JP4709343B2 - Use of chitinous substances to inhibit cellular nitric oxide production - Google Patents

Description

【００３０】
表１からわかるように、コラーゲンで被覆したウェルは活性化したRAW264.7細胞のＮＯの生産に際だった影響を与えなかったのに対して、キチンおよび/またはキトサンはＮＯの生産を著しく減少させた。
【００３１】
キチンの誘導体またはキチンおよびキトサンのモノマーもまたＮＯの生産を減少させるかどうかを試験するために、活性化したRAW264.7細胞を、N-アセチルグルコサミン(NAGA；キチンポリマーの単量体単位）、グルコサミン（キトサンの単量体単位）、およびキチンのオリゴ糖誘導体の存在下で培養した。結果を下記の表２にまとめた。
【００３２】
【表２】

【００３３】
表２からわかるように、キチンおよびキトサンモノマー、ならびにキチンの短い（1-3残基の）オリゴ糖誘導体は、活性化したRAW264.7細胞のＮＯの生産に実質的に影響を与えなかった。けれども、より長いオリゴ糖誘導体（キトペントースおよびキトヘキソース）は、ＮＯの生産を実質的に減少させ、キトヘキソースはキトペントースよりも一層強い阻害剤であった。グルコサミン、NAGA、およびすべての試験したキチンのオリゴ糖誘導体は上記の細胞毒性試験法を用いて測定したところ、RAW264.7細胞に対して細胞毒性を示さなかったことを記載しておく。
【００３４】
（その他の実施の形態）
本発明について、その詳細な説明と共に記載してきたが、上記の記載は例示を目的とするものであって、特許請求の範囲によって定義される発明の範囲を限定するものではない。他の態様、利点、および変更は本発明の範囲に含まれる。[0001]
BACKGROUND OF THE INVENTION
The present invention reduces nitric oxide production by cells in vitro or by cells in mammals by contacting the cells with an amount of chitosan or a derivative thereof effective to reduce nitric oxide production in the cells. Regarding the method.
[0002]
[Prior art]
Nitric oxide (NO) is associated with a series of different biological phenomena such as inflammation, septic shock, the detrimental consequences of ischemia and reperfusion injury, hypotension, cellular development, and apoptosis It is believed that Inflammation of tissues in vivo is often characterized by the invasion or presence of activated macrophages, so that activated macrophages produce NO, which is a mediator of vasodilation during the process of inflammation. Thus, inflammation can be reduced by reducing NO production by cells such as macrophages.
[0003]
Chitosan is an acid-soluble polymer of β- (1,4) -D-glucosamine. Chitin is an acetylated form of chitosan that is relatively insoluble. Chitin, chitosan, and derivatives thereof are utilized in many industrial applications, such as the production of viscosity modifiers, adhesives, chromatographic carriers, paper strengtheners, flocculants, food additives, pharmaceuticals and cosmetics.
[0004]
The present invention is based on the surprising result that chitosan and its derivatives can inhibit cellular NO production. This result was unexpected because it was reported that chitosan promotes NO production by rat macrophages in vitro (Peluso et al., Biomaterials 15: 1215-1220, 1994).
[0005]
Problems to be solved by the invention and means for solving the problems
Thus, the present invention identifies a subject (eg, a mouse, mammal, or human) for which a decrease in NO production is desired, and an amount of chitosan sufficient to reduce NO production in that subject. Characterized by a method for reducing NO production in a subject by administering to the subject a composition comprising. The composition may be locally (eg, at a site of inflammation in a subject such as an arthritic joint), systemically (eg, intravenous), or part of an implantable device (eg, for wound use). It can be applied as a surgical prosthesis, such as a bandage, or as an instrument for making the composition sustained release. It is desirable to reduce NO production when NO production is abnormally increased, or when NO production is normal but it is advantageous for the mammal to have a lower level of NO production than normal.
[0006]
The present invention also includes a method for reducing NO production in a cell by contacting the cell with a composition comprising an amount of chitosan sufficient to reduce the production of NO in the cell. .
[0007]
As used herein, the term “composition comprising chitosan” refers to (1) chitosan; (2) chitosan that is partially acetylated, chitosan that is fully acetylated to form chitin, and polysaccharides (eg, Derivatives of chitosan, including chitosan covalently linked to 5 to 10, or 5 or 6 sugar residues; and (3) (1) or (2) or both, and one or more additions Means a mixture comprising an agent (eg, carrier) or other biologically active compound as described below. In general, chitosan is produced by deacetylation of chitin. However, for convenience, the above definition is used to include both chitin and chitosan.
