JPH04149198A - Calcitonin derivative - Google Patents

Abstract

NEW MATERIAL:A calcitonin derivative wherein at least one residue of lysine residue at 11 position, glutamic acid residue at 15 position and lysine residue at 18 position is replaced with glutamine residue. USE:A treating agent of osteoporosis, Bechet's disease and hypercalcemia. PREPARATION:Solid-phase method is utilized for example, p- methylbenzhydrylamine (BHA) resin is used as an insoluble resin, and is bound with an amino acid containing a protected alpha-amino group and a protected side chain functional group from C end side successively along with the amino acid sequence of the objective calcitonin derivative in order to synthesize a protected peptide resin, the peptide is eliminated from the prepared peptide resin and deprotected. When the objective peptide contains an intramolecular disulfide bond, the peptide is oxidized with air, the prepared crude peptide is purified by high performance liquid chromatography to give a calcitonin derivative.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing a calcitonin derivative having biological activity.

[Conventional technology]

Calcitonin (hereinafter referred to as CT) is a peptide hormone produced in the thyroid gland of mammals or the catfish gland of fish and birds. This hormone is known to suppress the release of calcium from bones by suppressing osteoclasts and lower blood calcium levels, and is used to treat osteoporosis, Beget's disease, and hypercalcemia. It is widely used clinically as a medicine. To date, the amino acid sequences of human, cow, pig, sheep, rat, chicken, salmon, and eel OCT have been revealed, and all of these consist of 32 amino acids, with cysteine at positions 1 and 7. They have in common that they form a disulfide bond and have a prolineamide at the carboxy terminus. Compared to CT derived from mammals, CT derived from the catfish gland has higher activity and also has higher amino acid sequence homology. Many derivatives of these catfish hind knee-derived CTs have been synthesized (U.S. Patent No. 4,239,
680°Nα4,397,780. k 4.414,
149. No. 4,444.68L No.

4.451,395. No. 4,495,097
．． k 4,497,731. No.

4.528,132. Nct 4.537,716
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6. & 4.604,237. Nil 4,6
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388. Floor 4.632.978. Sou 4.639.5
09. Number 4,639,510. 4.639,511.
No. 4,659,804. ll&14,732
,969. k4, 732.969. Seed 4.746
, 728. No. 4.764,589. Floor 4.76
4,591. NIC14,804,742,EPN
n 2B8,058. EPNI290.029, JP 51-128993, JP 52-59178),
Structural factors involved in the expression of physiological activity are being searched for. Generally, the activity of peptide preparations largely depends on their stability in vivo. It is generally known that peptide bonds adjacent to charged residues in peptides are susceptible to hydrolysis by proteases [Japanese Biochemistry and Biochemistry Biochemistry Data Book I Tokyo Kagaku Doujin pp. 341-414 (19
81) ), salmon calcitonin, eel calcitonin, and tricalcitonin, examples of charged amino acid residues include a lysine residue at position 11, a glutamic acid residue at position 15, and a lysine residue at position 18.

[Problem to be solved by the invention]

Therefore, the present invention focuses on lysine and glutamic acid, which are charged amino acid residues, and various calcitonin derivatives in which lysine and/or glutamic acid are substituted with uncharged amino acid residues, or specific lysine residues and their C
The present invention aims to provide various calcitonin derivatives with terminal residues removed.

[Means to solve the problem]

The above problem is solved by a calcitonin derivative in which at least one of the lysine residue at position 11, the glutamic acid residue at position 5, and the lysine residue at position 18 is replaced with a glutamine residue [substituted derivative]; and amino acid residues from positions 16 to 17 and from positions 19 to 23, from positions 16 to 23
This problem can be solved by providing calcitonin derivatives [truncated derivatives] that are deleted from the amino acid residues up to position 1, the amino acid residues from position 18 to position 22, and the amino acid residues from position 18 to position 23.

[Specific description]

Calcitonin or its derivative, which is the basis for designing the calcitonin derivative of the present invention, has a lysine residue at position 11,
Any calcitonin or derivative thereof having a glutamic acid residue at position 15 and a lysine residue at position 18 can be used.

