JPH0940698A - Modified polypeptide compound and its use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a modified polypeptide compound, effective in preventing and treating calcium metabolic diseases and orally administrable without being influenced with a proteolytic enzyme present in a mucous membrane of an alimentary canal by partially replacing an amino acid sequence of a natural type calcitonin. SOLUTION: This new modified polypeptide compound has an amino acid sequence of the formula (residues Cys which are an amino acid residue at the 1st and 7th positions is bonded to disulfide bonds; Hse.NH2 is homoserinamide) and an <=18C acyl group bonded to α-amino group of Cys at the N-terminal. The polypeptide is obtained by replacing the prolinamide at the C-terminal of a natural type calcitonin with a homoserinamide and effective in preventing and treating calcium metabolic diseases and can orally be administered. The compound is obtained by synthesizing a peptide chain having an amino acid sequence of calcitonin according to a peptide solid-phase synthetic method, transducing the homoserinamide group into the C-terminal and further transducing an acyl group such as hexanoyl group into the N-terminal.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel modified polypeptide compound and its use. The modified polypeptide compound according to the present invention exhibits the same biological activity as the natural calcitonin and the synthetic calcitonin analog, and is superior in stability to the natural calcitonin and the synthetic calcitonin analog, and therefore, as a pharmaceutical ingredient, the calcium metabolism system. The prevention and treatment of diseases include, for example, reduction of pain and suppression of bone loss in osteoporosis, prevention of fracture in osteogenesis imperfecta, hypercalcemia and Paget's disease of bone.
It can be used for prevention and treatment of (Paget's disease).

[0002]

2. Description of the Related Art Calcitonin is a peptide hormone having an action of lowering blood calcium concentration. Alcohols derived from salmon, eel, stingray, chicken, pig, rat, sheep and human have been extracted and purified, and their primary amino acids have been extracted. The sequence has been revealed. All of these structural features common to calcitonin are composed of 32 amino acids, and Cys at positions 1 and 7 have a disulfide bond to form a cyclic structure, and the C-terminal is proline amide. Is. Although chemical synthetic methods have been mainly used to obtain these calcitonins, it is extremely difficult to provide the calcitonins inexpensively and in large quantities because the reaction steps are many and the synthetic reaction and purification require a long time. Met. Therefore, the method developed by researchers of the applicant company
(JP-A-4-59795), that is, a DNA fragment corresponding to the leader peptide is placed on the N-terminal side, while
On the C-terminal side, genes corresponding to a plurality of calcitonins or analogs thereof are tandemly linked via a DNA fragment encoding a spacer peptide, if necessary, to form a double-stranded DNA fragment in this way. Is incorporated into a vector such as a plasmid, and a microorganism such as Escherichia coli is transformed with this gene recombinant vector, and this transformant is cultured to produce a fusion protein or inclusion body, and treated with bromocyanine to produce methionine. C-terminal homoserine is obtained by cleaving with (Met) part to separate into individual fragments and changing the C-terminal of each fragment to homoserine (Hse) or homoserine lactone residue, followed by acid treatment and reaction with ammonia. The residue is amidated (Hse
· NH ₂₎ using the method of forming the calcitonin analog having the desired biological activity, it became clear that can inexpensively and mass production.

Therefore, the inventors of the present invention have described the above-mentioned Japanese Patent Laid-Open No. 4-5.
As a result of various studies on calcitonin analogs that can be synthesized by the gene recombination technology disclosed in 9795, surprisingly, the C-terminal proline amide (Pro. The following [Hse ³² · NH ₂ ] -eel calcitonin (corresponding to SEQ ID NO: 1) in which NH ₂ ) is replaced with homoserine amide is compared with the natural eel calcitonin, that is, proline amide at the 32nd position. It has already been clarified that the physiological activity is extremely high (Japanese Patent Application No. 6-138618). 1 5 10 15 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu- 20 25 30 Leu-His-Lys-Leu-Gln-Thr-Tyr -Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly- Thr-Hse ・ NH ₂ (In the above sequence, the first and seventh amino acid residues Cys are disulfide-bonded)

On the other hand, regarding the clinical application of calcitonin, since calcitonin is a peptide compound, it is highly likely to be decomposed by proteolytic enzymes present in the mucous membranes of the living body such as the lung, digestive tract and nasal cavity, and therefore injected. The main method of administration was. Therefore, in order to overcome this administration problem, a method of simultaneously adding a protease inhibitor [Morita, T. et al ., "Pharamaceutical Re
search ", Vol. 11, page 909 (1994); Kobayashi, S. et
al ., "Pharamaceutical Research", Vol.11, page 123
9 (1994); JP-A 2-115130, 3-48627, 3-
170438, 3-246233, 4-74133, 6-321804
Etc.] or a method for stabilizing calcitonin by chemically modifying it with a fatty acid or the like [Patent Documents 1 to 104098 and 1 to 29]
4695 Gazette, Tadashi Fujita et al. "Drug Delivery System", Vol.
9, page 179 (1994), etc.], administration methods other than injection have been investigated.