[0008]
The methods of the invention can be used to treat or prevent diseases or conditions where NO production or the resulting physiological effects are undesirable. Such diseases or symptoms will be described later. Furthermore, the methods of the present invention can be used in in vitro or in vivo animal models to test or screen the effectiveness of chitosan or its derivatives as drugs to treat various conditions such as inflammation. Examples of successful screening for effective or ineffective derivatives of chitin or chitosan are described in the examples below (see, eg, Tables 1 and 2).
[0009]
Other features or advantages of the invention will be apparent from the following detailed description and from the claims.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to administering to a subject a composition comprising chitosan for the treatment or prevention of a condition or disease mediated by cellular NO production. Various methods for practicing the invention are discussed below.
[0011]
Specific diseases or disorders that can be treated by the composition include inflammatory diseases or disorders, hypotension, septic or traumatic shock, chronic hypotension or persistent erectile dysfunction. Therefore, in the method of the present invention, a certain amount of a vasoconstrictor having an effect of increasing blood pressure can be administered together with chitosan or a derivative thereof. Preferred vasoconstrictors include epinephrine, norepinephrine, vasopressin, ^NG -monomethyl-L-arginine, ^NG- nitroarginine methyl ester and prostaglandins. The methods of the invention can also be used to inhibit smooth muscle cell relaxation that occurs in response to NO. Thus, in general, inhibition of NO production has many therapeutic effects as a major active ingredient in drug therapy or as an adjunct to other drugs such as steroids, antibodies, or peptide hormones. . Furthermore, the compositions of the present invention can be administered when NO production is normal, but it is advantageous for the mammal to have a lower level of NO production than normal. For example, as the patient dehydrates, it is desirable to reduce the normal vasodilation of the endothelium in the renal tubule.
[0012]
Inflammation includes a cellular immune response with the release of toxic molecules such as NO. A cellular immune response is beneficial, for example, to kill infectious microorganisms such as bacteria and parasites and to eliminate cancerous or infected cells. However, inflammation can be chronic, autoimmune, or harmful, such as asthma, cirrhosis, inflammatory bowel disease, and arthritis.
[0013]
NO is thought to be associated with the deleterious effects of ischemic symptoms. Ischemia or hypoxia becomes a particularly serious problem when it occurs in the heart, for example, as a result of myocardial infarction or after balloon angioplasty. Ischemia in the brain is also significant, but generally symptoms are associated with seizures. Therefore, in the method of the present invention, a drug designed to remove an obstruction that causes ischemia (eg, tissue plasminogen activator or streptokinase) can also be administered.
[0014]
NO is also an active neurotransmitter. Excessive production or activity of NO can cause neurological diseases, particularly those affecting the brain. Therefore, neurological diseases can be treated by administration of the composition used in the method of the present invention. When the composition is administered intravenously to facilitate crossing the blood brain barrier, chitosan or a derivative thereof is covalently linked to a hydrophobic component or protein known to cross the barrier. You may let them. Alternatively, the composition may be administered intracranial or intraventricularly.
[0015]
Chitin can be produced by deproteinizing and demineralizing crab or shrimp shells (see, eg, US Pat. No. 3,903,268). Subsequently, chitosan can be obtained by deacetylating the chitin with a hot alkaline solution. Alternatively, chitin can be isolated from various fungal species such as those belonging to the genus Actinomucor. In addition, chitosan and its derivatives can be obtained from known chemical vendors such as Sigma Chemical Co. (St. Louis, MO).
[0016]
The compositions of the present invention can be administered by any suitable route, for example, intravenous, intraarterial, topical, by infusion, intraperitoneal, intrapleural, oral, subcutaneous, intramuscular, sublingual, intraepidermal or intrarectal. Can be administered. The composition of the present invention can be formulated as a solution, suspension, suppository, tablet, granule, powder, capsule, ointment, or cream. To prepare these compositions, solvents (eg, water or saline), solubilizers (eg, ethanol, Polysorbates, or Cremophor EL7), reagents for providing isotonicity, preservatives, antioxidants Agents, excipients (eg lactose, starch, crystalline cellulose, mannitol, maltose, calcium hydrogen phosphate, light anhydrous silicic acid or calcium carbonate), binders (eg starch, polyvinylpyrrolidone, hydroxypropylcellulose, ethylcellulose, Carboxymethylcellulose, or gum arabic), lubricant (eg, magnesium stearate, talc, or hydrogenated oil), or stabilizer (eg, lactose, mannitol, maltose, polysorbate, macrogol, or polyoxyethylene hydrogenated castor oil) ) Can be added. If necessary, basic substances such as glycerin, dimethylacetamide, 70% sodium lactate, surfactant, or sodium hydroxide, ethylenediamine, ethanolamine, sodium bicarbonate, arginine, meglumine, or trisaminomethane Can be added. Formulations such as solutions, tablets, granules or capsules can be formed by adding these ingredients.
