 1                                                                  
       Val    Leu     Gly  Lys     Leu  Ser   Gln                         
Human  Met    &#34;       &#34;    Thr     Tyr  Thr   &#34;                           
Murine &#34;      &#34;       &#34;    &#34;       &#34;    &#34;     &#34;                           
Bovine Val    &#34;       Ser  Ala     &#34;    Trp   Lys                         
Porcine                                                                   
       &#34;      &#34;       &#34;    &#34;       &#34;    &#34;     Arg                         
Ovine  &#34;      &#34;       &#34;    &#34;       &#34;    &#34;     Lys                         
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Position:                                                                 
       15     16      17   18      19   20    21                          
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Salmon 1                                                                  
       Glu    Leu     His  Lys     Leu  Gln   Thr                         
Human  Asp    Phe     Asn  &#34;       Phe  His   &#34;                           
Murine &#34;      Leu     &#34;    &#34;       &#34;    &#34;     &#34;                           
Bovine &#34;      &#34;       &#34;    Asn     Tyr  &#34;     Arg                         
Porcine                                                                   
       Asn    &#34;       &#34;    &#34;       Phe  &#34;     &#34;                           
Ovine  Asp    &#34;       &#34;    &#34;       Tyr  &#34;     &#34;                           
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Position:                                                                 
       22     23      24   25      26   27    28                          
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Salmon 1                                                                  
       Tyr    Pro     Arg  Thr     Asn  Thr   Gly                         
Human  Phe    &#34;       Gln  &#34;       Ala  Ile   &#34;                           
Murine &#34;      &#34;       &#34;    &#34;       Ser  &#34;     &#34;                           
Bovine &#34;      Ser     Gly  Met     Gly  Phe   &#34;                           
Porcine                                                                   
       &#34;      &#34;       &#34;    &#34;       &#34;    &#34;     &#34;                           
Ovine  Tyr    &#34;       &#34;    &#34;       &#34;    &#34;     &#34;                           
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Position:                                                                 
       29     30      31   32                                             
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Salmon 1                                                                  
       Ser    Gly     Thr  Pro--NH.sub.2                                  
Human  Val    &#34;       Ala  &#34;                                              
Murine &#34;      &#34;       &#34;    &#34;                                              
Bovine Pro    Glu     Thr  &#34;                                              
Porcine                                                                   
       &#34;      &#34;       &#34;    &#34;                                              
Ovine  &#34;      &#34;       &#34;    &#34;                                              
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     The structural features responsible for the increased potency of ultimobranchial calcitonins relative to calcitonins of mammalian origin have not yet been fully determined. The removal of the C-terminal amide group to a proline moiety in human calcitonin results in a drastic loss of the activity, from 100% to 0.06%. The deletion of the entire prolinamide moiety also gives a drastic loss in activity, from 100% to 0.12% (Rittel, W. et al. (1976) Experientia 32, 246-248). 
     U.S. Pat. No. 4,469,632 shows the replacement of naturally occurring Arg 24  with D-Arg 24  in salmon 1 calcitonin, with increased activity. On the other hand, U.S. Pat. No. 4,414,149 shows a reduction in activity of salmon 1 calcitonin when L-Val 8  and L-Arg 24  are respectively replaced with L-Gly 8  and D-Arg 24 . 
     SUMMARY OF THE INVENTION 
     We have discovered that D-amino acid substituents at the C-terminal portion in synthetic salmon 1 calcitonin and other calcitonins provide calcitonin analogs having biological activity of the same type as known calcitonins (e.g., lowering plasma calcium levels). In our new peptides, the amino acid sequence contains at least one D-amino acid residue at position 31 or position 32 or both. The new peptides have good potency and quality when compared to known calcitonins. The introduction of D-amino acids which results in increased bioactivity can be due to increased stability and/or to specific structural features of the peptide analog. 
     The calcitonin analogs may be those of salmon, eel, chicken, bovine, porcine, ovine, murine or human. Preferably, a D-amino acid residue is at position 31 only. Preferred D-amino acid substituents are D-Ala, D-Val, D-Leu, D-Ile, D-Ser, D-Thr, D-Asp and D-Asn. A preferred analog is substituted salmon 1 calcitonin of the formula: ##STR2## in which X or Y or both are, independently, D-Ala, D-Val, D-Leu, D-Ile, D-allo-Ile, D-Ser, D-Thr, D-allo-Thr, D-Asp, D-Asn, D-Glu, D-Gln, D-Met, D-Met(SO), D-Met(SO 2 ), D-Pro, D-His, D-Phe, D-Tyr, D-Hypro, D-Lys, D-Arg, or the corresponding normally occurring L-amino acids, at least one of X or Y being a D-amino acid residue. 