Such natural calcitonin includes the following calcitonin: Salmon I C3NLSTCVLGKLSQELHKLQ
TYPRTNTGSGTP-NHz Salmon II C5N
LSTCVLGKLSQDLHKLQTFPRTNdingG
AGVP-NHz Salmon I[I C3NLSTCMLG
KLSQDLI()[LQTFl'RTNTGAGVP
-NH.

Eel C5NLSTCVLGKLSQELHKLQT
YPRTDVGAGTP-NHZ Tri CASLST
CVLGKLSQELHKLQTYPRTDVGAGT
P-NHt can be mentioned.

Furthermore, as a non-natural calcitonin derivative that is the basis for designing the calcitonin derivative of the present invention, among the calcitonin derivatives described in the documents listed in the prior art section, there is a lysine residue at the 11th position and a glutamic acid residue at the 15th position. base, 1
Examples include those having a lysine residue at the 8th position.

In the calcitonin derivative of the present invention, A, and the corresponding basic calcitonin in which A2 and X + X s are defined as described below, are all already known.

The substituted calcitonin derivative has, for example, the following amino acid sequence: A1χIXzLSTAzVLGQLSQELH[,QT
YPRTXJaGXsGTP-NHz.

A, χ+XzLSTAzVLGKLSQQLHKLQT
YPRTXsχ4GXsGTP-NHzA+×1χ, L
STA2νLG) [LSQELHQL(lTYPRTX
iX4GXsGTP-NHz.

A IX IXzLSTAzVLGQLSQQLHKL
QTYPRTXsXnGXsGTP-Nl2.

A I X I X zLsTA 2 VLGQLSQ
ELHQLQTYPRTXsX, GX 5GTP-N
H! .

A + X + X zLSTA zVLGKLsQQ
LHQLQTYP RTX sXa GX 5GTP
-NH! , or A I X I X 2LSTA ZVLGQLSQQ
LHQLQTYPRTX 3X4GX 5GTP-NH
! , (wherein A1 and A2 are each independently cysteine or 5-n-alkylcystine, or together they are 1,7-cystine or 1,7-α-
Consists of aminosperinic acid: X is S or A and lX2 is N or S; X is N or D;
4 is T or ■; and X5 is S or A).

Further, as the truncated calcitonin derivative, for example, the following amino acid sequence: A1χ+XzLSTAzVLGKLSQEKRTXJ4
GXsGTP-N)lx.

AIx+XtLSTAzVLGKLSQERTX3χ4
Gχ5GTP-Nl2゜A+X+XzLSTlhVLG
KLSQELHPRTXJJXsGTP-NHz, or A,X+XzLSTAzVLGKLSQELH
RTXJaGχ5GTP-Nl2 (wherein A1 and A2
are each independently cysteine or 5-n-alkylcystine, or together they are 1.7
- constitutes cystine or 1,7-α-aminosperinic acid; Xl is S or A; x2 is N or S; χ is N or D; X4 is T or ■; and X is S or A).

As a more specific example, the following amino acid sequence:
Drunkenness - 10,000 A I C3NLSTCVLGKLSQELHQL
QTYPRTDVGAGTP-NHzA 2 C5
NLSTCVLGQLSQELHKLQTYPRTDV
GAGTP-NH! A 3 C3NLSTCVL
GKLSQQLHKLQTYPRTDVGAGTP-N
) lx (natural C5NLSTCVLGKLSQELH
Mention may be made of calcitonin derivatives derived from the catfish gland of eel having KLQTYPRTDVGAGTP-NH.

A case in which a target peptide is synthesized by a solid phase method will be described below. That is, the target CT derivative is sequentially bonded to the insoluble resin starting from the C-terminal amino acid. The insoluble resin may be any one known in the art, but P-methylbenzhydrylamine (B ) IA) Resin is preferred. Amino acids with protected functional groups are used, but it is necessary to select an appropriate protecting group depending on the condensation method used and the method of elimination from the resin. After removing and deprotecting the peptide from the obtained protected peptide resin, if the target peptide has an intramolecular disulfide bond, it is then formed by an oxidation reaction such as air oxidation or potassium ferricyanide oxidation. In order to isolate the target peptide from the obtained crude peptide fraction, it is effective to use high performance liquid chromatography such as reverse phase, ion exchange, and gel filtration. The structure of the final specimen can be confirmed by amino acid sequence analysis and amino acid composition analysis.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the Examples are not intended to limit the scope of the present invention in any way.

