JP2006347956A - Anandamide formation inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new agent using CAP11 (a 11 kDa basic antibacterial polypeptide) as active component. <P>SOLUTION: The anandamide formation inhibitor comprises a dimer of peptide comprising an amino acid sequence expressed by sequence No 1, as active component. The peptide dimer comprising the amino acid sequence expressed by sequence No 1 preferably has a disulfide bonding formed between cycteine residueses of the 41th amino acid in the sequence No 1. The formation of anandamide is preferably formation of the anandamide by cells. The formation of anandamide is preferably formation of the anandamide induced by a lipopolysaccharide. The agent is usable for not only test reagents, such as medical, cytobiology etc., but also for a preventive of endotoxin shock, a deterioration preventive, an alleviation (amelioration of disease) agent, remedy, etc. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel agent that suppresses the formation of anandamide.

First, abbreviations used in this specification will be described.
CAP11: 11 kDa basic antibacterial polypeptide of 11 kDa
FACS: fluorescence-activated cell sorter
FCS: fetal calf serum
HPLC: high performance liquid chromatography
LPS: Lipopolysaccharide. Also called endotoxin.
PBS: phosphate-buffered saline
SS bond: disulfide bond
SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis
CAP11 is an antibacterial polypeptide found from neutrophils of guinea pigs, and is a homodimer in which peptides consisting of 43 amino acids are linked by an S—S bond (Non-patent Document 1). And it is known that CAP11 not only shows a strong bactericidal action against Gram-positive bacteria and negative bacteria, but also neutralizes the biological activity of Gram-negative bacteria LPS (Non-patent Documents 2 and 3).

Momogida, S. (Yomogida, S.) et al., 1996, Archives of Biochemistry and Biophysics, 328, p.219-226. Nagaoka, I.I. (Nagaoka, I) et al., 2000, Inflammation Research, 49, p73-79. Nagaoka, I.I. (Nagaoka, I) et al., 2001, Journal of Immunology, Vol. 167, p3329-3338. Anandamide is an unsaturated fatty acid ethanolamide that is present in the brain, testis, spleen, etc. It has been isolated and identified as an endogenous ligand of the "cannabinoid receptor". It is known that this substance inhibits vas deferens contraction by electrical stimulation, and body temperature lowering, exercise decrease, catalepsy, etc. are observed by systemic administration.

  An object of the present invention is to provide a novel agent containing CAP11 as an active ingredient.

  As a result of intensive studies to solve the above problems, the present inventors have found that CAP11 has an action of suppressing the production of anandamide, and have reached the present invention.

That is, the present invention provides an anandamide production inhibitor (hereinafter referred to as “the inhibitor of the present invention”) comprising a peptide dimer (CAP11) having the amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.
This CAP11 is preferably one in which an SS bond is formed between the cysteine residues of amino acid number 41 in SEQ ID NO: 1. The anandamide generation is preferably anandamide generation by cells. The anandamide generation is preferably anandamide generation induced by LPS.

  Since the inhibitor of the present invention can thereby suppress the formation of anandamide, not only test reagents for medicine and cell biology, but also preventive agents for endotoxin shock, preventive agents for deterioration, mitigation (improving symptoms), therapeutic agents, etc. It is also very useful.

  Hereinafter, the present invention will be described in detail by embodiments of the invention.

  The inhibitor of the present invention is an anandamide generation inhibitor containing CAP11 as an active ingredient. CAP11 is a peptide dimer consisting of the amino acid sequence shown in SEQ ID NO: 1.

  The production method of “CAP11”, which is an active ingredient of the inhibitor of the present invention, is not particularly limited, and for example, one extracted from neutrophils of guinea pigs may be used, which corresponds to the amino acid sequence described in SEQ ID NO: 1. A base produced by genetic engineering using a polynucleotide (DNA or RNA) may be used, or a base chemically synthesized according to the amino acid sequence shown in SEQ ID NO: 1 may be used.