     Preferred calcitonin analogs are [D-Ser 31  ] calcitonin, [D-Thr 31  ] calcitonin and [D-Asn 31  ] calcitonin. 
     Particularly preferred peptides of the invention are the salmon 1 analogs, especially [D-Ser 31  ] Salmon 1 calcitonin, [D-Thr 31  ] Salmon 1 calcitonin and [D-Asn 31  ] Salmon 1 calcitonin. 
    
    
     DESCRIPTION OF THE INVENTION 
     Resin Peptide Synthesis 
     The synthesis of calcitonin analogs may follow the stepwise solid phase strategy reported in Merrifield, R. B. (1963) J. Am. Chem. Soc. 85, 2149-2154, the teachings of which are incorporated herein by reference. The acid labile tert-butyloxycarbonyl (Boc-) group may be used for temporary alpha-N protection and the more acid stable groups may be used for protection of the side chains of the amino acids. Amino acid derivatives are listed in Table 1 and abbreviations are listed in Table 2. Attachment of the peptide chain to a copolymer matrix of styrene and 1% divinylbenzene may employ a benzhydrylamine type &#34;handle&#34; as reported in Pietta, P. G. et al. (1970) Chem. Commun. 650-651; Hruby, V. J. et al. (1977) J. Org. Chem. 42, 3552-3556; and Tam, J. P. et al. (1981) Tetrahedron Lett. 22, 2851-2854, which teachings also are incorporated by reference. All amino acids may be incorporated following a double coupling protocol with some modifications for particular amino acids. For all reactions, except for arginine, asparagine and glutamine, the first coupling employs the preformed symmetric anhydride method (Hagenmaier, H. and Frank, H. (1972) Hoppe-Seyler&#39;s Z. Physiol. Chem. 353, 1973-1976) in dichloromethane and the second coupling employs the preformed hydroxybenztriazole ester method (Konig, W. and Geiger, R. (1970) Chem. Ber. 103, 788-798) in dimethyl formamide (DMF). For Boc-Arg(Tos), standard DCC coupling conditions are employed to reduce the risk of lactam formation. The second coupling is done with the active HOBt ester method in DMF. Boc-Asn and Boc-Gln were exclusively coupled as HOBt esters in DMF to reduce nitrile and amidine formation (Mojsov, S. et al. (1980) J. Org. Chem. 45, 555-560). N-epsilon-(2-Chlorobenzyloxycarbonyl)lysine, Lys(ClZ), is used because it is more stable than the benzyloxycarbonyl derivative to the acid deprotection steps and it also avoids side chain branching (Erickson, B. W. and Merrifield, R. B. (1972) J. Am. Chem. Soc. 95, 3757-3763). The beta-cyclohexyl ester (cHex) of Boc-Asp-OH is used; it is also more stable to acids and thus minimizes aspartimide formation (Tam, J. P. (1979) Tetrahedron Lett. 4033-4036). The quantitative ninhydrin reaction is routinely used throughout the synthesis to monitor the extent of coupling after each cycle (Sarin, V. K. et al. (1981) Anal. Biochem. 117, 147-157). 
     
                       TABLE 1                                                     
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Amino acid derivatives for synthesis of salmon 1                          
calcitonin substitution analogs at position 31.                           
