JPH07267988A - New peptide - Google Patents

Abstract

PURPOSE:To obtain a new peptide, having a specific amino acid sequence containing a cyclic structure, capable of manifesting the affinity for neuropeptide Y receptors and acting on the central or peripheral nervous system and useful for preventing and treating, etc., diseases concerned therein. CONSTITUTION:This new peptide is expressed by the formula [A1 and A2 each is D-Cys or L-Cys, with the proviso that A2 is D-Cys when A1 is D-Cys; X1 and X2 each is Gly, Ala, Val, Leu or Ile; (m) and (n) each is 0 or 1; Y is Gly, Ala, Val, Ser or Thr; either of Z1 and Z2 is Arg and the other is Arg, Hrg, His, Lys, Nle, Leu or Val; Ac is acetyl], has the affinity for neuropeptide Y receptors and is capable of acting on the central or peripheral nervous system and useful for preventing and treating, etc., various diseases concerned therein. The peptide is obtained by synthesizing a peptide chain according to a peptide solid-phase synthetic method using a solid-phase support and then carrying out the oxidizing cyclization using hydrogen peroxide, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel peptide having an affinity for a neuropeptide Y receptor. It also relates to a medicament containing the peptide as an active ingredient.

[0002]

2. Description of the Related Art Neuropeptide Y (NPY) is a peptide consisting of 36 amino acid residues belonging to pancreatic peptides.
Widely distributed in central and peripheral tissues of humans and animals.
In the central nervous system, NPY has a feeding promoting action, an anticonvulsant action, a learning promoting action, an anxiety action, an antistress action and the like. Further, it is known that in depression, Alzheimer's disease and Parkinson's disease, a decrease in the amount of NPY in the cerebrospinal cord results in an increase in the amount of NPY in the brain with head injury. On the other hand, in peripheral tissues, NPY is also called a vasoconstrictor peptide, which regulates contraction of smooth muscle such as blood vessels.
It regulates cardiac contractility and secretion of blood pressure regulating hormones such as renin.

NPY receptor affinity substances (NPY receptor agonists and antagonists) are various diseases related to the physiological actions of NPY, namely bulimia nervosa and anorexia nervosa, unstable neuropathy, senile dementia, Parkinson's. disease,
It is considered to be useful as a therapeutic drug for diseases represented by depression, hypertension and hypotension. NPY is the C
It is known that the terminal side is essential for the expression of physiological activity (affinity). The smallest active fragment is NPY _29-36
However, its activity is very weak.

The smallest NPY fragment that has been reported to date is the NPY _27-36 fragment consisting of 10 amino acid residues by Tatemoto et al. [Proc. Nat].
I. Acad. Sci. USA, 89, 1174 (1992)].

[0005]

However, when considering stability in vivo and transferability into the brain, known amino acid fragments are not practically sufficient as pharmaceuticals and have high affinity for receptors. The development of NPY derivatives having high stability and stability is expected. Therefore, it is an object of the present invention to provide a peptide having a short chain and a remarkably excellent receptor affinity as compared with a known NPY fragment. further,
It is an object of the present invention to provide a physiologically active peptide useful for treating a disease associated with NPY.

[0006]

Means for Solving the Problems As a result of intensive studies on the NPY derivative, the present inventors have found a cyclic novel peptide containing two cysteines unlike the conventional NPY fragment, and completed the present invention. That is, the present invention has the general formula

[0007]

[Chemical 5]

(The symbols in the formulas have the following meanings: A ₁ , A ₂ ; D-Cys or L-Cy, which may be the same or different.
s, but when A ₁ = D-Cys, A ₂ = D-Cys X ₁ , X ₂ ; same or different Gly, Ala, Val, Leu
Or Ile m, n; same or different, 0 or 1 Y; Gly, Ala, Val, Ser or Thr Z ₁ , Z ₂ ; either one is Arg, the other is Arg, Hrg,
His, Lys, Nle, Leu or Val) is a novel peptide or salt thereof.

The compound of the present invention will be described in detail below. The symbols of amino acid residues in the compounds of the present invention have the following meanings. Cys Cysteine Tyr Tyrosine Gly Glycine Ala Alanine Val Valine Leu Leucine Ile Isoleucine Ser Serine Thr Threonine Arg Arginine Hrg Homoarginine His Histidine Lys Lysine Nle Norleucine

Further, these amino acid residues may be D-type amino acid residues or L-type amino acid residues unless otherwise specified. In the compound (I) of the present invention,-[(X ₁ )
_{In m-} (X ₂ ) _n -Y]-, X ₁ and X ₂ are Gly, Ala, Va
l, Leu or Ile, and in Y, Gly, Ala, Val, Se
r or Thr. m and n mean that either one or both of X ₁ and X ₂ may be absent. Preferably X ₁ and X ₂ are both present,
X ₁ is Leu and X ₂ is Ile. In that case, Y is T
hr is preferred.