[0017]
Chitosan or its derivatives can also be added to compositions containing other drugs. For example, in Knapczyk et al., “Pharmaceutical Dosage Forms with Chitosan”, edited by Skjak-Break et al., “Chitin and Chitosan”, Elsevier Applied Science, 1998, pp665- See 669. This study and others, and the examples below, indicate or suggest that chitosan and its derivatives are well tolerated and not cytotoxic in vivo. Thus, the dose of chitosan or a derivative thereof can be increased to an effective level with little need to worry about toxicity.
[0018]
The specific dose of the composition of the present invention is determined in consideration of the results of animal experiments and various conditions. More specific doses are clearly apparent; dosing regimen; age, weight, sex, sensitivity, food eaten, dosing interval, conditions of the subject such as drugs administered in combination; and NO-induced abnormalities Varies with the cause, severity, and extent of The optimal dose and frequency of administration under a given condition must be determined by appropriate dosage tests based on the above guidelines by medical professionals.
[0019]
In general, it is useful to test the effectiveness of a composition, material, or medical device comprising chitosan or a derivative thereof before administering the composition to a human patient. For example, Minami et al., “Application of Chitin and Chitosan in Large Animal Practice”, edited by Brine et al., “Advances in Chitin and Chitosan” Test chitinous compositions for the ability to promote or treat a number of symptoms using calves or cows according to the method described in Elsevier Applied Science, New York, 1992, pp 61-69 be able to. Trials of efficacy using small animals are described in Okamoto et al., “Application of Chitin and Chitosan in Small Animals”, edited by Brine et al., “Advances in Chitin and Chitosan”. Chissan), Elsevier Applied Science, New York, 1992, pp70-78; Johnson et al., “In vivo Tissue Response to Implanted Chitosan Glutamate”, edited by Brine et al., “Chitin”. In the Advances in Chitin and Chitosan, Elsevier Applied Science, pp 3-8, 1992; or Su et al., Biomaterials 18: 1169-1174, 1997. . For wound healing studies, after forming chitosan or its derivatives into a surgical prosthetic sheet, this sheet can be used, for example, to close a hernia or replace a skin patch. Su et al., Supra; Austin et al., Science 212: 749-753, 1981; Su et al., Biomaterials 20: 61-68, 1999; and Kifune et al., “Clinical application of chitin artificial skin (Beschitin W)” (“Clinical Application of Chitin Aritificial Skin (Beschitin W) ”), edited by Brine et al.,“ Advances in Chitin and Chitosan ”, see Elsevier Applied Science, pp 9-15, 1992. In another method, chitosan or derivatives thereof can be used to produce sutures that promote healing or reduce inflammation. For example, Tachibana et al., Jap. J. Surg. 18: 533-539, 1988; and Biagini et al., “Biological Materials for Wound Healing”, edited by Brine et al., “Chitin and Chitosan. "Advances in Chitin and Chitosan", see Elsevier Applied Science, pp 16-24, 1992. These test methods are easy to specifically detect the amount of NO in a biopsy of the affected site or the implanted device using known methods or the assays described in the examples below. Can be corrected.
[0020]
Without further elaboration, it is believed that one skilled in the art can fully utilize the present invention based on the above disclosure and the following description. The following examples are to be construed as merely illustrative of how those skilled in the art may practice the invention, and are not intended to limit the other parts of the disclosure in any way. All documents cited herein are hereby incorporated by reference.
[0021]
【Example】
The NO-inhibitory activity of chitosan and its derivatives was found using the following materials and methods.
Cell culture : NIH / 3T3 (mouse embryo fibroblasts, CCRC 60008) and RAW264.7 (mouse monocytes / macrophages, CCRC 60001) cell lines were obtained from Culture Collection and Research Center (CCRC), Taiwan. Both cell lines were incubated at 37 ° C in Dulbecco's modified essential medium (DMEM, GIBCO BRL, Rockville, MD) containing 1.5 g / L NaHCO ₃ and supplemented with 10% fetal calf serum (Hyclone, Logan, UT). and cultured 5% CO ₂ and incubated in. Cell cultures were tested for mycoplasma contamination using the fluorescent stain Hoechst 33258 (Hay et al., ATCC Quality Control Methods for Cell Lines, American Type Culture Collection, Manassas, VA, 1992, pp23-33). All cultures described in this example were free of mycoplasma.
