Calcitonin analogue and use thereof

The invention provides a polypeptide of calcitonin analogue and use thereof. The polypeptide has such an amino acid sequence that proline-amide residue (Pro-NH.sub.2) at C-terminal of native eel calcitonin is substituted to a homoserine amide residue (Hse.multidot.NH.sub.2).

BACKGROUND OF THE INVENTION 
(1) Field of the invention 
The invention relates to a polypeptide of calcitonin analogue and use 
thereof. 
The polypeptide can be used to prevent and/or cure calcium metabolic 
diseases such as reduction of ache in osteoporosis, prevention of fracture 
in osteogenesis imperfecta, and curing of hypercalcemia and Paget's 
disease. 
(2) Related arts 
Calcitonin was firstly found by D. H. Copp et al. in the year of 1961 from 
human jugular vein blood as one of peptide hormones, which shows an 
activity of reducing a calcium concentration in blood. 
The calcitonin has been constructed from 32 amino acid residues having a 
sequence (substantially corresponding to SEQ ID NO: 1), as shown below. 
##STR1## 
In the amino acid sequence, Pro.multidot.NH.sub.2 means proline amide 
residue and Cys residues in the 1st and 7th positions are linked by a 
disulfide bond. 
Thereafter, peptide hormones having biological activities similar to the 
calcitonin have been extracted from various animals such as salmon, eel, 
chicken, pig, rat, sheep, cow, rabbit and stingray, and amino acid 
sequences of such native calcitonins have been determined. 
Structural features of calcitonins include a constant chain length of 32 
amino acids, a disulfide bridge between the cysteine residues in positions 
1 and 7, forming a ring of seven amino acid residues at the N-terminal, 
and a carboxy terminal proline amide. However, amino acid residues common 
to all calcitonins are those in 1, 4th-7th, 28th and 32nd positions, only 
(7 amino acid residues in total). It has been considered that the proline 
amide at C-terminal, common to all calcitonins, is indispensable for 
developing biological activities thereof [J. T. Potts, Jr. et al., 
"Calcium, Parathyroid Hormone and the Calcitonins", page 121 printed by 
Excerpta Medica, Amsterdam (1971); P. Sieber, "Calcitonin 1969", page 28, 
Proc. 2nd Symp., printed by Medical Books, London (1970); and W. Rittel et 
al., "Experientia", Vol. 32, page 246 (1976)]. 
It has been known that the calcitonins show biological activities of 
reducing calcium concentration in blood [P. F. Hirsch et al., "Science", 
Vol. 146, page 412 (1963)], inhibition of feeding [W. J. Freed et al., 
"Science", Vol. 206, page 850 (1979)], and inhibition of gastric secretion 
[R. D. Hesch et al., "Horm. Metab. Res.", Vol. 3, page 140 (1971)]. 
For obtaining the calcitonins, extraction from mammalian blood and tissue 
samples was firstly tried and then chemical synthesis has been employed. 
The former has disadvantages of limitation from availability of the raw 
material and troublesome in purifying operations, and the latter has also 
disadvantages of that synthetic operations are troublesome, since all of 
calcitonins are constructed from 32 amino acid residues, and that long 
period of time is required for synthesis per se and purification, so that 
it has been quite difficult to provide the calcitonins in a large amount 
with reasonable cost. 
In the recent years, the genetic engineering has remarkably developed and 
such a process utilizing techniques in the genetic engineering has been 
proposed, which comprises steps of synthesizing a single-stranded DNA 
fragment encoding one of calcitonins by using a DNA synthesizer and its 
single-stranded cDNA, preparing a double-stranded DNA fragment from the 
single-stranded DNA fragments, purifying the double-stranded DNA fragment, 
inserting the purified double-stranded DNA fragment into a vector, 
transforming Escherichia coli by the recombinant vector, cultivating the 
transformant to express a polypeptide encoding the calcitonin, and 
separating and purifying the expressed polypeptide. In this case, however, 
a polypeptide with a C-terminal proline amide and showing the desired 
biological activities can not be expressed by Escherichia coli and thus 
the C-terminal amidating treatment is required during the 
separating-purifying step, but a C-terminal amidating enzyme is expensive, 
the enzymatic treatment requires special techniques and a skill, and the 
enzymatic process makes yield of the objective substance low. Therefore, 
this conventional process can not be said as one suitable for industrial 
scale production of calcitonins, from view points of cost on the reagent 
and yield. 