The amino acid residues of X ₁ , X ₂ and Y are L
Either type or D type may be used. L type is preferable. _One of the amino acid residues in Z ₁ and Z ₂ is
Arg, and the other is selected from Arg, Hrg, His, Lys, Nle, Leu and Val. The amino acid residues of Z ₁ and Z ₂ may be L-type or D-type. L type is preferable. Preferably both Z ₁ and Z ₂ are L-Arg. In addition, Ac means an acetyl group. Typical examples of the compound of the present invention included in the general formula (I) are as follows.

[0012]

[Chemical 6]

[0013]

[Chemical 7]

[0014]

[Chemical 8]

The compound of the general formula (I) can form salts with acids or bases, and the peptide derivative of the present invention includes these salts. Here, as salts with acids, mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid,
Examples thereof include acid addition salts with organic acids such as succinic acid, tartaric acid, carbonic acid, picric acid, methanesulfonic acid, ethanesulfonic acid and glutamic acid.

As the salt with a base, an inorganic base such as lithium, sodium, potassium, magnesium, calcium and aluminum, an organic base such as methylamine, ethylamine and ethanolamine, and a basic amino acid such as lysine and ornithine. Alternatively, an ammonium salt may be used. The object compound of the present invention can be produced by various methods. When the synthetic method is used, first, a peptide compound consisting of the constituent amino acid sequences is produced, and then SH of two Cys in the obtained peptide compound is produced. It is preferred to form disulfide bonds in the group.

Production method of peptide compound: The production of the peptide compound of the present invention is a coupling method, a C-terminal end activation method, which is carried out by making full use of various protecting groups, coupling reagents and the like, which are one of peptide synthesis methods. It is of course possible to synthesize using a liquid phase method such as N-terminal activation method, but a solid phase method [Merrifield; J. Am. Chem.
c., 85,2185 (1963), which was first introduced and then improved, is advantageous. When synthesizing a peptide by the solid phase method, an excellent automatic peptide synthesizer; for example, a peptide synthesizer 430A manufactured by Applied Biosystems is commercially available, and it may be performed according to a standard operation program of this apparatus. It goes without saying that the method for producing the compound of the present invention should not be limited to the application of currently commercially available devices. The synthesis by these peptide synthesizers is rarely accompanied by racemization, and the target compound in which the configuration of the starting amino acid is maintained can be obtained.

In order to further enhance the degree of purification, the synthesized peptide is advantageously purified by preparative reverse phase high performance liquid chromatography or the like. In addition, in the synthesis using an automatic peptide synthesizer, the amino protecting group is generally removed with trifluoroacetic acid, so the final product is isolated as the trifluoroacetic acid salt. It can be isolated. In order to maintain the purity and stability of the synthetic peptide, freeze-drying is preferable. The peptide synthesizing procedure by the automated peptide synthesizing machine is performed as follows.

1) The solid-phase resin is condensed with the C-terminal α-amino protected amino acid A of the desired polypeptide. 2) Removal of the protecting group of α-amino protected amino acid A-washing-
Perform the neutralization-wash operation. 3) The α-amino protected amino acids constituting the desired polypeptide are sequentially condensed. 4) The operations of removing the protective group of the α-amino protected amino acid, washing, neutralizing and washing are performed. 5) After synthesizing the desired polypeptide, the N-terminal amino group is acetylated. 6) The target peptide is cleaved from the solid phase resin, and the protecting groups bonded to all side chains of amino acid residues are removed. 7) An oxidative cyclic reaction is carried out using the two thiols in the free peptide obtained in 6). Further, as another method for producing a peptide compound, it is also possible to carry out by a genetic engineering method using a DNA encoding this peptide.

Formation of disulfide bond: Next, in order to form an intramolecular disulfide bond in the SH group of two Cys in the peptide compound obtained in the above, an oxidative treatment may be performed. For example, the peptide synthesized by the solid phase method is cleaved from the solid phase resin by the above method and the protecting groups attached to all side chains of amino acid residues are removed, and then the free thiol group is removed in an ammonium acetate buffer solution. It can be carried out by adding hydrogen peroxide and oxidizing it.

Isolation / purification is carried out by various chromatography,
The salting out method, the crystallization method, the filtration method, the concentration method or the centrifugation method is used. The structure confirmation method of the compound of the present invention is
Examples include LC, amino acid analysis, elemental analysis, microquenching or peptide mapping methods.

