JPH09249698A - Human calcitonin derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new human calcitonin derivative, having a variation of an amino acid residue at a specific site of a natural type calcitonin, having higher activities and less adverse effects than those of the natural type, strong lowering actions on calcium in blood and useful for treatment, etc., of osteoporosis, pain, etc. SOLUTION: This new human calcitonin derivative (salt) is a derivative in which at least one position of the 17-, 22- and 24-positions and optionally an amino acid residue at the 15-position in the natural type calcitonin having an amino acid sequence represented by the formula are substituted by other amino acid residues; Asp at the 15-position is substituted by Glu; Asn at the 17-position is substituted by His; Phe at the 22-position is substituted by Tyr and Gln at the 24-position is substituted by Arg. The calcitonin derivative (salt) has higher activities and less adverse effects than those of the natural type and is useful as a therapeutic agent, etc., for osteoporosis, pain, etc. The derivative is obtained by synthesizing the amino acid sequence in which the 15-, 17-, 22- and 24-positions, etc., of the natural type calcitonin by using a peptide solid- phase synthetic method, etc., utilizing a polymer resin.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel human calcitonin derivative having biological activity.

[0002]

2. Description of the Related Art Known natural calcitonin is eel, salmon,
Those derived from chicken, pig, human, bovine, sheep, rat and the like are known, and they are all composed of 32 amino acids. Calcitonin originally possessed by these humans and animals is called natural calcitonin.

On the other hand, JP-A-63-2767698, JP-A-63-284198, JP-A-63-287800, J. Bi.
ochem.Vol.159 P125 (1986), Endocrinology Vol.117 P8
0 (1987), J. Biochem. Vol. 162 P399 (1987), etc.
A derivative obtained by substituting at least one amino acid residue of natural calcitonin with another amino acid residue is disclosed.

Calcitonin derived from mammalian thyroid gland represented by natural human calcitonin has lower blood calcium lowering action than calcitonin derived from gill gland typified by natural salmon calcitonin and natural eel calcitonin. Is generally known. Due to its strong blood calcium lowering effect, natural salmon calcitonin and eel calcitonin derivatives have been used as therapeutic agents for osteoporosis or pain improvement, but side effects such as antigenicity and nausea have become a problem. Although natural human calcitonin does not cause these side effects, it requires a large amount of drug because of its low blood calcium lowering effect.

Accordingly, there is a need for calcitonin which has less side effects and is more active than natural human calcitonin. The amino acid sequence of natural human calcitonin is shown in chemical formula 1 below.

[0006]

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[0007]

The present invention is the following invention relating to a human calcitonin derivative which is highly active as compared with natural human calcitonin.

A human calcitonin derivative obtained by substituting at least one position selected from the 17-position, 22-position and 24-position of natural human calcitonin and optionally at the 15-position with another amino acid residue, The substitution at position 15 is a glutamic acid residue, the substitution at position 17 is a histidine residue, the substitution at position 22 is a tyrosine residue, and the substitution at position 24 is an arginine residue. A human calcitonin derivative or a pharmaceutically acceptable salt thereof.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION As shown in the amino acid sequence of natural human calcitonin, the amino acid residues at positions 15, 17, 22, and 24 of natural human calcitonin are Asp (aspartic acid) and Asn, respectively. (Asparagine), Phe (phenylalanine), Gln (glutamine)
Is the residue of. The human calcitonin derivative of the present invention has the 17-position asparagine residue substituted with a histidine residue, the 22-position phenylalanine residue substituted with a tyrosine residue, or the 24-position glutamine residue with an arginine residue. Is replaced with. Furthermore, in addition to this one substitution, the 15-position may be substituted with a glutamic acid residue.

Further, the human calcitonin derivative of the present invention has 2 to 4 substitutions thereof at the same time.
That is, the 17-position is replaced with a histidine residue and the 22-position is replaced with a tyrosine residue (in addition, the 15-position may be replaced with a glutamic acid residue), and the 17-position is replaced with a histidine residue and 24 Substitutes with arginine residue at position (additionally, 15-position may be replaced with glutamic acid residue), 22-position with tyrosine residue and 24-position with arginine residue (additional And 15-position may be substituted with a glutamic acid residue), and 17-position may be replaced with a histidine residue, 22-position with a tyrosine residue and 24-position with an arginine residue (in addition to , 15-position may be substituted with a glutamic acid residue).

The following chemical formula 2 shows the amino acid sequence of the human calcitonin derivative of the present invention obtained by performing three substitutions at the 17-position, 22-position and 24-position at the same time. As the human calcitonin derivative of the present invention, the human calcitonin derivative represented by the amino acid sequence of the following chemical formula 2 is preferable, and the human calcitonin derivative is 10 of natural human calcitonin.
The blood calcium lowering effect is about twice as high, and the side effect is also low.

