JPS59204159A - (met(o)8,18)hpth-(1-34)peptide - Google Patents

Abstract

NEW MATERIAL:A peptide of the formula H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met(O)- His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met(O)-Glu-Arg-Val-Glu-Trp-Leu-Arg -Lys- Lys- Leu-Gln-Asp-Val-His-Asn-Phe-OH or a salt thereof. USE:A medicine having reducing action on calcium in urine. PREPARATION:A well-known hPTH-(1-34)peptide is oxidized with an oxidizing agnet, e.g. hydrogen peroxide, in an aquous solution at room temperature, and water is added thereto to freeze-dry the oxidation product. Thus, the aimed peptide of the formula is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel human parathyroid hormone (hPTl).
() Regarding peptide derivatives. More specifically, the present invention provides CMet(0)' 18]h P T H-(1,-3
4) It is a salt of a peptide. The above peptide is represented by the formula % formula % and is known as hPTH-(1-34) pef.

Tido [proc, Nat, Acad, Sci,
U.S.A., 71.384 (1974), Endo
crinology, 98.1294 (1976)
, Hoppe-5eyler's Z,' Phys
iol, Ch, am,, 335°415 (197
4):l] methionine at positions 8 and 18 (Net)
is an oxidized derivative.

h P T I-I is P1J consisting of 84 amino acids
Bev.

Its biological activities include increasing blood power, lowering blood phosphorus, lowering urinary calcium, urinary phosphorus, increasing urinary 0-AMP, and vitamin activity in the kidneys. The hPTH-(1-34) peptide has effects such as increasing the enzyme activity of D-1 hydroxyl, and hPTH-(1-34) peptide has all of these effects.

8.18 However, the present invention [:Met(0) l]
h PTH-(1-34,) peptide is characterized by having only the urinary Cal/Rum lowering action among the above actions, and having no other actions, and does not require only the urinary Cal/Rum lowering action. It is a substance that is useful for certain diseases.

The peptide [1] of the present invention can be obtained in the form of a base or a salt thereof depending on the conditions of the method. Salts include inorganic acid salts, formic acid, acetic acid, propionic acid, glycolic acid, co-phosphoric acid,
It is a salt of organic acids such as malic acid, tartaric acid, and citric acid. Kasudamoto's peptide [1] can form a complex by adding certain inorganic or organic substances. By complex is meant a compound that is formed upon addition and provides a sustained action to the peptide.

In order to produce the peptide [1] of the present invention, a known hP
'l'LI-(1-34) peptide CI(oppe-
5eyJer'8 z, PhysiOl, Chem
,, 335.415. (1974)] in an aqueous solution,
This is carried out by oxidation with a mild oxidizing agent such as hydrogen peroxide. The above oxidation reaction is usually fully reliable at room temperature. The desired peptide [1] is obtained by adding water to the obtained reaction solution and freeze-drying it.

In addition, the peptide [1] of the present invention can be obtained by condensing individual protected amino acids and/or protected lower peptides to the amino acid order represented by formula [1] by a liquid phase synthesis method, and in the final step of the condensation reaction. It can also be obtained by another method in which the protecting group for the N-terminal amino group and the protecting group for the side chain functional group are removed by acid decomposition.

The condensation reaction itself is carried out by repeating the attachment and detachment of a protecting group and the condensation reaction in accordance with conventional methods for peptide synthesis. That is, the various protecting groups used in the production of the raw material and all intermediates of the present peptide [1] are those known in peptide synthesis. Such protecting groups are described in the literature and reference books in the field of peptide synthetic chemistry. A benzyloxycarbonyl group is used to protect the ε-amine group of lysine, an O-chlorobenzyloxycarbonyl group is used to protect the α-carboxyl group, and a methyl ester group is used to protect the α-carboxyl group.
For the benzyl ester group '/', the benzyl ester group is used to protect the carboxyl group of the side chain, the benzyl group is used to protect the hydroxyl group of serine, and the menthylene-2-sulfonyl group is used to protect the amine group in the guanidino group of arginine. In the synthesis of the target compound [1], the condensation of individual amino acids and lower peptides is carried out, for example, by forming a protected α-amino group and an activated terminal carboxyl group. This can be carried out by reacting an amino acid spanning peptide with an amino acid type or peptide having a free α-amino group and a protected terminal carboxyl group.