[0005]

However, although the addition of a protease inhibitor suppresses the degradation of calcitonin, it affects the absorption of other substances present on the mucous membrane, and also has a toxic effect. No effective inhibitor has been found which may result in expression and is therefore safe and selectively inhibits only calcitonin degradation. Regarding the chemically modified calcitonin analog, in addition to the above reports, Japanese Patent Publication No. 49-194, Japanese Unexamined Patent Publication Nos. 62-132898 and 63-2842.
98, 63-287800, 64-64800, JP 1-
230598, Same 1-503390, Same 2 -17200, Same 3-505589
Although disclosed in the publication, USP 4,804,742, etc., there is no description regarding stability improvement in these documents, and further, the disclosed modified calcitonin analog is chemically synthesized and therefore inexpensive. It is unsuitable for mass production in large quantities.

Therefore, the object of the present invention is to obtain a Japanese Patent Application No. 6-13861.
8 A modified calcitonin that is as excellent in productivity and physiological activity as the calcitonin analog disclosed in the specification and is designed to be more stable on the living mucosa than the basic calcitonin analog. -To provide a modified polypeptide compound that is an analog. An additional but important object of the present invention is to provide a preventive and / or therapeutic agent for a calcium metabolism system disease, which comprises such a modified polypeptide compound as an active ingredient.

[0007]

[Means for Solving the Problems and Achieving the Purpose] The inventors of the present invention have conducted extensive studies to achieve the above-mentioned object, and as a result, based on the calcitonin derivative described in the above-mentioned Japanese Patent Application No. 6-138618. As a skeleton, Cys α- at the N-terminus
Regioselective introduction of acyl group to amino group, and further 1
Position to 31-position has a native eel, amino acid sequence identical to the calcitonin from salmon or chicken, 32 is succeeded in synthesizing a novel modified polypeptide compounds which are homoserine amide (Hse-NH ₂₎ In addition, the compound shows high stability in the gastrointestinal mucosa homogenate,
Therefore, they have found that they are unlikely to be affected by the proteolytic enzyme present in the mucosa, and have completed the present invention.

That is, the modified polypeptide compound according to the present invention has an amino acid sequence (corresponding to SEQ ID NO: 1) of 1 5 10 15 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-. Leu-Ser-Gln-Glu- 20 25 30 Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly- Thr-Hse ・ NH ₂ or amino acid Sequence (corresponding to SEQ ID NO: 2) is 1 5 10 15 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu- 20 25 30 Leu-His -Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly- Thr-Hse ・ NH ₂ or amino acid sequence (corresponding to SEQ ID NO: 3) is 1 5 10 15 Cys -Ala-Ser-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu- 20 25 30 Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg- Thr-Asp-Val-Gly-Ala-Gly- Thr-Hse ・ NH ₂ (in each of the above sequences, the first and seventh amino acid residues Cys are disulfide-bonded), The number of carbon atoms in the α-amino group of Cys at the N-terminal is
It is characterized in that 18 or less acyl groups are bonded.

The preventive and therapeutic agent for calcium metabolism system disease according to the present invention is characterized by containing the above-mentioned modified polypeptide compound as an active ingredient.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will now be described in more detail and specifically with reference to Production Examples, Production Examples, Biological Activity Test Examples, Stability Test Examples and Formulation Examples. Reference Production Example 1 (Synthesis of [Hse ³² · NH ₂ ] -eel calcitonin) A calcitonin analog having the following amino acid sequence (corresponding to SEQ ID NO: 1) and disclosed in Japanese Patent Application No. 6-138618. Was synthesized according to the method described herein and using an Applied Biosystems Peptide Synthesizer 430A, and then purified. Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Se
r-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-
Asp-Val-Gly-Ala-Gly-Thr-Hse ・ NH ₂ (In the above sequence, Hse ・ NH ₂ means an amidated homoserine residue, and the first and seventh amino acid residues Cys is a disulfide bond)