Therefore, researchers in the assignee company have energetically studied 
and investigated on a process utilizing genetic recombination techniques 
to find that various calcitonin analogues can be obtained without use of 
the expensive C-terminal amidating enzyme, each of which analogues 
includes no methionine residue (Met) in its amino acid sequence thereof 
and C-terminal structure is proline.sup.32 -homoserine.sup.33 amide 
(Pro.sup.32 -Hse.sup.33 -NH.sub.2), a primary alkyl, 1-20 carbon atoms, 
amide of homoserine.sup.33 , or an optional polypeptide chain and 
containing homoserine amide at C-terminal [Jap. Pat. Hei 4 (A. D. 1992) - 
59795(A) which corresponds to EP-A-0 464 549]. The process described in 
these patent literatures is somewhat similar to a process proposed by the 
assignee company on motilin analogues as one of peptide hormones and 
disclosed in Jap. Pat. Hei 3 (A.D. 1991) - 80096(A) which corresponds to 
EP-A-0 355 720. Namely, according to the process described in the patent 
literatures, each of the calcitonin analogues can be prepared by 
synthesizing a single-stranded DNA fragment consisting of a leader 
polypeptide at N-terminus and with Met residue at its C-terminus, a 
polypeptide encoding the calcitonin analogue gene and with Met residue at 
its C-terminus, a spacer peptide with Met residue at its C-terminus, a 
polypeptide encoding the calcitonin analogue gene with Met residue at its 
C-terminus (the spacer peptide and calcitonin analogue gene being arranged 
in plural); synthesizing a single-stranded DNA fragment complementary to 
the first single-stranded DNA fragment; preparing a double-stranded DNA 
fragment by the single-stranded fragments; inserting the double-stranded 
fragment into a vector such as a plasmid to obtain a recombinant vector; 
transforming by the recombinant vector a microorganism such as Escherichia 
coli or mammalian cell such as CttO cell; cultivating the transformant to 
express the polypeptide as an inclusion body or fused protein in the 
transformant; treating the inclusion body with cyanogen bromide to cleave 
at each position of Met and modify the amino acid residue of Met at 
C-terminal into Hse or Hse-lactone residue; treating the reaction solution 
with an acid, for instance 0.1N HCl solution (at 30.degree. C. for 3 
hours) to convert all of individual polypeptides into those with 
Hse-lactone residue at C-terminus; lyophilizing the same; and then 
treating the same with ammonia solution or reacted with a primary alkyl 
amine. 
The method disclosed in said patent literatures makes possible the 
preparation of various calcitonin analogues in a large amount and with 
reasonable cost, but a biological activities of those, for instance salmon 
I calcitonin-Hse.sup.33 -NH.sub.2 is substantially same with the native 
Salmon I calcitonin. 
SUMMARY OF THE INVENTION 
One of the objects of the invention is to provide a calcitonin analogue 
which is excellent in productivity as in said patent literatures and shows 
remarkably higher biological activities than its basic native type 
calcitonin. 
An additional but important object of the invention is to provide a 
pharmaceutical composition for preventing and/or curing calcium metabolic 
diseases, which contains the calcitonin analogue as an effective 
ingredient.

DETAILED DESCRIPTION OF THE INVENTION 
The inventors have energetically studied and discovered that a substitution 
of Pro.sup.32 -NH.sub.2 at the C-terminal by Hse.sub.32 -NH2 remarkably 
enhances the biological activities, although the structure of the 
C-terminal Pro-NH.sub.2 common to all of native calcitonins has been 
considered as essential for developing the biological activities thereof, 
so that the invention was established. 