In this case, the carboxyl group can be, for example, an acid azide, an acid anhydride, an acid imidazolide or an active ester, such as N-anomethyl ester, p-nitrophenylnisder, N-hydro-nitroimide ester, 2,4,5-triacid imide ester, Single carbodiimides, such as N,N'-di7chlorohexylcarbodiimide, N-ethyl-N'-3-dimethylaminepropylcarbodiimide, N'-carbodiimide, which can be activated by converting to chlorophenyl esters etc. carbonyldiimidazole, isoxo IJ
can be activated by reaction using a condensing agent such as a Woodward reagent.

Practical condensation methods in the present invention include the AND method, active ester method, and Wünsch method [Z, Naturfors
Ch.

21, 426 (1966)] 'The Geiger method [
Chem-Ber, + month) 3,788 (19
70)] etc. to make the power of ＼ru kukodō. The condensation order can be synthesized in any order as long as it is the amino acid jl direction shown by formula [1], and it is best to connect amino acids and (or peptides) sequentially from the C-terminal side.

Methionine can be oxidized by itself to form a protective group for the α-amine group in the above condensation reaction, such as t-butyloxycarbonyloacetic acid. The α-carboxyl attachment group, e.g. methyl ester, can be decomposed with dilute sulfur hydroxide solution and converted into a hydratide or a protected hydratide such as trichloroethoxycarbonyl hydratide. .

In this way, a protected tetratriacontapeptide having an -N-terminal α-amino group, an ε-amino group, a C-terminal carboxyl group, a side chain carboxyl group, a guanidino group and a (-1: bamboo) hydroxyl group is obtained.

These protecting groups are preferably removed in one step by acid decomposition, eg anhydrous hydrogen fluoride.

The peptide [1] thus obtained can be separated and purified by known means for purifying peptides or proteins. For example, it can be carried out by gel filtration using a gel filtration agent such as Sephadex G-25, Sephadex G-50, or Sephadex L H-20, or by column chromatography using carboquine methyl cellulose, ion exchange resin, etc. can.

Next, the biological activity of the peptide [1] of the present invention will be described.

(1) hPTH-(1-34) and [Met(o)
8' 18] Effect of hPTH-(1-34) on blood calcium, phosphorus, urinary calcium, phosphorus and C-AMP Test method Sprangue weighing 300 g fed standard diet -
A cannula was inserted into the femoral artery of a Dawley (S, D,) rat (male), and 4% glucose, 5 mM
act2.5 m M Mg Cl3.20 m MN
aCl, 3 ml/nutrient solution containing 2.5 mM KCt
hPTH-(1-34) peptide and [Met(0)' 18]hPTI-I-(1-34) respectively at the rate of time.
) Continuous infusion with peptide 2 μ2/h. Both peptides were administered for 1 hour each. Calcium concentrations in the blood and urine were measured using an atomic absorption spectrometer (Hitachi 208).
emistry, 12.871 (1966)], and urinary C-AMP was measured by the method of Honma et al. [
Biochem, Med, 18.257 (197
7)].

■Measurement results h P'I'' Blood calcium, phosphorus, urinary calcium, phosphorus and C-AM in r-1-(1-34)
The results of measuring J] are shown in Figure 1, and include blood calcium elevation, blood phosphorus-lowering, urinary calcium-lowering, urinary phosphorus-raising, and urinary C-AMP.
Has an elevating effect. On the other hand, [Met(0)8°18
] Blood energy, phosphorus, urinary calcium, phosphorus and C-AMP in hP'Ll'IT-(1-34)
The results of the measurement are shown in Figure 2, which shows that it has only a urinary calcium lowering effect and has no other effects.

(2) hPTH-(1-34) and [IMet(0
) 8・181hl) Tf(-(1-34)) has no effect on 25-hydroxyvitamin D3-1 hydroxylase in the kidney (1) Test method
Deficiency diet (contains 0.45% calcium, 0.3% phosphorus)
After 6 weeks, pendoparbital (50my/
The patient was anesthetized by intravenous injection of 1 kg body weight, and thyroid and parathyroidectomy was performed. Next, insert a cannula into the left femoral artery and apply the nutrient solution at 31n/! h4pT respectively at the rate of /time
Store t-34) and [Net(0)"'18"'J
h P T H-(1-34) ], 5 μ7/h in continuous infusion. Both peptides were administered for 1 hour each, and 24 hours after the start of the injection, [3HI'25-hydroxyvitamin D3 (9C1/mM) 0.5/i
Ci was injected intravenously, and 6 hours later, blood was collected from the femoral artery. Blood is plasma separated, Blight and Dy
er method (Can, J., Biochem, phy
siol, 37, 911 (1959)],
The amount of [H]1.25-dihydroxy/vitamin D3 in the blood was determined.