Reference Production Example 2 (Synthesis of [Hse ³² .NH ₂ ] -salmon calcitonin) It has the following amino acid sequence (corresponding to SEQ ID NO: 2) and is disclosed in Japanese Patent Application No. 6-138618. Calcitonin analogs were synthesized according to the methods described herein and using an Applied Biosystems Peptide Synthesizer 430A and then purified. Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Se
r-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-
Asn-Thr-Gly-Ser-Gly-Thr-Hse ・ NH ₂ (In the above sequence, Hse ・ NH ₂ means an amidated homoserine residue, the first and seventh amino acid residues. Cys is a disulfide bond)

Reference Production Example 3 (Synthesis of [Hse ³² .NH ₂ ] -chicken calcitonin) It has the following amino acid sequence (corresponding to SEQ ID NO: 3) and is disclosed in Japanese Patent Application No. 6-138618. Calcitonin analogs were synthesized according to the methods described herein and using an Applied Biosystems Peptide Synthesizer 430A and then purified. Cys-Ala-Ser-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Se
r-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-
Asp-Val-Gly-Ala-Gly-Thr-Hse ・ NH ₂ (In the above sequence, Hse ・ NH ₂ means an amidated homoserine residue, and the first and seventh amino acid residues Cys is a disulfide bond)

Production Example 1 (N-α-hexanoyl- [Cys ¹ , Hse
Synthesis of ³²・ NH ₂ ] -eel calcitonin) 2 mg of [Hse ³²・ NH ₂ ] -eel calcitonin obtained in Reference Production Example 1 was weighed out, and 0.1% trifluoroacetic acid was added.
It was dissolved in 2 ml of (TFA) and freeze-dried. This freeze-dried powder was dissolved in 1 ml of dry dimethylformamide (DMF), 2 equivalents of hexanoic anhydride were added, and the solution was added at 90 ° C at 90 ° C.
Allowed to react for minutes. Then add 9 ml of anhydrous ether,
The precipitate was collected by cooling on an ice bath and centrifuged to collect the precipitate. The resulting precipitate was collected on a Waters μ-bondersphere reverse-phase C-18 column (19
(mm x 15 cm) and purified by HPLC under the following conditions. Eluent: 30% to 60% 0.1% T in 0.1% TFA in water
FA Acetonitrile solution linear gradient, 30 minutes, flow rate: 7.0 ml / min, detection wavelength: 210 nm. By recovering the main peak fraction by HPLC and freeze-drying, the target product, N-α-hexanoyl- [Cys ¹ , Hse ³²
1 mg of NH ₂ ] -eel calcitonin was obtained. The obtained modified polypeptide compound was identified by performing FAB-MS under the following conditions. As a result, it was confirmed from the analysis of fragment ions that it had a structure represented by the following formula. Analytical conditions: Matrix: metanitrobenzyl alcohol, inlet selection: direct, ion mode: FAB ⁺ FAB-MS, (M + H) ⁺ : 3515.8 (theoretical value), 3515.6 (actual value). CH ₃ (CH ₂ ) ₄ C (O) -Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-
Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Ty
In r-Pro-Arg-Thr- Asp-Val-Gly-Ala-Gly-Thr-Hse · NH 2 ( above sequence, Hse · NH ₂ means an amidated homoserine residue, 1st And the seventh amino acid residue, Cys, are disulfide-bonded)

Production Example 2 (N-α-nonanoyl- [Cys ¹ , Hse ³²
Synthesis of [NH ₂ ] -eel calcitonin) [Hse ³² · NH ₂ ] -eel calcitonin obtained in Reference Production Example 1 under the conditions shown in Production Example 1, except that nonanoic acid anhydride was used instead of hexanoic acid anhydride. Process using
Then, it was purified by HPLC. The obtained modified polypeptide compound was identified by FAB-MS under the conditions shown in Production Example 1, and as a result, it was confirmed that it had a structure represented by the following formula. FAB-MS, (M + H) ⁺ : 3557.8 (theoretical), 3557.8 (actual). CH ₃ (CH ₂ ) ₇ C (O) -Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-
Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Ty
In r-Pro-Arg-Thr- Asp-Val-Gly-Ala-Gly-Thr-Hse · NH 2 ( above sequence, Hse · NH ₂ means an amidated homoserine residue, 1st And the seventh amino acid residue, Cys, are disulfide-bonded)