The calcitonin analogue according to the invention is, therefore, 
characterized in that an amino acid sequence (substantially corresponding 
to SEQ ID NO: 2) thereof is 
##STR2## 
In the amino acid sequence, Hse.multidot.NH.sub.2 means homoserine amide 
and Cys residues in the 1st and 7th positions 1 and 7 are linked by a 
disulfide bond. 
The amino acid sequence is same with that for eel calcitonin excepting that 
the amino acid residue at C-terminal in the latter is 
Pro.multidot.NH.sub.2. 
The pharmaceutical composition according to the invention and for 
preventing and/or curing calcium metabolic diseases is characterized by 
containing as an effective ingredient the calcitonin analogue having said 
amino acid sequence. 
The calcitonin analogue according to the invention can be prepared by 
synthesizing by a peptide synthesizer a polypeptide encoding said amino 
acid sequence but having rise residue at the 32nd position, oxidizing 
thiol group in Cys at 1st and 7th positions by potassium ferricyanide to 
yield a disulfide linkage between two Cys residues, treating the 
polypeptide with an acid to form a polypeptide having Hse-lactone residue 
at C-terminal, and treating the polypeptide with an ammonia to amidate 
C-terminal thereof. 
For obtaining the calcitonin analogue in a large amount, the techniques as 
disclosed in said Jap. Pat. Hei 3 (A.D. 1991) - 80096(A) corresponding to 
EP-A-0 355 720 have been utilized. Namely, the calcitonin analogue is 
prepared by synthesizing a single-stranded DNA fragment consisting of a 
leader polypeptide at N-terminus and with Met residue at its C-terminus, a 
polypeptide encoding the calcitonin analogue gene and with Met residue at 
its C-terminal of the 32nd position, a spacer peptide with Met residue at 
its C-terminus, a polypeptide encoding the calcitonin analogue gene with 
Met residue at its C-terminal (the spacer peptide and calcitonin analogue 
gene being arranged in plural); synthesizing a single-stranded DNA 
fragment complementary to the first single-stranded fragment; preparing a 
double-stranded DNA fragment by the single-stranded fragments; inserting 
the double-stranded fragment into a vector such as a plasmid to obtain a 
recombinant vector; transforming by the recombinant vector a microorganism 
such as Escherichia coli or mammalian cell such as CHO cell; cultivating 
the transformant to express a protein as an inclusion body or fused 
protein in the transformant; treating the inclusion body with cyanogen 
bromide to cause a cleavage at each position of Met and modify the amino 
acid residue of Met at C-terminus into Hse or Hse-lactone residue; and 
then treating the reaction solution with the potassium ferricyanide, acid 
and ammonia, as referred to above. 
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
The invention will now be further explained with reference to a Reference 
Example, Example, Biological Test Example and Medicine Preparation 
Example. 
Reference Example (Synthesis of native type eel calcitonin) 
A polypeptide having following amino acid sequence (corresponding to SEQ ID 
NO: 3) was synthesized by a peptide synthesizer (Mode 431A manufactured by 
Applied Biosystems Co.), and then removals of protective groups and 
cleavage of the linkage between the polypeptide and resin peptide was 
carried out by trifluoromethanesulfonic acid (TFMSA) procedure. In the 
synthesis, methylbenzhydrylamine resin was employed to provide a 
C-terminal amide peptide. 
Cys-Set-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu 
-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr -Pro 
The synthesized polypeptide was purified by reverse phase high performance 
liquid chromatography (rp-HPLC) by using .mu.-bondasphere (Trademark of 
Waters Co.) C-18 column (19.times.150 mm), under following conditions and 
fraction in a main peak was recovered and lyophilized. 
Elute: Linear gradient of 20-40% acetonitrile in 0.012N HCl (45 minutes) 
Flow rate: 7.0 ml/min. 
