JPH0482899A - Peptide and salt thereof and grave's disease diagnostic or remedy containing the same - Google Patents

Abstract

NEW MATERIAL:A peptide (salt) of the formula. USE:A diagnostic or remedy for Grave's disease. PREPARATION:For example, a phenylacetoamidomethyl resin (Pam resin) as a solid phase carrier is condensed with an amino acid having a protected alpha-amino group and a protected side chain functional group from the C-terminal of an peptide in the presence of a condensing agent such as carbodiimide according to the amino acid sequence of the peptide by a solid phase synthetic method to form a peptide bond, followed by removing the protecting groups. The processes using the next protected amino acids are repeated to prepare a protected peptide resin. The protected peptide is cleaved from the carrier resin with trifluoroacetic acid, etc., and the protecting groups are removed to prepare the crude peptide, which is purified by high performance chromatography to provide the peptide of the formula.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a novel peptide or a salt thereof, and also relates to a thyroid-stimulating antibody, which is a type of TSH receptor autoantibody considered to be a cause of hyperthyroidism. (TSAb: Th
yroid stimulating antibod
The present invention relates to a peptide or a salt thereof useful as a diagnostic or therapeutic agent for y).

[Technical background of the invention and its problems]

The TSAb, which is said to be the cause of hyperthyroidism, is directly related to the etiology and pathological condition of the disease, and its quantitative changes are considered to be an important marker for understanding the pathological condition. Conventionally, the autoantibodies have been measured using a stimulation assay method using the production of cAMP (cAMP) through activation of adenylate cyclase as an indicator.
ion assay method), “5I-T S H (Thy
(roid stimulating hormone)
A binding assay method using Radioimmunoassay is known.

In the stimulation assay method, Kosugi
The method of Endocrinology
Vol. 125° No. 1.410 pages (1989) is known. In this method, rat thyroid cell line 1i
Using RT I, 5, azonyl! - Measure the amount of cAMPO produced by activating cyclase, and use it as an index to measure the activity of TSAb. On the other hand, in the hinding assembly method, Smith (Smith, B.
R) et al.'s method (Clinical Endocr.
inology Vol, 17. 409 pages (1
9B2)) is known, and in that method +251T
T S H solubilized from pig thyroid using S H
The degree of inhibition of the binding between the receptor and ``1-TSH by the TSH receptor autoantibody is measured.
SH receptor autoantibodies include TSAb and TBIT (TSI).
Binding-inhibiting immunoglobulin ('I''S f (bindi)
ng 1nhibitory immunoglobl
in) ), but the results of this method show that TS
It is unclear whether either or both of the autoantibodies bound to the l+ receptor.

Since conventional methods require complicated operations for measurement, there is a desire for a simpler method.

The present inventors have discovered that this peptide specifically recognizes TSAb and has the effect of inhibiting the binding between TSAb and TSH receptors, and has found that this peptide can be used in the diagnosis of hyperthyroidism. The present invention was completed based on the finding that 1iJi is useful as a drug or therapeutic agent.

[Disclosure of the invention]

The synthetic peptide of the present invention is a peptide represented by -formation: % formula % or a pharmacologically acceptable salt.

Examples of the pharmacologically acceptable salts include alkali metal salts such as sodium and potassium, alkaline earth metal salts such as magnesium and calcium, monomethylamine,
Examples include amine salts such as 1-ethylamine, and quaternary ammonium salts such as trimethylethylammonium and tetramethylammonium.

In addition, the abbreviations described throughout this specification have the following meanings.

Pha; f, phenylalanine T
yr; L-tyrosine Val;
L-valine Glu F L-glutamic acid Gln
; L-glutamine AS+) + L-aspartic acid 11e i L-isoleucine Boc
; t-butoxycarbonyl Fmoc;
9-Fluorenylmethyloxycarbonyl TFA; ) Lifluoroacetic acid TFMSA
;) Lifluoromethanesulfonic acid The peptide in the present invention can be obtained by conventional methods such as liquid phase method and solid phase method, but more conveniently, it can be obtained using a commercially available peptide synthesizer using the same phase method. I can do things.

In the solid phase method, amino acids corresponding to the amino acid residues of the peptide are condensed from the C-terminal side of the peptide to a polymeric support to form a peptide bond. As the condensation method, for example, a dicyclocarbodiimide (DCC) method, an acid anhydride method, an activated ester method, or the like can be used. As a protecting group for α-amino group, Boc group or Fm
An oc group can be used. For example, when synthesizing using an automatic peptide synthesizer, it is preferable to select the amino acid in accordance with the manual instructions of the apparatus, taking into consideration the properties of the amino acid.