Production Example 3 (N-α-dodecanoyl- [Cys ¹ , Hse
³² · NH _2] - Synthesis of eel calcitonin) obtained in Reference Example _{^{1 [Hse 32 · NH 2]}} - eel calcitonin was weighed 2 mg, 0.1% trifluoroacetic acid
It was dissolved in 2 ml of (TFA) and freeze-dried. This lyophilized powder was dissolved in 1 ml of dry dimethylformamide (DMF), 2 equivalents of dodecanesanoic anhydride were added, and the mixture was added at 25 ° C.
Allowed to react for 120 minutes. Then, it was treated under the conditions shown in Production Example 1 and purified by HPLC. The resulting modified polypeptide compound was identified under FA conditions under the conditions shown in Production Example 1.
As a result of B-MS, it was confirmed to have a structure represented by the following formula. FAB-MS, (M + H) ⁺ : 3599.9 (theoretical), 3599.9 (actual). CH ₃ (CH ₂ ) ₁₀ C (O) -Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu
-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-T
yr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Hse ・ NH ₂ (In the above sequence, Hse ・ NH ₂ means an amidated homoserine residue, and the first And the seventh amino acid residue, Cys, are disulfide-bonded)

Production Example 4 (N-α-octadecanoyl- [Cys ¹ ,
Synthesis of Hse ³² · NH ₂ ] -eel calcitonin) [Hse ³² · NH ₂ ] -eel calcitonin obtained in Reference Preparation Example 1 was used under the conditions shown in Preparation Example 3, except that octadecanoic acid was used instead of dodecanoic anhydride. Treated with anhydride and then purified by HPLC. The resulting modified polypeptide compound was identified by FAB- under the conditions shown in Production Example 1.
As a result of MS, it was confirmed that the compound had a structure represented by the following formula. FAB-MS, (M + H) ⁺ : 3684.0 (theoretical value), 3684.0 (actual value). CH ₃ (CH ₂ ) ₁₆ C (O) -Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu
-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-T
yr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Hse ・ NH ₂ (In the above sequence, Hse ・ NH ₂ means an amidated homoserine residue, and the first And the seventh amino acid residue, Cys, are disulfide-bonded)

Production Example 5 (N-α-propanoyl- [Cys ¹ , Hse
³² · NH _2] - Synthesis of salmon calcitonin) obtained in Reference Example _{^{2 [Hse 32 · NH 2]}} - propionic anhydride instead of under the conditions indicated salmon calcitonin in Production Example 1, except hexanoic acid anhydride The product was purified by HPLC. The resulting modified polypeptide compound was identified by FAB-MS under the conditions shown in Production Example 1.
As a result, it was confirmed to have a structure represented by the following formula. FAB-MS, (M + H) ⁺ : 3490.7 (theoretical), 3490.8 (actual). CH ₃ CH ₂ C (O) -Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly
-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-P
ro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Hse ・ NH ₂ (In the above sequence, Hse ・ NH ₂ means an amidated homoserine residue. (The seventh amino acid residue, Cys, is disulfide-bonded.)

Production Example 6 (N-α-nonanoyl- [Cys ¹ , Hse ³²
· NH _2] - Synthesis of salmon calcitonin) Reference obtained in Production Example _{^{2 [Hse 32 · NH 2]}} - under the conditions indicated salmon calcitonin in Production Example 1, except nonanoic acid anhydride in place of hexanoic acid anhydride And then purified by HPLC. The obtained modified polypeptide compound was identified by FAB-MS under the conditions shown in Production Example 1, and as a result, it was confirmed that it had a structure represented by the following formula. FAB-MS, (M + H) ⁺ : 3574.8 (theoretical value), 3574.8 (actual value). CH ₃ (CH ₂ ) ₇ C (O) -Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-
Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Ty
In r-Pro-Arg-Thr- Asn-Thr-Gly-Ser-Gly-Thr-Hse · NH 2 ( above sequence, Hse · NH ₂ means an amidated homoserine residue, 1st And the seventh amino acid residue, Cys, are disulfide-bonded)