When the peptide chain represented by SEQ ID NO: 1 (monomer of peptide constituting CAP11) is obtained, CAP11 is obtained by oxidizing the monomer to dimerize. Can do. This CAP11 is preferably a homodimer in which the peptide consisting of 43 amino acids represented by SEQ ID NO: 1 forms an SS bond at the cysteine residue of amino acid number 41.
The chemical synthesis of “the peptide monomer that constitutes CAP11” here is, for example, a liquid phase synthesis method or a solid phase synthesis method (Nobuo Izumiya, Tetsuo Kato, Toshihiko Aoyagi, Michinori Waki, “Basics and Experiments on Peptide Synthesis” "1985, see Maruzen Co., Ltd.).

  The produced “monomer of peptide constituting CAP11” and CAP11 can be purified by protein isolation and purification methods generally known in the field of protein chemistry. Specifically, for example, extraction, recrystallization, salting out with ammonium sulfate (ammonium sulfate) or sodium sulfate, centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography Processing operations such as chromatography, reverse phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution, and combinations thereof.

  The produced “peptide monomer constituting CAP11” and CAP11 can be hydrolyzed with an acid such as hydrochloric acid or methanesulfonic acid, and the amino acid composition can be examined by a known method. It can be confirmed whether or not the “peptide monomer” and CAP11 are correctly produced.

  More precisely, by determining the amino acid sequence of the produced peptide by a known amino acid sequencing method (for example, Edman degradation method, etc.), whether “the monomer of the peptide constituting CAP11” or CAP11 was produced correctly. You can check whether or not.

  In addition, the form of a salt is also included by CAP11 which is an active ingredient of this invention inhibitor. When the inhibitor of the present invention is used for pharmaceutical use, this salt is preferably a pharmaceutically acceptable salt.

  CAP11 can form a salt by acid addition. Examples of such salts include inorganic acids (hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, etc.) or organic carboxylic acids (acetic acid, propionic acid, maleic acid, succinic acid, malic acid, citric acid, tartaric acid, Salicylic acid, etc.), acidic sugars such as glucuronic acid, galacturonic acid, gluconic acid, ascorbic acid, acidic polysaccharides such as hyaluronic acid, chondroitin sulfate, alginic acid, or organic sulfonic acids (methanesulfonic acid, p-toluenesulfonic acid, etc.) Is exemplified. Of these salts, pharmaceutically acceptable salts are preferred.

CAP11 can form a salt with a basic substance. Examples of such salts include salts with inorganic bases such as alkali metal salts (sodium salts, lithium salts, potassium salts, etc.), alkaline earth metal salts, ammonium salts, or organic compounds such as diethanolamine salts and cyclohexylamine salts. Examples thereof include salts with bases. Of these salts, pharmaceutically acceptable salts are preferred.
The inhibitor of the present invention suppresses anandamide production by cells, particularly blood cells (particularly macrophages). The anandamide generation to be suppressed is preferably anandamide generation induced by LPS.

  The inhibitor of this invention should just contain CAP11 at least. For example, the inhibitor of the present invention may be composed only of CAP11, or may be in the form of a composition composed of CAP11 and other components.

The inhibitor of the present invention can be used not only as an experimental reagent but also as a medicament for the purpose of suppressing anandamide formation. For example, it may be aimed at suppressing anandamide production in endotoxin shock.
When the inhibitor of the present invention is used as a medicine, in addition to CAP11, excipients, binders, lubricants, coloring agents, disintegrating agents, buffering agents, isotonic agents, preservatives, painlessness that can be used in the pharmaceutical field An agent and the like can also be contained. When the inhibitor of the present invention is used as a medicine, depending on the purpose of the treatment, injection (subcutaneous, intradermal, intravenous, intraperitoneal, etc.), eye drop, instillation, transdermal, oral, inhalation, etc. An administration method can be selected. Depending on the method of administration, injections (solutions, suspensions, emulsions, solid preparations for use, etc.), tablets, capsules, granules, powders, solutions, lipolytic agents, ointments, gels, An external powder, spray, inhalation powder, eye drop, eye ointment, suppository, pessary and the like can be appropriately selected and formulated.