A method for synthesizing ECT derivatives having the sequences of AI, A2 and A3 is shown. BHA resin 0.5 mmol by peptide synthesis $143OA manufactured by Abrid Biosystems
Synthesized by solid phase method using This device synthesizes peptides by condensing t-Boc amino acids using the DCC-HOBi method. All synthetic reagents and reaction conditions were supplied by the company.

From the obtained protected peptide resin, the target peptide was removed and deprotected using trifluoroacetic acid (TFA)/trifluoromethanesulfonic acid (TFMSA) in the presence of thioanisole and ethanedithiol, and the peptide was recovered by recrystallization in ether. do. This is dissolved in 10% acetic acid and the low molecular weight reagent is removed using a Bio Gel P 2 (Bio-Rad Laboratories) column, followed by freeze-drying. The resulting pale yellow flocculent solid is dissolved in a 6M guanidine hydrochloride solution at a concentration of 200 μ/d, and stirred at room temperature for 2 hours to generate intramolecular cystine bonds. After removing guanidine by dialysis or gel filtration, the peptide solution was freeze-dried and subjected to reverse phase HPLC using a 00S-80TrI column (Tosoh), ion exchange HPLC using a B5-502C column (Asahi Kasei), and G-2000S-column (Tosoh). The target peptide was isolated and purified using gel filtration FIPLC. By the above method, BH
Approximately 301J from synthesis using A resin 0.5mho]
A final purified sample of g can be obtained.

Sequence confirmation and purity test of the final preparation were performed by amino acid sequence analysis and composition analysis. Table 1 shows derivatives AI, A2
, shows the sequence analysis results of A3.

Table 1: Amino acid composition analysis of ECT derivatives The biological activity of each CT derivative was determined by the growth inhibitory effect (T, MDay) in the pig kidney epithelial-derived cell line LLCPL.
r et al., J. Ce1l Biol, 9L 195-
200.1981) or adenylate cyclase activation effect (A.

Wohlwend et al., Biochem, Biophys
,1lies,Commun++ 13L537-54
2) was evaluated. The results are shown in Table 2.

Biological activity of Daiichi Shi-tomo ECT derivatives -9.29 9.35 -9.19 -9.13 As is clear from Table 2, all the derivatives have ECT activity, of which A1 has ECT activity. It had slightly stronger activity than natural ECT.

Claims (1)

[Scope of Claims] A calcitonin derivative in which at least one of the lysine residues at positions 1 and 11, the glutamic acid residue at position 15, and the lysine residue at position 18 is replaced with a glutamine residue. 2. The calcitonin derivative according to claim 1, wherein the calcitonin derivative is a derivative of salmon calcitonin, eel calcitonin or tricalcitonin. 3. The following amino acid sequence: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ or ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, are A_1 and A_2 each independently cysteine or S-n-alkylcysteine? , or they together form 1,7-cystine or 1,7-α-
constitutes aminosuberic acid; X_1 is S or A; X_2 is N or S; X_3 is N or D; X_4 is T or V; and X_5 is S or A.
The calcitonin derivative according to claim 1 or 2, which has the following. 4, amino acid residues from positions 16 to 17 and positions 19 to 23, amino acid residues from positions 16 to 23, 18
Amino acid residues from position to position 22, or from position 18 to position 2
A calcitonin derivative lacking amino acid residues up to the 3rd position. 5. The calcitonin derivative according to claim 4, wherein the calcitonin derivative is a derivative of salmon calcitonin, eel calcitonin or tricalcitonin. 6. The following amino acid sequence: ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ or ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, are A_1 and A_2 each independently cysteine or S-n-alkylcysteine? , or together these can be 1,7-cystine or 1,7-α
- Consists of aminosuberic acid; X_1 is S or A
The calcitonin derivative according to claim 4 or 5, wherein X_2 is N or S; X_3 is N or D; X_4 is T or V; and X_5 is S or A.