[0012]

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In synthesizing the human calcitonin derivative of the present invention, a solid phase peptide synthesis method using a polymer resin or a liquid phase synthesis method which is a general organic synthesis method can be used. Resins used for solid phase peptide synthesis include, for example,
Benzhydrylamine resin, 4- (2,4-dimethoxyphenyl-aminomethyl) -phenoxyacetamide-
Aminomethyl resin, 4- (2,4-dimethoxyphenyl-aminomethyl) -phenoxyacetamide-4-methylbenzhydrylamine resin, 4- (2,4-dimethoxyphenyl-aminomethyl) -phenoxy resin and the like can be used.

As the amino acid, an amino acid derivative having a protected functional group can be used, if necessary. Examples of the α-amino protecting group for amino acids include benzyloxycarbonyl group (Z), 9
-Fluorenylmethyloxycarbonyl group (Fmo
c), a tertiary butyloxycarbonyl group (Boc), a formyl group (HCO), an acetyl group (Ac), etc. can be used. Examples of the α-carboxy protecting group of amino acid include benzyl group (Bzl), tert-butyl group (tBu), methyl group (Me), ethyl group (Et), phenacyl group (Pac).
Etc. are available. In addition, the inside of () shows the symbol of the group usually used, and it is the same also in the following groups.

Further, as the amino acid side chain functional group protecting group,
Benzyl group (Bzl), p-toluene sulfonyl group (To
s), p-nitrophenoxy group (NOp), benzhydryl group (Bzh), acetamide group (Acm), tert-butyl group (tBu), tert-butyloxycarbonyl group (B)
oc), cyclohexyl group (cHex), benzyloxymethyl group (Bom), third butoxymethyl group (Bu
m), tert-butylthio group (tButhio), dinitrophenyl group (Dnp), 9-fluorenylmethyloxycarbonyl group (Fmoc), 2,2,5,7,8-pentamethylchroman-6-sulfonyl group ( Pmc), a trityl group (Trt), etc. can be used. One or two or more of these protecting groups can be arbitrarily selected and used depending on the purpose.

For the peptide extension reaction, that is, the sequential addition reaction of amino acids or amino acid derivatives, a dehydration condensation method using carbodiimide or an active ester method can be used.

In the dehydration condensation method, as a condensing agent, dicyclohexylcarbodiimide (DCC), 1-ethyl-3-
(3-Dimethylaminopropyl) carbodiimide (WS
C), 2- (1H-benzotriazol-1-yl)-
1,1,3,3-Tetramethyluronium hexafluorophosphate (HBTU), benzotriazole-1-
Il-oxy-tris (dimethylamino) -phosphonium hexafluorophosphate (BOP) and the like can be used. In the active ester method, N-hydroxysuccinimide ester (HONSu), pentafluorophenyl ester (OPfp), dihydroxybenztriazine ester (ODhbt) and the like can be used. In addition, the abbreviations of commonly used compounds are shown in parentheses.

As the reaction solvent, N, N-dimethylformamide, N, N-dimethylacetamide, tetrahydrofuran, chloroform, methylene chloride, ethyl acetate,
1,4-dioxane, dimethyl sulfoxide, N-methylpyrrolidone, pyridine, water and the like can be used.

As the releasing agent for the functional group-protecting group of amino acid or peptide, hydrogen fluoride, trifluoroacetic acid, trifluoromethanesulfonic acid, ammonia /
Methanol, hydrogen bromide / acetic acid, hydrogen / palladium on carbon,
Mercury acetate, acetic acid / zinc powder, alkali / water / methanol, etc. can be used.

As a purification method during or in the course of synthesis, various kinds of chromatography such as reverse phase chromatography, normal phase chromatography, ion exchange chromatography, gel filtration and recrystallization can be used. Further, as the disulfide bond forming method, a method by air oxidation, a method using potassium ferricyanide, iodine, or the like can be used.

[0021]

EXAMPLES Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited thereto.

[Example 1] Synthesis of [17-histidine, 22-tyrosine, 24-arginine] -human calcitonin (human calcitonin derivative having the amino acid sequence of the above-mentioned chemical formula 2): Synthesis was carried out by an automatic peptide synthesizer. It was done by law.

(1) Introduction of proline into benzhydrylamine resin: 5 g of benzhydrylamine resin (-NH
₂ : 0.5 mmol / g) was suspended in 50 ml of N, N-dimethylformamide (DMF) and swollen. After removing the supernatant, add 50 ml of DMF, and then add Boc
Proline 10 mmol (2.15 g), dicyclohexylcarbodiimide 20 mmol, and N-hydroxybenzotriazole 12 mmol (1.62 g) were added, respectively, and the mixture was stirred overnight at room temperature. The resin was filtered through a glass filter and washed well with methylene chloride.

Next, a solution of acetic anhydride in methylene chloride (10 v
/ V%) 200 ml and stirred at room temperature for 1 hour to block unreacted amino groups. After the reaction was completed, it was washed with methylene chloride and dried under reduced pressure. After drying, 50 ml of a methylene chloride solution of trifluoroacetic acid (50 v / v%) was added, and the mixture was stirred at room temperature for 1 hour. It was filtered with a glass filter, washed with methylene chloride, methanol, methylene chloride, and triethylamine in this order, and then dried under reduced pressure.

(2) Sequential amino acid addition reaction: 1 g of the resin obtained in (1) was packed in a column of an automatic synthesizer and the amino acids were sequentially added under the following conditions.