When the inhibitor of the present invention is used as a pharmaceutical, the dosage is the purpose of the administration (prevention, maintenance (prevention of deterioration), reduction (improvement of symptoms) or treatment), the type of the disease intended to suppress anandamide production, and the patient's symptoms It should be individually set according to sex, age, administration method, etc., and is not particularly limited. For example, in the case of an intravenous injection for the purpose of suppressing anandamide generation in endotoxin shock, once an adult The amount of CAP11 can be 1 mg to 10 mg as a standard.

Hereinafter, the present invention will be described more specifically with reference to examples.
<1> Preparation of CAP11, etc. Entrusted to Juntendo University School of Medicine Central Instrumental Analysis Room, “monomer of peptide constituting CAP11” shown in SEQ ID NO: 1 (consisting of 43 amino acids and having no S—S bond) Was prepared by a solid-phase synthesis method (Fmoc / PyBop (benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate) method). Hereinafter, this substance is simply referred to as “monomer”.

CAP11 is dissolved in a Tris buffer containing 0.5 M guanidine hydrochloride, 400 μM oxidized glutathione is added thereto, and allowed to stand at room temperature for 2 to 3 days to induce oxidation and dimerize. It was manufactured by. This was subjected to reverse phase HPLC using a capsule pack C18 column (manufactured by Shiseido Fine Chemical Co., Ltd.), eluted and purified by a linear concentration gradient with water and acetonitrile, and confirmed to have a molecular weight of about 11 kDa by SDS-PAGE. Therefore, it was used in the following pharmacological test. CAP11 prepared by this method is a homodimer in which a peptide consisting of 43 amino acids shown in SEQ ID NO: 1 forms an SS bond at the cysteine residue of amino acid No. 41.
<2> Pharmaceutical pharmacology test Measurement of amount of anandamide in cells and culture medium Macrophage cell RAW264.7 was cultured in a dish having a diameter of 150 mm for 2 days. Thereafter, the cells were washed with serum-free RPMI-1640 medium (manufactured by SIGMA-ALDRICH), and then cultured at 37 ° C. for 2 hours in RPMI-1640 medium (containing 2% FCS) containing the following substances.
(1) No processing (n = 3)
(2) LPS with final concentration of 10 ng / ml (n = 6)
(3) LPS having a final concentration of 10 ng / ml and CAP11 having a final concentration of 100 ng / ml (n = 3)
(4) LPS with a final concentration of 10 ng / ml and CAP11 with a final concentration of 1 μg / ml (n = 3)
Cells and culture medium were collected, and four 5 ml suspensions were prepared for each sample. To each of these, 18.75 ml of chloroform / methanol mixture (1: 2 (v / v)) was added and stirred for 30 seconds, and then 6.25 ml of chloroform was further added and stirred for 30 seconds. Each of these was then added with 6.25 ml of water and centrifuged at 1000 × g for 15 minutes at 4 ° C.

  After centrifugation, the organic solvent phase was collected and dried under a stream of nitrogen. The dried residue was redissolved in 1 ml of chloroform, transferred to a new 1.5 ml tube, and dried again under a nitrogen stream.