■Measurement results The measurement results are shown in Table 1, and Th p 'p
H-(, ], -34) has the effect of increasing vitamin D 1-hydroxylase activity in the kidney, but [Met(0)8.
18] hP-DinH-(1-34) does not have the above-mentioned effects.

Table 1 Next, production examples of the present invention will be described with reference to Examples.

Example I CMet(0)' 18]hP TH-(1-34)
;■1-Ser-Val'-Ser-Glu
-11e -Gln -Leu -Met(0) -
1(is -Aen -Leu -Gly -Lys
-I-1is -Leu -Asn -Ser -Me
t(0) -Glu-Arg-Va, -Glu--T
rp-Leu-A, rg-Lye-Lys-Leu
-Gln -Asp-Val -IIs -Asn
-Phe -ORh P TH- (1-34);
H-8er-Val-8er-Glu-11e
-Gln-Leu -Met -Hls -A
sn -Leu-Gly-Lys-Hls
-Leu -Asn-8er -Met -Glu
-Arg-” Val-Glu-T
rp-Lbu-Arg-Lys-Lys-
Leu-Gln-A, 5p-Val-1is-A
Dissolve sn-Phe -OH10tn9 in 2.5 ml of water, add 250 μt of approximately 1% hydrogen peroxide solution, and
The mixture was stirred at 7°C for 30 minutes. Add 2.25 ml of water to the reaction solution and lyophilize to obtain [IMet(0,)'':lhP
T H-(]-34) was obtained. Yield amino acid analysis.

(1) Acid hydrolysis (hydrolysis in 0.5 ml of 6N hydrochloric acid containing 2% thioglycolic acid at 105°C for 24 hours);
Asp 4.11 (4), Ser 2.46 (3
), Glu5.10(5), Gly1.02(1
), Val 2.83(3), Met 1.97(
2], 11e 0.97 (1), Leu 5.00
(5), Phe O,95(1), Lys 3.11
(3), His 2.44 (3), Argl, 9
As
p 1.25 (1), Asn 3.29 (3)
, 5er2.82 (3), Glu 3.00 (3)
, Gln 1.27 (2) , Glyl, 26 (1)
, Val 3,27 (3) , Met (,O) 2
．． 57 (2), 11e 1.15 (1), Leu
5.00 (5), Phe 1.10 (1), Lys
3.22 (3), l-1is 2.35 (3)
, Argl, 90 (2), 'f'rp 0.71 (
], ) Thin layer chromatography [Carrier: Melli cellulose, developing solvent: butanol-pyridine-acetic acid-water (15:
10:3:1.2);Rf=0.50[hl)'I"l
-1-(]-34); Rf=0.591

[Brief explanation of drawings]

FIG. 1 shows curves showing the effects of known hPTH-(1-34) peptides on blood Cal/Rum, blood phosphorus, urinary Cal/Um, urinary phosphorus, and urinary C-AMP. Figure 2 shows the [Met(0)8.18]hPTIl-
(,1-34) Peptide blood calcium, blood phosphorus,
Urinary calcium, urinary phosphorus and urinary C-, A M P
represents a curve showing the influence on Representative: Tetsu I Naota Figure 2 Procedural amendment dated July 9, 1939 Otsu Indication of the case Showa 5g patent application No. 73-Otsu 07! Name of the invention CMet (0)8118:)'hPTH-(/-3'
l) Bepti 1-3 Relationship with the case of the person making the amendment Voluntary submission by the patent applicant j Changed "Hidouchi F" in the second line of page 7 of the specification in the detailed description of the invention of the specification to be amended to "Hidouchit" ” he corrected. "Atom" in Hiroyuki's specification section, page g, is corrected to "atomic.""(9ci/mM) J on page 10, line g of the specification is corrected to r (9Ci/mM) J.""Amino acid analysis;" on page 12, line 1 of the specification is changed to "amino acid analysis; / Om 'j J. Procedural amendment C'° / 1920/1920 Y/) 73rd, Director General of the Patent Office Mr. Kazuo Wakasugi / Indication of the case 1925 Patent Application No. 75 Otsu No. 07 Invention Name CMet, (0)”' ) h P TH-(/-3
'l) Peptide 3 Relationship with the case of the person making the amendment Patent applicant's address: 632 Mifuku, Ohito-cho, Tagata-gun, Shizuoka Prefecture /l Date of amendment order (voluntary)! Detailed explanation of the invention in the specification subject to amendment Column B Contents of the amendment In the second line of page 7 of the specification, "decomposed with a sodium solution" has been replaced with "can be decomposed with a sodium solution to form a free carboxyl group." and correct it.゛－゛－

Claims (1)

[Claims] 1), formula %formula% (0) Peptide matakesono salt represented by.