1) Reaction: room temperature 30 minutes, solvent: DMF, 2) washing: room temperature 10 minutes, solvent: DMF, 3) Fmoc removal: room temperature 10 minutes, solvent: DM of piperidine
F solution (20 v / v%), 4) Washing: room temperature 10 minutes Solvent; DMF.

By repeating steps 1) to 4), the proline and the subsequent 3
One amino acid was added sequentially. The amino acid derivatives used are shown in Tables 1 and 2 below.

[0028]

[Table 1]

[0029]

[Table 2]

After completion of the reaction, the peptide was eliminated from the resin and the protecting group with hydrogen fluoride and washed with ether. The precipitate was dissolved in an aqueous acetic acid solution (50 v / v%), impurities were filtered off, and the filtrate was freeze-dried to obtain about 600 mg of a crude peptide.

(3) Purification and formation of disulfide bond: About 500 mg of the crude peptide was added to an aqueous solution of trifluoroacetic acid (0.
1v / v%) and HPLC using ODS column
And purified. A liquid / B liquid = 20%, A liquid / B liquid = 3
Elution was performed in the order of 0%, A solution / B solution = 40%, and 30% elution fractions were collected and lyophilized.

46 mg of the freeze-dried peptide was dissolved in 50 ml of an aqueous acetic acid solution (0.05 v / v%), and the pH was adjusted to 8.5 with 3M aqueous ammonia. Then 0.1 M K ₃
1.5 ml of Fe (CN) ₆ was added and stirred at room temperature for 30 minutes to form a disulfide bond. PH with 50% acetic acid
After adjusting to 5.0, anion exchange resin (Cl ⁻ type) was added and stirred for 20 minutes, then the resin was filtered off.

After the filtrate was concentrated, it was added again to high performance liquid chromatography using an ODS column and purified again by the same method as described above. Fractions eluted at 30% were collected and lyophilized to obtain 28 mg of the title peptide.

[Example 2] Measurement of biological activity: The human calcitonin derivative obtained in Example 1 was dissolved in 10 mM acetate buffer (pH 4.2) containing 0.1% BSA (bovine serum albumin), and T47D cells ( CAM using human breast cancer cells)
The activity was determined by measuring the amount of P (Amersham: cAMP measurement kit). The measurement result is shown in the graph of FIG. In the graph, hCT represents natural human calcitonin.
As the measurement results show, the human calcitonin derivative obtained in Example 1 has 10 times the activity of natural human calcitonin.

[Example 3] Measurement of biological activity: The human calcitonin derivative obtained in Example 1 was dissolved in physiological saline to obtain 40
A human calcitonin derivative of 00 μg / kg was intramuscularly administered to rats, and the food intake and body weight of the rats after the administration were measured. Similarly, natural salmon calcitonin was administered instead of the human calcitonin derivative (dose 100 μg / k
g), and physiological saline alone was administered as a control group. The results are shown in the graphs of FIG. 2 (food intake) and FIG. 3 (body weight). In the graph, sCT represents natural type salmon calcitonin. In the case of natural salmon calcitonin, the food intake and body weight were decreased, but the human calcitonin derivative was not different from the control group.

[0036]

INDUSTRIAL APPLICABILITY The human calcitonin derivative of the present invention has high activity as compared with natural human calcitonin and has less side effects as compared with conventional highly active calcitonin.

[0037]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 32 Sequence type: Amino acid Sequence type: Synthetic peptide Sequence characteristics: Disulfide bond between positions 1 and 7 Cys. Carboxyl group at Pro position 32 is amidated. Sequence: Cys Gly Asn Leu Ser Thr Cys Met Leu Gly Thr Tyr Thr Gln Asp Phe 1 5 10 15 His Lys Phe His Thr Tyr Pro Arg Thr Ala Ile Gly Val Gly Ala Pro 20 25 30

[Brief description of drawings]

FIG. 1 is a graph showing the results of measurement of biological activity between a human calcitonin derivative and natural human calcitonin in Example 2.

FIG. 2 is a graph showing the results (food intake) of the biological activity (side effects) of the human calcitonin derivative and natural salmon calcitonin in Example 3.

FIG. 3 is a graph showing the results (body weight) of the measurement of biological activity (side effect) between the human calcitonin derivative and natural salmon calcitonin in Example 3.

Claims (2)

[Claims] 1. A human calcitonin derivative obtained by substituting the amino acid residue of at least one position selected from the 17-position, 22-position and 24-position of natural human calcitonin and optionally the 15-position with another amino acid residue. The substitution at the 15th position is a glutamic acid residue, the substitution at the 17th position is a histidine residue, the substitution at the 22nd position is a tyrosine residue, and the substitution at the 24th position is an arginine residue. A human calcitonin derivative or a pharmaceutically acceptable salt thereof, which is a substitution with a group. 2. A natural human calcitonin in which the 17th amino acid residue is replaced with a histidine residue, the 22nd amino acid residue is replaced with a tyrosine residue, and the 24th amino acid residue is replaced with an arginine residue. A human calcitonin derivative or a pharmaceutically acceptable salt thereof.