As the protecting group for the protected amino acid used in the solid phase method, those commonly used in peptide synthesis are used. For example, when a Boc group is used as the α-amino group, the carboxyl group of Asp and Glu is a benzyl group protected by an ester crystal, the cycloh:1-syl group is used, and the bitroxyl group of 'l' y r is , 2-bromobenzyloxycarbonyl group are used, respectively. In addition, when using Fmoc group as the α-amino group, Asp, , G
Tertiary butyl ester is used for the carboxyl group of lu and the hydroxyl group of ``)'yr, respectively.

When synthesizing by the liquid phase method, it can be carried out according to the usual peptide synthesis method as well as the solid phase method. As a known method, for example, Nobuo Izumiya et al., Peptide Synthesis, Marukubi Co., Ltd., 1975.
Examples include the methods described in 2010.

Next, when a Boc group is used as the α-amino group protecting group, removal from the protecting group and support is carried out using hydrogen fluoride, TFMSA, etc. containing a scavenger. Scavengers include thioanisole, ethanedithiol,
Dimethyl sulfide and the like are used. Furthermore, when Fmoc group is used as the α-amine group protecting group, a mixture containing phenol and TFA can be used.

Ion exchange resins, partition chromatography, gel chromatography, commonly used for peptide purification
This can be carried out by using reverse phase liquid chromatography or the like alone or in combination. The amino acid composition of purified peptides can be easily measured using an amino acid analyzer.

Since the peptide of the present invention is useful as a means to easily understand quantitative changes in TSAb, it can be used as a diagnostic agent for hyperthyroidism, and also has the effect of inhibiting the binding between TSAb and TST-f receptor. It can also be used as a treatment for hyperthyroidism.

When the peptide of the present invention is used as a therapeutic agent, the peptide can be used in the form of a suitable formulation mixed with a pharmaceutically acceptable carrier. Dosage forms for oral administration include tablets, capsules, granules, and powders. Alternatively, dosage forms for parenteral administration include injections and the like. The dose of the drug (■) depends on the severity of the patient's symptoms, age and weight, administration method, etc., and must be determined by a specialist's test to determine the appropriate dose.

〔Example〕

Using 658 mg (isoleucine content 0.76 mmol/g) of N-t-butoxycarbonyl-isoleucine phenylacetamide 1'' methyl resin (PAM-resin: installed in the automatic peptide synthesizer described later) as a raw material, the automatic peptide synthesizer [Applied Biosystems] This peptide II-Ty
r-Val-Phe-Phe-Glu-Glu-Gln
-Glu85p-Glu-11e-Off was synthesized.

According to the conventional method, 11 ■ are sequentially added from the C-terminal Tie according to the above amino acid sequence.

It reacts with 2 mmol of each amino acid in the body.

1.5 g of protected peptide resin was obtained by this.

The side chain functional group of the amino acid used in this reaction was protected by Asp.
,, The carbonyl group of Glu was protected with cyclohexyl ester, and the hydroxyl group of Tyr was protected with 2-bromobenzyloxycarbonyl ester.

After drying, 1 g of this protected peptide resin was stirred with a mixture of 500 μl of ethanedithiol and 1 ml of thioanisole for 10 minutes under ice cooling, and then 10 ml of TFA was added.
Stir for 10 minutes. In this mixture, l me T F
Add MSA, stir for 10 minutes, and then stir for 50 minutes at room temperature.
Stir for a minute.

Thereafter, 50 mff1 of anhydrous ether was added to precipitate the crude peptide, and the crude peptide and the detached resin were filtered using a glass filter. 5 ml of TFA was added to the crude peptide on the glass filter and dissolved to obtain a TFA solution.

Anhydrous ether was added to this to obtain a crude peptide precipitate. After separation, this product was washed several times with anhydrous ether and then dried in a vacuum oven. Peptide 15 obtained in this way
0 mg, high performance liquid chroma I/graph (HPLC)
It was purified under the following conditions. The column was a reverse-phase Asahipack ODP-90 (manufactured by Asahi Kasei Corporation) containing 20mM ammonium acetate buffer (A) (pH
8,5) and acetonitrile (B) [60
(B) 100% in minutes, flow rate 10mA! /min]. After concentrating the necessary elution fractions, 50 mg of the target peptide was obtained by freeze-drying.