[0022]

INDUSTRIAL APPLICABILITY The compound of the present invention is NPY derivative as NP
For various diseases related to the physiological functions of Y, namely hyperphagia, anorexia nervosa, obesity, epilepsy, anxiety, senile dementia, depression, Parkinson's disease, brain tissue degeneration associated with head trauma, and stress. It is useful as a therapeutic drug for various physical symptoms, hypertension, hypotension, heart failure, angina, asthma, diarrhea, hormonal abnormalities, etc. The compound of the present invention is also useful as an adjunct to diet therapy in diabetic patients and as a drug used for blood pressure control during surgery, after surgery, during shock, or during pheochromocytoma. The action of the compound of the present invention was confirmed by the following test methods.

Membrane preparations prepared from SK-N-MC cells derived from human neuroblastoma cells or porcine hippocampus were prepared from the compounds of the invention and 50 pM [ ¹²⁵ I] -peptide YY (PY.
Y), 50 mM containing 10 mM magnesium chloride, 1 mM phenylmethylsulfonyl fluoride, 1 mg / ml bacitracin, and 5 mg / ml bovine serum albumin
180 ° C in M Tris-HCl buffer (pH 7.4) at 25 ° C
After incubation for 1 minute, the membrane-bound radioactivity was separated by centrifugation at 10,000 xg for 3 minutes. Non-specific binding was measured in the presence of 1 μM NPY.

In this pharmacological test, the compound of the present invention was N
It showed a high affinity for the PY receptor. In particular, the compound of the above formula (1) showed a good affinity for the NPY receptor. A preparation containing one or more of the compound of the present invention or a salt thereof as an active ingredient is prepared by using an injection, an inhalation agent, a suppository, which is prepared by using a commonly used carrier or excipient for preparation and other additives. , Transdermal solution, ointment, transdermal patch,
It is preferable that it is prepared into a transmucosal patch (for example, oral patch), a transmucosal solution (for example, nasal solution), and administered parenterally.

Examples of carriers and excipients for the preparation include solid or liquid non-toxic medicinal substances. Examples of these include lactose, magnesium stearate, starch, talc, gelatin, agar, pectin, gum arabic, olive oil, sesame oil, cocoa butter, ethylene glycol, and other commonly used substances. The clinical dose of the compound of the present invention is appropriately set in consideration of the disease, weight, age and sex of the patient to whom it is applied, administration route, etc., but is usually 1 to 500 mg per day for an adult, It is administered in 2 to 4 divided doses.

[0026]

[Manufacturing Method] The present invention will be described in more detail with reference to examples, but the invention is not limited to these examples. Example 1

[0027]

[Chemical 9]

1) Solid phase synthesis of peptide by t-Boc (tert-butoxycarbonyl group) method Apparatus: Peptide automatic synthesizer 430A manufactured by Applied Biosystems Co., Ltd. The synthesis procedure was performed according to the software installed in the synthesizer. Amino acid derivative and resin (manufactured by Peptide Institute) MBHA resin (p-methylbenzhydrylamine resin) Boc-Tyr (BrZ), Boc-Arg (Tos), Boc-Thr (Bzl), Boc-Ile, Boc
-Leu, Boc-D-Cys (4-MeBzl) (Boc is tert-butoxycarbonyl group, Z
Is a benzyloxycarbonyl group, Tos is a tosyl group, B
zl means a benzyl group, respectively. The same applies hereinafter) Reaction auxiliary reagents Dichloromethane (DCM), Dimethylformamide (D
MF), diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), dicyclohexylcarbodiimide (DCC), acetic anhydride

2) Synthetic method The synthesis is carried out starting from the C-terminal toward the N-terminal. First act
In the ivation tank, the first Boc-Tyr (BrZ) is fully activated by DCC and then reacted with the MBHA resin in the reaction tank. After the reaction is completed, TF is added to condense the next amino acid.
A treatment is performed to remove the Boc group on the resin, and DI
After neutralization with EA, the following amino acid derivative (Boc-Arg (To
s)) is condensed with DCC. This operation was automatically repeated with the N-terminal amino acid acid according to the program, and finally, the Boc group was removed by TFA treatment and washed with DIEA, and then the N-terminal amino group was acetylated by acetic anhydride treatment.

3) Cleavage of peptide from solid phase resin p-Cresol was added to the synthetic protected peptide resin and the mixture was set in a hydrogen fluoride (HF) treatment device, then at -2 to -5 ° C, 6
After treatment for 0 minutes, the peptide was cleaved from the solid-phase resin, and at the same time, the entire side chain protecting groups of amino acids were removed. After the reaction, HF was distilled off under reduced pressure, the crude peptide was extracted with a 50% aqueous acetic acid solution, the resin was filtered off, and freeze-dried to obtain a powder. Analytical RP-HPLC gave a clear main peak. 4) Oxidative cyclization reaction The crude thiol-free peptide obtained above is dissolved in ammonium acetate buffer (if it is difficult to dissolve, urea is added), hydrogen peroxide is added to adjust the pH to 8, and the reaction is allowed to proceed.
After checking by P-HPLC and confirming that the reaction was completed, stop the reaction by adding ascorbic acid, make the pH acidic, and directly subject to preparative purification by RP-HPLC to isolate the target peak. went.