Production Example 7 (N-α-nonanoyl- [Cys ¹ , Hse ³²
· NH _2] - Synthesis of chicken calcitonin) Reference obtained in Production Example _{^{3 [Hse 32 · NH 2]}} - under the conditions indicated chicken calcitonin in Preparation 1, except nonanoic acid anhydride in place of hexanoic acid anhydride And then purified by HPLC. The resulting modified polypeptide compound was identified by FAB-MS under the conditions shown in Production Example 1.
As a result, it was confirmed to have a structure represented by the following formula. FAB-MS, (M + H) ⁺ : 3514.8 (theoretical value), 3514.8 (actual value). CH ₃ (CH ₂ ) ₇ C (O) -Cys-Ala-Ser-Leu-Ser-Thr-Cys-Val-Leu-
Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Ty
In r-Pro-Arg-Thr- Asp-Val-Gly-Ala-Gly-Thr-Hse · NH 2 ( above sequence, Hse · NH ₂ means an amidated homoserine residue, 1st And the seventh amino acid residue, Cys, are disulfide-bonded)

Production Example 8 (N-α-2-methylpropanoyl- [C
ys ¹ , Hse ³² · NH ₂ ] -Synthesis of chicken calcitonin) [Hse ³² · NH ₂ ] -chicken calcitonin obtained in Reference Preparation Example 3 under the conditions shown in Preparation Example 1 except that hexanoic anhydride was used. Was treated with 2-methylpropionic anhydride and then purified by HPLC. The obtained modified polypeptide compound was identified by FAB-MS under the conditions shown in Production Example 1, and as a result, it was confirmed that it had a structure represented by the following formula. FAB-MS, (M + H) ⁺ : 3444.8 (theoretical), 3444.6 (actual). (CH ₃ ) ₂ CHC (O) -Cys-Ala-Ser-Leu-Ser-Thr-Cys-Val-Leu-G
ly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr
-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Hse ・ NH ₂ (In the above sequence, Hse ・ NH ₂ means an amidated homoserine residue, and the first and (The 7th amino acid residue, Cys, is a disulfide bond)

Test Examples (Measurement of Stability against Proteolytic Enzymes Present in Rat Intestinal Mucosa) Modified and unmodified polypeptide compounds (calcitonin analogs) obtained in Production Examples and Reference Production Examples, and commercially available natural salmon And the stability of eel calcitonin in rat intestinal mucosa homogenate was determined.
Wistar male rats (6 weeks old) were fasted for 24 hours and then sacrificed, and the intestinal tract from the gastric pylorus to the cecal-colon junction was removed. The inside of the obtained intestinal tract was washed with physiological saline, opened with scissors, the mucous membrane was scraped off with a glass plate, and added to the physiological saline. Then, treat with a Polytron homogenizer (Kinematica) for 60 seconds on an ice bath, and then centrifuge at 4 ° C (3600g x 30 minutes).
Then, the obtained supernatant was used as an intestinal mucosa homogenate. 20 mM of this homogenate (protein concentration: 27 μg / ml) 200 μl
250 μl of phosphate buffer (pH 6.8) was added, and the mixture was incubated at 37 ° C. for 1 minute. Then, 50 μl of a polypeptide compound (1.0 mg / ml) as a sample was added and incubated at 37 ° C. Collect 40 μl each before and 2 hours after the start of incubation, and immediately add 10 μl of 0.13 mg / ml benzalkonium chloride solution and 40 μl of ethanol and mix, and then mix the resulting sample solution with Waters μ-Bondas. Reverse phase
HP using C-18 column (3.9mm x 15cm) under the following conditions.
It was analyzed by LC. Eluent: 0.1% from 30% to 60% in 0.1% TFA in water
TFA acetonitrile solution linear gradient, 20 min. Flow rate: 1.0 ml / min, detection wavelength: 210 nm. The survival rate was determined from the area value in the chromatogram obtained for each polypeptide compound, and the elimination half-life was calculated from the change over time. The results are shown in Table 1 below, and the modified calcitonin compounds according to the present invention are commercially available natural salmon calcitonin and eel calcitonin, and unmodified as disclosed in Japanese Patent Application No. 6-138618.
[Hse ³² · NH ₂ ] -was found to be significantly superior in stability when compared to eel, salmon and chicken calcitonin.

[Table 1]

Formulation Example 1 (Injection) Modified polypeptide compound 100I obtained in Production Example 1
A human serum albumin solution was added to U to dissolve it, followed by freeze-drying. This freeze-dried preparation is dissolved in physiological saline before use. The activity unit of the modified polypeptide compound used can be set within the range of about 10-400 IU, and the above-mentioned serum albumin should be replaced with other additives generally used for peptide hormone-based preparations. You can also

Formulation Example 2 (suppository) Modified polypeptide compound 100I obtained in Production Example 2
U was added to cacao butter or Witepsol (mark), mixed by heating, and processed by a conventional method to prepare a suppository. still,
The activity unit of the modified polypeptide compound used is 10-
It can be set within the range of about 400 IU.