The residue after drying was dissolved in 40 μl of acetonitrile and subjected to reverse phase chromatography (80% acetonitrile, isocratic). Fractions corresponding to the anandamide retention time (already confirmed) were collected and dried under a stream of nitrogen. Residue after drying was re-dissolved in 50 μl of acetonitrile, and an equivalent amount of 4- (N-chloroformylmethyl-N-methyl) amino-7-N, N-dimethylaminosulphonyl-2,1, 3-benzoxadiazole (DBD-COCl) Acetonitrile solution (10 mM) was added and incubated at 60 ° C. for 2 hours for fluorescent derivatization.
The derivatized anandamide was subjected to reverse phase HPLC using TSK-GEL ODS-80Ts (manufactured by Tosoh Corporation) and analyzed with a fluorescence detector set at excitation: 450 nm and detection: 560 nm.
The measured amount of anandamide was calculated as a ratio to the amount of unstimulated cells (sample (1)). The results are shown in FIG. In FIG. 1, “resting” is the sample of (1), “LPS” is the sample of (2), “+ CAP11 100 ng / ml” is the sample of (3), and “+ CAP11 1 μg / ml” is the above Samples of (4) are shown respectively. From FIG. 1, it was shown that CAP11 suppresses the production amount of anandamide induced by LPS. It has also been shown that this suppression is dependent on the dose of CAP11.

2. Measurement of binding amount of LPS to CD14 on cells Macrophage cell line RAW264.7 was cultured in a dish having a diameter of 100 mm for 2 days. Thereafter, the cells were detached with ice-cold PBS and collected. The cells were then suspended at a concentration of 1 × 10 ⁶ in RPMI-1640 medium (SIGMA-ALDRICH, containing 10% FCS) containing the following substances, and pretreated at 37 ° C. for 10 minutes.
(1) No processing
(2) No processing
(3) CAP11 with a final concentration of 100 ng / ml
(4) CAP11 with a final concentration of 1 μg / ml
(5) Anti-CD14 neutralizing antibody with a final concentration of 4 μg / ml (BD PharMingens)
After the treatment, Alexa-labeled LPS (manufactured by Molecular Probes) at a final concentration of 100 ng / ml was added to each sample except the above (1), and further treated at 37 ° C. for 15 minutes. Thereafter, the cells were washed with ice-cold PBS and analyzed by FACS. The population of cells used for the analysis is shown in FIG. 2 (circled in FIG. 2), and the analysis results are shown in FIG. In FIG. 3, the horizontal axis represents Alexa fluorescence intensity, and the vertical axis represents the number of cells.

  From FIG. 3, it was shown that CAP11 suppresses the binding of LPS to CD14 on the cells (contrast (2) and (4) in FIG. 2). It was also shown that this suppression depends on the dose of CAP11 (contrast (3) and (4) in FIG. 2). Furthermore, the observed binding of LPS was shown to be via CD14 (contrast of (2) and (5) in FIG. 2).

From the above results, it was suggested that inhibition of LPS binding to CD14 and the suppression of anandamide formation by this were involved as one of the protective mechanisms of CAP11 against endotoxin shock.
<5> Formulation Examples Formulation examples of the inhibitor of the present invention are shown below, but these are merely examples and are not limited thereto.
(1) Injection CAP11 10mg
The above components were dissolved in 2 mL of a 5% mannitol aqueous solution, subjected to sterile filtration, and then sealed in an ampoule.
(2) Injection for dissolution
(A) CAP11 (lyophilized body) 10 mg (enclosed in an ampoule)
(B) 2 mL of sterile filtered PBS (enclosed in an ampoule)
The above-mentioned (A) and (B) were used as one set to produce an injection for dissolution at the time of use. In use, (A) can be dissolved in (B) and used.

It is a figure which shows suppression of the cell anandamide production | generation (caused by LPS) by CAP11. It is a figure which shows the population of the cell used for FACS analysis. It is a figure which shows the binding suppression of LPS with respect to CD14 on a cell by CAP11.

Claims (4)

An anandamide production inhibitor comprising, as an active ingredient, a peptide dimer comprising the amino acid sequence represented by SEQ ID NO: 1. The “dimer of a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1” is characterized in that a disulfide bond is formed between the cysteine residues of amino acid No. 41 in SEQ ID NO: 1. The inhibitor described. The inhibitor according to claim 1 or 2, wherein the anandamide production is anandamide production by cells. The inhibitor according to any one of claims 1 to 3, wherein the anandamide generation is anandamide generation caused by lipopolysaccharide.

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