[D-Ser.sup.31 ]salmon 1 calcitonin                                        
cycl nr.                                 coup-                            
and                                      ling                             
amino                                    pro-                             
acid    protected amino acids                                             
                       MW     mmol  g    cedure                           
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32      Pro--benzhydryl       1     2                                     
        amine resin                                                       
31      Boc--D-Ser(Bzl)                                                   
                       259.1  8     2.08 A                                
                              4     1.04                                  
30,28,10                                                                  
        Boc--Gly       175.2  8     1.4  A                                
                              4     0.7                                   
29,13,5,2,                                                                
        Boc--Ser(Bzl)  259.1  8     2.08 A                                
                              4     1.04                                  
27,25,21,6                                                                
        Boc--Thr(Bzl)  309.1  8     2.48 A                                
                              4     1.24                                  
26,3    Boc--Asn       232.2  4     0.93 B                                
24,     Boc--Arg(Tos)  442.5  4     1.77 C                                
23,     Boc--Pro       215.1  8     1.72 A                                
                              4     0.86                                  
22,     Boc--Tyr(Cl.sub.2 Bzl)                                            
                       441.2  8     3.53 A                                
                              4     1.76                                  
20,14   Boc--Gln       246.3  4     0.98 B                                
19,16,12,9,4,                                                             
        Boc--Leu       249.2  8     2.0  A                                
                              4     1.0                                   
18,11   Boc--Lys(C1--Z)                                                   
                       314.8  8     2.5  A                                
                              4     1.26                                  
17,     Boc--His(Tos)  409.2  8     3.28 A                                
                              4     1.64                                  
15,     Boc--Glu(OcHex)                                                   
                       342.4  8     2.74 A                                
                              4     1.37                                  
8,      Boc--Val       217.1  8     1.74 A                                
                              4     0.87                                  
7,1,    Boc--Cys(4-Me--Bzl)                                               
                       352.2  8     2.6  A                                
                              4     1.3                                   
[D-Thr.sup.31 ]salmon 1 calcitonin                                        
31      Boc--D-Thr(Bzl)                                                   
                       309.1  8     2.48 A                                
                              4     1.24                                  
[D-Asn.sup.31 ]salmon 1 calcitonin                                        
31      Boc--D-Asn     232.2  4     0.93 B                                
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     TABLE 2 
     Abbreviations (Biochem Biophys. Acta 133, 1-5 (1967)) 
     Boc=tert-butyloxycarbonyl 
     Bzl=benzyl 
     Tos=tosyl 
     Cl 2  Bzl=2,6-dichlorobenzyl 
     Cl-Z=o-chlorobenzyloxycarbonyl 
     OcHex=gamma-cyclohexyl ester 
     4-Me-Bzl=4-methylbenzyl 
     HOBt=N-hydroxybenztriazole 
     DIEA=diisopropylethylamine 
     DCC=dicyclohexylcarbodiimide 
     DMF=N,N-dimethylformamide 
     CM=carboxymethyl 
     TFA=trifluoroacetic acid 
     HPLC=high performance liquid chromatography 
     MRC units=Medical Research Council units standard 
     Pro=L-prolyl 
     Ser=L-seryl 
     Gly=L-glycyl 
     Thr=L-threonyl 
     Asn=L-asparaginyl 
     Arg=L-arginyl 
     Tyr=L-thyronyl 
     Gln=L-glutaminyl 
     Leu=L-leucyl 
     Lys=L-lysyl 
     His=L-histidyl 
     Glu=L-glutaminyl 
     Val=L-valyl 
     Cys=L-cysteinyl 
     D-Ser=D-seryl 
     D-Thr=D-threonyl 
     D-Asn=D-asparaginyl 
     Resin Peptide Cleavage and Purification 
     Cleavage of the peptides from the resin and removal of all the remaining protecting groups is accomplished by treatment with anhydrous hydrogen fluoride in the presence of anisole (Yamashiro, D. and Li, C. H. (1978) J. Am. Chem. Soc. 100, 5174-5179). Crude peptide is removed from the resin by washing with 10% aqueous acetic acid. After lyophilization, the residue may be treated with dithiothreitol (Cleland, W. W. (1964) Biochemistry 3, 480-482) in sodium phosphate buffer at pH 7.5. The intramolecular disulfide bond in calcitonin between cysteine residues 1 and 7 can be formed by diluting the solution several-fold and adding potassiumferricyanide in aqueous solution. The resultant peptide solution is then concentrated by passing it through a CM-Sephadex, C-25 column and then eluting with a linear gradient of sodium chloride from zero to 0.3 molar in the same phosphate buffer (Live, D. H. et al. (1977) J. Org. Chem. 42, 3556-3561; Moe, G. R. and Kaiser, E. T. (1985) Biochemistry 24, 1971-1976). The sample is finally desalted by gel filtration, concentrated and isolated by HPLC. 