Detection wave length: 220 nm 
A part (60 mg) of the lyophilized polypeptide was taken to dissolve the 
same in 0.05% acetic acid solution (60 ml). After adjusted pH of the 
solution to 8.5 by 3M aqueous ammonia solution, 0.1M potassium 
ferricyanide (1.8ml) was added and the mixture was stirred for 30 minutes 
at room temperature to form a disulfide bridge between the Cys residues in 
positions 1 and 7. The reaction solution adjusted its pit to 5.0 by adding 
50% acetic acid solution was subjected to gel-filtration using a Sephadex 
(Trademark of Pharmacia AB) G-15 column to remove excess potassium 
ferricyanide, and then a peptide fraction was lyophilized. 
The resulting lyophilized polypeptlde was subjected again to HPLC under the 
same conditions as above by using the .mu.-bondasphere (Trademark of 
Waters Co.) C-18 column (19.times.150 mm) to obtain a desired natlye type 
eel calcitonin. 
Identification of the polypeptlde was carried out by FAB-MS analysis to 
confirm that the polypeptide was rightly synthesized. 
Example (Synthesis of [Hse.sup.33 -NH.sub.2 ] eel calcitonin) 
A polypeptide having following amino acid sequence (corresponding to SEQ ID 
NO: 4) was synthesized by a peptide synthesizer (Model 431A manufactured 
by Applied Biosystems Co.), and then removals of protective groups and 
cleavage of the linkage between the polypeptide and resin peptide was 
carried out by TFMSA procedure. In the synthesis, aminomethylated 
polystyrene.multidot.HCl resin was employed and 
N-Boc-0-benzyl-L-homoseryl-4-(oxymethyl)phenylacetic acid was employed as 
Hse source (Boc: tert-butyloxycarbonyl). 
Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-GlY-Lys-Leu-Ser-Gln-Glu-Leu 
-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-AsP-Val-Gly-Ala-Gly-Thr 
-Hse.multidot.OH 
The synthesized polypeptide was purified by HPLC [Waters .mu.-bondasphere 
C-18 column (19.times.150 mm)], under following conditions and fraction in 
a main peak was recovered and lyophilized. 
Elute: Linear gradient of 20-40% acetonitrile in 0.012N HCl (45 minutes) 
Flow rate: 7.0 ml/min. 
Detection wave length: 220 nm 
A part (60 mg) of the lyophilized polypeptide was taken to dissolve the 
same in 0.05% acetic acid solution (60 ml). After adjusted pH of the 
solution to 8.5 by 3M aqueous ammonia solution, 0.1M potassium 
ferricyanide (1.8 ml) was added and the mixture was stirred for 30 minutes 
at room temperature to form a disulfide bridge between the Cys residues in 
positions 1 and 7. The reaction solution adjusted its pH to 5.0 by adding 
50% acetic acid solution was subjected to gel-filtration using a Sephadex 
(Trademark of Pharmacia AB) G-15 column to remove excess potassium 
ferricyanide, and then a peptide fraction was lyophilized. 
The resulting lyophilized polypeptide was purified by reverse phase HPLC 
under the same conditions as above. The purified polypeptide having a 
C-terminal Hse residue was lyophilized and lactonized by treating in 0.1N 
HCl at 30.degree. C. for 3 hours and lyophilized. 
An ammonia solution (manufactured by Adrich Co., 2.0M solution in methanol) 
was added to the lyophilized polypeptide by 0.5 ml per 1 g of the 
polypeptide to stir for 1 hour at 37.degree. C. to convert the Hse-lactone 
to the corresponding primary amide and then dried in vacuo to obtain a 
desired [Hse.sup.32 -NH.sub.2 ]-eel calcitonin. 
Identification of the polypeptide was carried out by FAB-MS analysis to 
confirm that the polypeptide was rightly synthesized. 