Purification by RP-HPLC Column ODS reverse phase column Analysis 4.6 × 150 mm, s-5 120A ODS preparative 30 × 250 mm, s-5 120A ODS eluent A: 0.1% TFA water B: 0 1% TFA acetonitrile analysis 25 minutes preparative 60 minutes detection 220nm

Physicochemical properties Appearance: White powder Amino acid analysis value Hydrolysis condition: 6N HCl, 110 ° C., 22h Thr (1) 0.90 Ile (1) 0.97 Leu (1) 1.01 Tyr (1) 0.94 NH ₃ (1) 1.18 Arg (2) 2.00 Cys (2) 1.87 Purity (HPLC): 93.6% Liquid chromatography (HPLC) conditions Column: YMC Pac ODS-AM (4.6mmI.D. × 150mm) Eluent: 0.1% TFA gradient : CH ₃ CN 10% -60% (25 min.) Temperature: 50 ° C Flow rate: 1 ml / min. Detection: 220 nm or less, the compounds of Examples 2 to 3 were obtained by treating in the same manner as in Example 1 by using the amino acids constituting each compound instead of the amino acids used in Example 1. Example 2

[0033]

[Chemical 10]

Physicochemical properties Appearance: White powder Amino acid analysis value Hydrolysis condition: 6N HCl, 110 ° C, 22h Thr (1) 0.89 Ile (1) 0.95 Leu (1) 1.00 Tyr (1) 0.93 NH ₃ (1) 1.17 Arg (2) 2.00 Cys (2) 1.88 Purity (HPLC): 97.5% Liquid column chromatography (HPLC) conditions Column: YMC Pak ODS-AM (4.6 mm I.D. × 150 mm) Lot. 041516478 Eluent: 0. 1% TFA gradient: CH ₃ CN 10% -60% (25 min.) Temperature: 50 ° C. Flow rate: 1 ml / min. Detection: 220 nm Example 3

[0035]

[Chemical 11]

Physicochemical properties Appearance: White powder Amino acid analysis value Hydrolysis condition: 6N HCl, 110 ° C., 22h Thr (1) 0.90 Ile (1) 0.97 Leu (1) 1.01 Tyr (1) 0.96 NH ₃ (1) 1.13 Arg (2) 2.00 Cyr (2) 1.81 Purity (HPLC): 98.3% Liquid chromatography (HPLC) conditions Column: YMC Pak ODS-AM (4.6 mmI.D. × 150 mm) Lot.041516478 Eluent: 0.1 % TFA gradient: CH ₃ CN 10% -60% (25 min.) Temperature: 50 ° C. Flow rate: 1 ml / min. Detection: 220nm

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area A61K 38/00 ABN C07K 14/435 8318-4H A61K 37/02 ABN

Claims (7)

[Claims] 1. A general formula: (The symbols in the formulas have the following meanings: A ₁ , A ₂ ; D-Cys or L-Cy, which may be the same or different.
s, but when A ₁ = D-Cys, A ₂ = D-Cys X ₁ , X ₂ ; same or different Gly, Ala, Val, Leu
Or Ile m, n; same or different, 0 or 1 Y; Gly, Ala, Val, Ser or Thr Z ₁ , Z ₂ ; either one is Arg, the other is Arg, Hrg, Hi
s, Lys, Nle, Leu or Val), or a novel peptide or salt thereof. 2. _One of Z ₁ and Z ₂ is L-Arg and the other is L-Ar.
The novel peptide or salt thereof according to claim 1, which is g, L-Hrg, L-His, L-Lys, L-Nle, L-Leu or L-Val. 3.-[(X ₁ ) _m- (X ₂ ) _n -Y]-is -Leu-Ile-T
The novel peptide or salt thereof according to claim 2, which is hr-. 4. Z ₁ and Z ₂ are both L-Arg.
The novel peptide or salt thereof described. 5. A chemical formula: The novel peptide according to claim 4 or a salt thereof. 6. A chemical formula: (The symbols in the formula have the following meanings: A ₁ , A ₂ : D-Cys or L-Cys which are the same or different, but when A ₁ = D-Cys, A ₂ = D-Cys) Item 4. The novel peptide or salt thereof according to item 4. 7. embedded image A novel peptide or salt thereof