     While the D-amino acid substitutions in at least one of the positions 31 and 32 may be made in salmon, eel, chicken, bovine, porcine, murine, ovine and human calcitonin, for exemplification, the following detailed disclosure is directed to salmon 1 calcitonin. The formula for our new substitution analogs at position 31 and position 32 of salmon 1 calcitonin may be written as follows: ##STR3## in which X=D-Ala, D-Val, D-Leu, D-Ile, D-allo-Ile, D-Ser, D-Thr, D-allo-Thr, D-Asp, D-Asn, D-Glu, D-Gln, D-Met, D-Met(SO 2 ), D-Met(SO,), D-Pro, D-Hypro, D-Lys, D-Arg, D-His, or the corresponding L-amino acids; and 
     Y=D-Pro, D-Hypro, D-Phe, D-Tyr or the corresponding L-amino acids. At least one of X and Y is a D-amino acid residue. 
     As may be seen from the formula above, 32 amino acids are involved and in this formula, the positions are numbered according to the accepted procedure beginning at position 1 for the cysteine on one end of the chain and ending with proline amide at position 32 at the other end of the chain. For clarity of description, this same numbering system will be followed in referring to the cycles of the synthesis. The assembly of the amino acids begins with cycle 31 which involves the coupling of the amino acid to the proline moiety, followed by residue 30 and so on to the last amino acid. Protected amino acid derivatives that may be used in the synthesis of calcitonin analogs are given in Table 1. The resin which is functionalized with proline is available from chemical supply houses. 
     As indicated earlier, three types of coupling procedures are used, depending on the properties of reactants. In Table 1, the amino acid position and cycle number, type of coupling procedure, molecular weights and amount of reactants for the cycle are given. The details for each coupling protocol A, B and C are described below. 
     RESIN PEPTIDE SYNTHESIS 
     EXAMPLE 1 
     [D-Ser 31  ]salmon 1 calcitonin: Double coupling protocol using symmetric anhydride and active ester methods may be used to ensure as complete coupling as possible. The following protocol may be used for all amino acids except for arginine, asparagine and glutamine. The protocol is given for 2 g benzhydryl type resin functionalized with a total of 1 mMol of proline. 
     COUPLING PROCEDURE A 
     1. The resin is washed with dichloromethane, CH 2  Cl 2 , (30 mL, 6×1 min). 
     2. Removal of the Boc protecting group is done with 50% TFA in CH 2  Cl 2  (30 mL, 3×1 min) and with 30 mL for 20 min. 
     3. The reagent is then removed with CH 2  Cl 2  wash (30 mL, 6×1 min). 
     4. Traces of acid are finally removed with 5% DIEA in CH 2  Cl 2  (30 mL, 2×2 min). 
     5. A final wash is done before the coupling is completed, CH 2  Cl 2  (30 mL, 6×1 min). 
     6. 5 mg of the resin are removed for ninhydrin test. 
     7. The protected amino acid (listed in Table 1, 8 mMol) dissolved in 10 mL of CH 2  Cl 2  is treated with DCC (4 mMol, 825 mg) in 3 mL of CH 2  Cl 2 . After 10 min, the solution is filtered and added to the resin. The precipitate is washed with 10 mL of CH 2  Cl 2  and added to the reaction vessel which is then shaken for 2 h at room temperature. 
     8. The resin is washed with CH 2  Cl 2  (30 mL, 4×2 min). 
     9. The resin is washed with 5% DIEA in CH 2  Cl 2  (30 mL, 2 min). 
     10. The resin is washed with CH 2  Cl 2  (30 mL, 4×2 min). 
     11. Ninhydrin test is performed. 
     12. The resin is washed with DMF (30 mL, 2×2 min). 
     13. HOBt (4 mMol, 540 mg) in 7 mL of DMF at 0° C. is mixed with DCC (4 mMol, 825 mg) in 3 mL CH 2  Cl 2 . The protected amino acid (listed in Table 1, 4 mMol) dissolved in 6 mL of DMF is then added. The mixture is kept for 10 min at 0° C. and is then added to the resin. The mixture is shaken for 2 h at room temperature. 
     14. The resin is then washed with DMF (30 mL, 2×2 min). 
     15. The resin is washed with CH 2  Cl 2  (30 mL, 4×1 min). 
     16. The resin is washed with 5% DIEA in CH 2  Cl 2  (30 mL, 2 min). 
     17. The resin is washed with CH 2  Cl 2  (30 mL, 3×1 min). 
     18. Ninhydrin test is performed. 
     COUPLING PROCEDURE B 
     (Used for the amino acids asparagine and glutamine) 
     Steps 1-6 were the same as coupling procedure A. 