Collagen / chitin coated wells Two mixtures were prepared. A mixture of type I collagen (rat tail, Sigma, St. Louis, MO) was dissolved in 0.1 N acetic acid to make a 1 mg / ml collagen solution. Chitin was added to 0.1N acetic acid to make a 1 mg / ml mixture, which was sonicated for 3 minutes using a Labsonic U sonicator (Braun Biotech International, Germany). After autoclaving at 121 ° C. for 15 minutes, the chitin mixture became a fine Wires slurry. Microtiter tissue culture plates (96-well) were rinsed with phosphate buffered saline (D-PBS, GIBCO BRL) excluding Dulbecco's Ca ²⁺ and Mg ²⁺ . Then 5 μl of collagen solution or different amounts of chitin mixture was added to each well. The plates were then air dried in a laminar flow hood and stored at 4 ° C. for up to 1 week.
[0022]
Cytotoxicity test : For experiments on chitin cytotoxicity, cells were passaged by incubating at a density of 2000 cells / well for 48 hours in 96-well tissue culture plates pre-coated with different amounts of chitin or collagen. Cultured. For chitosan cytotoxicity experiments, cells were incubated overnight in subcultured 96-well tissue culture plates. The next day, the medium was discarded and replaced with fresh medium containing different amounts of chitosan. The culture was then further incubated for 48 hours. Cytotoxicity was determined using 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide dye (MTT; Merck Co., Germany) and Mosmann, J. Immunol. Methods 65:55 -63, 1983.
[0023]
Measurement of NO production by the detection of nitrite: Griess reagent and nitrite in using sodium nitrite medium as standard (NO ₂ ^-) accumulate indirectly the production of nitric oxide (NO) by measuring the Quantified (Green et al., Anal. Biochem. 126: 131-138, 1982; and Ignarro et al., Proc. Natl. Acad. Sci. USA 90: 8103-8107, 1993). For the nitrite assay, RAW264.7 cells were subcultured in coated 96-well tissue culture plates at 1 × 10 ⁵ cells / well for 24 hours. Medium contains 100 U / m interferon-γ (IFN-γ, recombinant mouse; GIBCO BRL) and 100 ng / ml lipopolysaccharide (LPS, Escherichia coli serotype O128: B12; Sigma), phenol red And DMEM without serum. The samples were then tested as described by Rollo et al., J. Leukoc. Biol. 60: 397-404, 1996.
[0024]
To determine the effect of chitin on NO production, cells were subcultured in 96-well tissue culture plates coated directly with chitin. After overnight incubation, the cells became attached to the bottom of chitin-coated wells. The medium was removed and replaced with medium supplemented with IFN-γ and LPS as described above. After incubation for 48 hours, according to the method of Rollo et al., 50 ml of the culture supernatant was sequentially added with the same amount of 60 mM sulfanilamide (Sigma) dissolved in 3N HCl and 4 mM N-1-naphthylethylenediamine (Sigma). After mixing, the mixture was shaken at room temperature for 5 minutes. The concentration of nitrous acid was measured by reading the absorbance of the sample at 540 nm using a microplate reader (MRX, Dynex Technologies, Inc., VA).
[0025]
Other chemicals : Chitin (molecular weight about 450 kDa) and chitosan (molecular weight about 150 kDa, 95% deacetylated) were obtained from Sun-Chiu Chemical Co., Taiwan. Glucosamine and N-cetylglucosamine were purchased from Sigma Chemical Co. Chitobiose (2-acetamido-2-deoxy-4-O- (2-acetamido-2-deoxy-bD-glucopyranosyl) -D-glucopyranose), chitotriose, chitotetraose, chitopentose, and chitohexose Purchased from Calbiochem Co. (La Jolla, Calif.).
[0026]
Cytotoxicity believed to be related to chitin and chitosan was evaluated for both mouse embryonic fibroblast NIH / 3T3 cells and mouse monocyte-macrophage RAW264.7 cells. Different volumes of chitin slurry solution were added to a 96-well tissue culture plate and allowed to air dry before cells were seeded and allowed to attach. After 48 hours of incubation, cell growth was assayed using MTT dye. When 1-2 μl of 1 mg / ml chitin mixture was added to the wells, chitin increased the growth of RAW264.7. The proliferation of NIH / 3T3 cells increased when 1 μl was added to the wells. For each cell type, above these doses, chitin inhibited cell growth depending on the dose (added to 20 μl per well). However, even at a dose of 5 μl / well, cell growth was only reduced by about 10%, indicating that the experimental conditions used below did not substantially inhibit cell growth.