Pharmacological Test Example (Measurement of biological activity) 
A solution of the polypeptide obtained by each of the Example and Reference 
Example in 1% sodium acetate buffer (pH 4) containing 0.1% bovine serum 
albumin (BSA) was administered to a Wister male rat (age of 6 weeks and 
fasted from the previous day) from its jugular vein. Serum calcium 
concentration at 1 hour after the administration was measured by the OCPC 
method using a marketed measuring kit ("Calcium C-Test Wako" manufacture 
by Wako Pure pharmaceuticals Co., Ltd., Japan). Activity of sample 
calcitonins expressed in unit numbers were obtained by using an eel 
calcitonin (manufactured by Penisula Lab. Inc., Lot No. 015405, 4500 
IU/mg) as a standard and 2--2 parallel dose examination method. The 
activity unit numbers of each sample polypeptide per 1 mg was obtained as 
a specific activity. 
Results are shown in following Table 1. As apparently seen therefrom, it 
has been found that the biological activity of the calcitonin analogue 
according to the invention ([Hse.sup.32 -NH.sub.2 ]-eel calcitonin) is 
remarkably higher than the native type eel calcitonin, wherein the 32nd 
amino acid is Pro-NH.sub.2. 
TABLE 1 
______________________________________ 
Sample Specific activity (IU/mg) 
______________________________________ 
Reference Example 
4500 
Example 6800 
______________________________________ 
Medicine Preparation Example 
A solution of the calcitonin analogue (100 IU) obtained by the Example and 
in refined water was aseptically charged into vials. After lyophilized, 
the vial was sealed to obtain a dry powdery medicine. The powdery medicine 
is dissolved in saline or the like for injection purpose, when it shall be 
used. 
The activity unit of the calcitonin analogue in a medicine can be changed 
in a range of about 10-about 200 IU. 
An additive or stabilizer widely accepted for peptide hormone preparations, 
such as serum albumin or the like peptide and a protein can be added to 
the solution of calcitonin analogue. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 4 
(2) INFORMATION FOR SEQ ID NO: 1 : 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 Amino acids 
(B) TYPE: Amino acid 
(C) STRANDEDNESS: Single 
(D) TOPOLOGY: Both 
(ii) MOLECULE TYPE: Peptide 
(iii) HYPOTHETICAL: No 
(iv) ANTI-SENSE: No 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 : 
CysGlyAsnLeuSerThrCysMetLeuGlyThrTyrThrGlnAspPheAsn 
51015 
LysPheHisThrPheProGlnThr AlaIleGlyValGlyAlaPro 
202530 
(2) INFORMATION FOR SEQ ID NO: 2 : 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 Amino acids 
(B) TYPE: Amino acid 
(C) STRANDEDNESS: Single 
(D) TOPOLOGY: Both 
(ii) MOLECULE TYPE: Peptide 
(iii) HYPOTHETICAL: No 
(iv) ANTI-SENSE: No 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 : 
CysSerAsnLeuSerThrCysValLeuGlyLysLeuSerGlnGluLeuHis 
51015 
LysLeuGlnTh rTyrProArgThrAspValGlyAlaGlyThrXaa 
202530 
(2) INFORMATION FOR SEQ ID NO: 3 : 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 Amino acids 
(B) TYPE: Amino acid 
(C) STRANDEDNESS: Single 
(D ) TOPOLOGY: Linear 
(ii) MOLECULE TYPE: Peptide 
(iii) HYPOTHETICAL: No 
(iv) ANTI-SENSE: No 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 : 
CysSerAsnLeuSerThrCysValLeuGlyLysLeuSerGlnGluLeuHis 
51015 
LysLeuGlnThrTyrProArgThrAspValGlyAlaGlyThrPro 
202530 
(2) INFORMATION FOR SEQ ID NO: 4 : 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 Amino acids 
(B) TYPE: Amino acid 
(C) STRANDEDNESS: Single 
(D) TOPOLOGY: Linear 
(ii) MOLECULE TYPE: Peptide 
(iii) HYPOTHETICAL: No 
(iv) ANTI-SENSE: No 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 : 
CysSerAsnLeuSerThrCysValLeuGlyLysLeuSerGlnGluLeuHis 
510 15 
LysLeuGlnThrTyrProArgThrAspValGlyAlaGlyThrXaa 
202530 
__________________________________________________________________________