     7. The resin is washed with DMF in CH 2  Cl 2  (1:2 v/v, 30 mL, 2×2 min). 
     8. To HOBt (4 mMol, 540 mg) in 7 mL DMF/CH 2  Cl 2  (1:1 v/v) at 0° C. is added DCC (4 mMol, 825 mg) in 3 mL of CH 2  Cl 2 . To that mixture is then added the protected amino acid (listed in Table 1, 4 mMol) in 6 mL of DMF/CH 2  Cl 2 . The reaction mixture is added to the resin after 10 min at 0° C. The resin is then shaken for 2 h at room temperature. 
     9. The resin is washed with DMF/CH 2  Cl 2  (1:2 v/v, 30 mL, 2×2 min). 
     The steps 8-18 described in coupling procedure A are then followed. 
     COUPLING PROCEDURE C 
     (Used for the amino acid arginine) 
     Steps 1-6 are the same as coupling procedure A. 
     7. The protected amino acid (listed in Table 1, 4 mMol) in 10 mL CH 2  Cl 2  is added to the resin. DCC (4 mMol, 825 mg) in 3 mL CH 2  Cl 2  is added after 5 min to the resin. The reaction mixture is then shaken for 2 h at room temperature. 
     The steps 8-18 described in coupling procedure A are then followed. 
     EXAMPLE 2 
     [D-Thr 31  ] salmon 1 calcitonin: The synthesis of this analog follows the same protocol as previously described for [D-Ser 31  ]salmon 1 calcitonin (Table 1). Boc-D-Thr(Bzl) is used in cycle 31 in the coupling to the proline residue linked to the resin. Coupling procedure A is used. The preceeding coupling reactions of residue 30 to 1 are the same as described previously for [D-Ser -  ]salmon 1 calcitonin. 
     EXAMPLE 3 
     [D-Asn -  ]salmon 1 calcitonin: Boc-D-Asn is used in cycle 31, and coupling procedure B is employed. The preceding couplings were the same as previously described (Table 1). 
     In each example, the addition of Cys 1  represents the completion of the solid phase synthesis. The Boc group is finally removed by steps 1-6 in coupling procedure A. The resin peptides are then removed from the reaction vessel and dried in vacuum. Cleavage and purification steps are carried out as follows: 
     RESIN-PEPTIDE CLEAVAGE 
     The dried resin peptide (2 g) and 2 mL of anisole are placed in a teflon reaction vessel which is cooled in a dry ice-acetone bath and about 15 mL of hydrogen fluoride gas is condensed into the vessel. The mixture is stirred at 0° C. in an ice bath for 45 min. The hydrogen fluoride is then evaporated under vacuum, using first a water aspirator and later a high vacuum pump. The residue is triturated with 5×30 mL of ethyl acetate, and the peptide was extracted from the resin beads with 100 mL of 10% aqueous acetic acid solution. The mixture was lyophilized to dryness. 
     PURIFICATION OF CRUDE PEPTIDES 
     A 100 mg sample of the lyophilized peptide is treated with excess dithiothreitol (5 mMol) in 5 mL of 50 mM sodium phosphate buffer at pH 7.5 for 1 h at room temperature. The intramolecular disulfide bond between cysteine residues 1 and 7 is formed by diluting the peptide solution to a volume of 1 liter in the same buffer. A solution of 20 mM K 3  Fe(CN) 6  is slowly added with stirring until a persistant yellow color is obtained. The resultant dilute peptide solution is concentrated by passing it through a CM-Sephadex, C-25 column and then eluting with a linear gradient of NaCl from zero to 0.3M employing the same buffer. Fractions from this column may be desalted on a Sephadex G-15 column, eluting with a 0.03M aqueous acetic acid solution. Samples for biological testing are isolated on an analytical HPLC (column: Altex ODS, 5 micron, 4.6×250 mm, flow 1.5 mL/min, gradient of 30-45% acetonitrile in 0.1 M ammonium acetate buffer at pH 5.5). The isolated samples may be quantified using salmon 1 calcitonin as reference sample. 
     The HPLC isolated samples are hydrolyzed with 5.5M hydrochloric acid, and amino acid analyses are performed to confirm the chemical composition. 
     The new polypeptides are useful in lowering the content of calcium in the plasma, as indicated by standard tests in rats (Kumar, M. A. et al. (1965) J. Endocrinology 33, 469-475). While only certain embodiments of our invention have been described in specific details, it will be apparent to those skilled in the art that many other specific embodiments may be practiced and many changes may be made, all within the spirit of the invention and the scope of the appended claims.