[0027]
Chitosan showed no measurable cytotoxicity to NIH / 3T3 cells at doses up to a concentration of 200 μg / ml in the medium. Low concentrations of chitosan (0.32 μg / ml) promoted the growth of RAW264.7 cells, but chitosan concentrations of 8 μg / ml and higher were also observed to slightly inhibit the growth of RAW264.7 cells.
[0028]
Macrophage RAW264.7 cells are stimulated to produce NO by IFN-γ or LPS (Rollo et al., Supra). However, chitosan inhibited NO production (measured as nitrite) in RAW264.7 cells activated by IFN-γ and LPS, depending on the dose, up to 200 μg / ml. At the highest dose (200 μg / ml), NO production was reduced by about 85%. Similarly, when grown on coated tissue culture plates supplemented with 5 μl / well of chitin, the NO production of activated RAW264.7 cells was reduced by approximately 52%. The results of NO inhibition are summarized in Table 1 below.
[0029]
[Table 1]

[0030]
As can be seen from Table 1, wells coated with collagen did not significantly affect NO production in activated RAW264.7 cells, whereas chitin and / or chitosan significantly reduced NO production. I let you.
[0031]
To test whether chitin derivatives or chitin and chitosan monomers also reduce NO production, activated RAW264.7 cells were treated with N-acetylglucosamine (NAGA; monomer unit of chitin polymer), The cells were cultured in the presence of glucosamine (a monomer unit of chitosan) and an oligosaccharide derivative of chitin. The results are summarized in Table 2 below.
[0032]
[Table 2]

[0033]
As can be seen from Table 2, chitin and chitosan monomers, and short (1-3 residue) oligosaccharide derivatives of chitin did not substantially affect the production of NO in activated RAW264.7 cells. However, longer oligosaccharide derivatives (chitopentose and chitohexose) substantially reduced NO production, and chitohexose was a stronger inhibitor than chitopentose. Note that glucosamine, NAGA, and all tested oligosaccharide derivatives of chitin were not cytotoxic to RAW264.7 cells as measured using the cytotoxicity test method described above.
[0034]
(Other embodiments)
While the invention has been described in conjunction with the detailed description thereof, the above description is for illustrative purposes and is not intended to limit the scope of the invention as defined by the claims. Other aspects, advantages, and modifications are within the scope of the invention.

Claims (17)

Asthma, cirrhosis, inflammatory bowel disease, arthritis, hypotension, septic or traumatic shock, chronic hypotension, persistent erection, ischemia, hypoxia, ischemic obstruction, or neurological disease Of chitosan in the manufacture of a medicament for reducing nitric oxide production in a living subject.   Use according to claim 1, wherein the chitosan is partially acetylated.   Use according to claim 1, wherein the chitosan is fully acetylated to form chitin.   Use according to claim 3, wherein the chitosan comprises a polysaccharide consisting of 5 to 10 sugar residues.   Use according to claim 4, wherein the polysaccharide is a pentasaccharide residue in length.   Use according to claim 4, wherein the polysaccharide is 6 saccharide residues in length.   Use according to claim 1, wherein the chitosan comprises a polysaccharide consisting of 5 to 10 sugar residues.   8. Use according to claim 7, wherein the polysaccharide is a pentasaccharide residue in length.   8. Use according to claim 7, wherein the polysaccharide is hexasaccharide residues long.   Use according to claim 1, wherein the medicament is in the form of an implantable device. Comprising contacting the composition with a cell containing a sufficient amount of chitosan to reduce the production of nitric oxide in the cell, a method of in vitro reducing the production of nitric oxide in cells, The method, wherein the cells are RAW264.7 cells activated by IFN-γ and LPS .   12. The method of claim 11, wherein the chitosan is partially acetylated.   12. The method of claim 11, wherein the chitosan is fully acetylated to form chitin.   14. The method of claim 13, wherein the chitosan comprises a polysaccharide consisting of 5 to 10 sugar residues.   15. The method of claim 14, wherein the polysaccharide is a pentasaccharide residue long.   15. The method of claim 14, wherein the polysaccharide is a hexasaccharide residue long.   12. The method of claim 11, wherein the chitosan comprises a polysaccharide consisting of 5 to 10 sugar residues.