Amino acid analysis of the peptide thus synthesized was performed. For amino acid analysis, use 6N hydrochloric acid at 110°C.
After hydrolysis for 20 hours, the amino acid analyzer L850
0 (newly manufactured by Hitachi Seisakusho).

Amino acid analysis results Tyr Val Phe Glu/
Gln Asp Ile measurement value 0.96
0.9B 1.98 5.03 1.02 1.0
2 Theoretical value 112511 The above amino acid analysis results revealed that the peptide that is the object of the present invention was obtained.

[Test example]

Test Example I The effect of the peptide on the binding between TSH receptor and "'I-TSH" is investigated.The measurement method is Schulling (St+
ewring, G) et al.'s method (CIinical mouth n
Docrinology Vol, 17.409 pages (
1982)] using a commercially available TSH receptor antibody measurement kit. The outline is shown below.

20ul of solution containing 0.1.1.0 or 10ug of peptide and 50u each of normal serum or TBIT positive serum
Put 1 into a test tube and add 125Il to it.

Add 100 μl of TSHI solution and incubate for 2 hours at 4°C. Thereafter, 50ul of solubilized porcine TSH receptor solution is added and incubated at 37°C for 90 minutes. Then 30% of polyethylene glycol
170 μ of buffer solution! Add and centrifuge (3000x
g for 30 minutes at 4°C) After obtaining the precipitate, measure the radioactivity of the precipitate using a scintillation counter. The results are shown in Figure 1.

The peptide can be used in both normal serum and TBI! In all tests using positive sera, +251-bTSH and T
It can be seen that the peptide does not affect the binding to the S H receptor, and in particular does not show binding to FBI+.

Test Example 2 The binding of the peptide to the TSH receptor autoantibody present in the serum of a patient with hyperthyroidism (TS Ab positive serum) and the serum of a normal person is examined.

Add 50 ul of patient serum or normal serum to iodine label (1
251) Add 20ul of the solution containing the peptide,
After incubation for 2 hours at 4°C, polyethylene glycol was added to a maximum of 15% flA degree and the precipitate was obtained by centrifugation (3000 x g for 30 min at 4°C). The precipitate was then released. IJ activity is measured using a scintillation counter. Patient serum shows 50-70% higher binding than normal serum.

From the results of Test Examples 1 and 2 above, this peptide is TSA
This peptide shows high binding to TSAb, but not to TBI■, indicating that this peptide specifically recognizes TSAb.

Test Example 3 Rat thyroid cells FRTI,5 were used to evaluate the effect of the peptide on the production of cAMP stimulated by TSAb using the method of Kosugi, S. et al.
rtmology Vol, 125. Na 1.4
10 (1989)). The outline is shown below.

Cells are pre-cultured FRTL5 cells I x 105 cells
Dispense 500 μβ of ls/rn1 into a 24-well multi-plate, culture in a 00g incubator for 6 days while changing the medium (excluding TSH), remove the medium, wash, and adjust the cells on the plate. and test it.

200 ul of TSAb positive serum and the peptide 1 or 1
The solution containing 0 ug is allowed to react in a test tube at 4° C. for 2 hours. This reaction solution is added to the previously prepared FRTL5 cells and incubated at 37°C for 3 hours.

Measurement of cAMP produced upon stimulation with TSAb was performed using the commercially available cAMP-radioimmunoassay (Y
SI-7701: Tested using Yamana Soy Sauce ■Manufactured. 3 cases of TSAb positive serum (sample A: ×, specimen B: ○
, Specimen C: ◆). As a result, the peptide suppresses cAMP production in a concentration-dependent manner.

The results are shown in Figure 2, and T
The activity of SAb is expressed in terms of the equivalent amount of b-T SH.

From the results of Test Example 3 above, it is clear that this peptide has TSAb and T
Indicates that it has the effect of inhibiting binding with S H receptors.

[Brief explanation of the drawing]

Figure 1 shows that the peptide is +2' to the TSH receptor.
This figure shows that the binding of I-bTSH is not inhibited, and the binding is not inhibited even when TBII-positive serum is used. It is a figure showing that this peptide suppresses.

Claims (1)

[Claims] 1) General formula: H-Tyr-Val-Phe-Phe-Glu-Glu
-Gln-Glu-Asp-Glu-Ile-OH or a salt thereof. 2), General formula: H-Tyr-Val-Phe-Phe-Glu-Glu
- A diagnostic or therapeutic agent for hyperthyroidism, comprising a peptide represented by -Gln-Glu-Asp-Glu-Ile-OH or a salt thereof.