Formulation Example 3 (Suppository) Modified polypeptide compound 100I obtained in Production Example 6
U was dissolved in purified water, uniformly dispersed in pre-melted macrogol (mark), and then treated by a conventional method to prepare a vaginal suppository. The activity unit of the modified polypeptide compound used can be set within the range of about 10 -400 IU.

Formulation Example 4 (nasal drops) 50 IU of the modified polypeptide compound obtained in Production Example 7
Was dissolved in purified water containing benzalkonium chloride to prepare a preparation for intranasal administration. The activity unit of the modified polypeptide compound used can be set within the range of about 10 -400 IU.

[0026]

INDUSTRIAL APPLICABILITY The modified polypeptide compound according to the present invention is stable in gastrointestinal mucosal homogenate in comparison with natural salmon calcitonin and eel calcitonin and unmodified [Hse ³² · NH ₂ ] -calcitonin analog. Since it is extremely high and is hardly affected by the proteolytic enzyme existing in the mucous membrane, it is possible to use an administration route other than injection. Furthermore, since the modified polypeptide compound according to the present invention has Cse at the Hse ³² · NH _2, genetic engineering techniques can be applied, and chemical modification at the N-terminus can also utilize a simple regioselective technique. It can be prepared inexpensively and in large quantities. Thus, the present invention is extremely excellent in providing an active ingredient of a preventive and / or therapeutic agent for calcium metabolism system diseases.

[Sequence list]

SEQ ID NO: 1 Sequence length: 32 Sequence type: Number of amino acid chains: Single chain Topology: Both forms Sequence type: Peptide Other information: Cys at positions 1 and 7 are disulfide-bonded Xaa homoserine amide _{(Hse · NH 2) 1 5} 10 15 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu- 20 25 30 Leu-His- Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly- Thr-Xaa SEQ ID NO: 2 Sequence length: 32 Sequence type: Number of amino acid chains: 1 Chain topology: Both forms Sequence type: Peptide Other information: Cys at positions 1 and 7 are disulfide-bonded Xaa is homoserine amide (HseNH ₂ ) 1 5 10 15 Cys-Ser-Asn-Leu- Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu- 20 25 30 Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly -Ser-Gly- Thr-Xaa SEQ ID NO: 3 Sequence length: 32 Sequence type: Number of amino acid chains: Single-stranded topology: Both forms Sequence type: Peptide Other information: 1 Cys at position 7 and 7 are disulfide-bonded Xaa is homoserine amide (HseNH ₂ ) 1 5 10 15 Cys-Ala-Ser-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu -Ser-Gln-Glu- 20 25 30 Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly- Thr-Xaa

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Noboru Tomiya 35, Higashi-Tobori-cho, Higashi-ku, Nagoya, Aichi Prefecture Sanwa Chemical Research Institute Co., Ltd.

Claims (4)

[Claims] 1. An amino acid sequence (corresponding to SEQ ID NO: 1) of 1 5 10 15 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-20 25 30 Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly- Thr-HseNH ₂ (In the above sequence, the first and the seventh The second amino acid residue, Cys, is a disulfide bond) and has 18 carbon atoms in the α-amino group of Cys at the N-terminus.
A modified polypeptide compound having one or less acyl groups attached. 2. An amino acid sequence (corresponding to SEQ ID NO: 2) of 1 5 10 15 Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-20 25 30 Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly- Thr-HseNH ₂ (In the above sequence, the first and the seventh The second amino acid residue, Cys, is a disulfide bond) and has 18 carbon atoms in the α-amino group of Cys at the N-terminus.
A modified polypeptide compound having one or less acyl groups attached. 3. An amino acid sequence (corresponding to SEQ ID NO: 3) of 1 5 10 15 Cys-Ala-Ser-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-20 25 30 Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly- Thr-HseNH ₂ (In the above sequence, the first and the seventh The second amino acid residue, Cys, is a disulfide bond) and has 18 carbon atoms in the α-amino group of Cys at the N-terminus.
A modified polypeptide compound having one or less acyl groups attached. 4. A preventive and therapeutic agent for a calcium metabolism system disease, which comprises the modified polypeptide compound according to any one of claims 1 to 3 as an active ingredient.