JP7233087B2 - antiarteriosclerotic - Google Patents

Description

The present invention relates to antiarteriosclerotic agents.

Arteriosclerosis is the terminal image of lifestyle-related diseases. By controlling risk factors such as diabetes, hypertension, dyslipidemia, chronic kidney disease, and smoking (Non-Patent Document 1), the probability of developing this disease can be controlled, or acute catastrophic medical conditions (myocardial infarction, brain infarction, severe lower extremity ischemia), make the most of medical resources (fire emergency system, acute hospital with equipment and personnel capable of performing acute endovascular treatment), and save lives. There is no way to suppress it. Risk control strategies lack certainty, and even in clinical trials, there is no other way than to judge efficacy based on relative risk reduction in population studies. Building a life-saving emergency system requires enormous costs in terms of personnel, time, and materials, and is not evenly distributed to all regions.

The cause of this disease is considered to be a high-risk condition due to lifestyle-related diseases, as well as pathological conditions derived from inflammation and immune response. According to studies including previous research by the inventors of the present application, lifestyle habits, especially dietary habits, and the chronicization and fixation of immune disorders and inflammation due to the antigenization of intestinal bacteria are one of the biological mechanisms of this disease. It is thought that The inventors of the present application have also conducted research using mouse arteriosclerosis onset models to date, demonstrating that the intestinal symbiotic microbiota promotes arteriosclerosis through the activation of the TLR signaling pathway in B2 cells. reported that depletion of B2 cells in the mouse body suppresses the onset of arteriosclerosis (Patent Documents 1 and 2, and Non-Patent Documents 2 and 3). Anti-CD23 antibody is expected as a positive therapeutic means for arteriosclerosis.

A disorder of the intestinal barrier, called leaky gut syndrome (LGS), is considered to be one of the pathologies that lead to the antigenization of intestinal bacteria. However, the relationship between impaired intestinal barrier and arteriosclerosis is completely unknown.

On the other hand, lubiprostone, a prostone derivative, is a compound that selectively activates the ClC-2 chloride channel. Improves constipation when the water content is low. Soft capsules of lubiprostone (trade name AMITIZA®) are already available in the United States, Switzerland, the United Kingdom, and Israel as a treatment for chronic idiopathic constipation. ) is marketed as a therapeutic drug for (Non-Patent Documents 4 and 5).

As a new use of lubiprostone, Patent Document 3 discloses an invention in which lubiprostone is used as an agent for preventing or improving renal dysfunction. However, the effect of lubiprostone on arteriosclerosis is completely unknown.

International Publication No. 2012/067165 International Publication No. 2018/034346 International Publication No. 2015/049876

Anderson KM. et al., Am Heart J, 1991;121:293-8 Ishigami T. et al., FASEB J, 2013;27:3437-3445 Chen L., Ishigami T., et al., EBioMedicine, 2016;13:237-247 Lacy BE, Levy LC. Clin Interv Aging. 2008; 3: 357-364. Pharmaceutical Interview Form, AMITIZA Capsules 12 μg/24 μg, December 2018 Revised 10th Edition

For the suppression and control of arteriosclerosis, the suppression and control of lifestyle-related diseases are regarded as important. Endovascular treatment using a catheter is nothing more than a stopgap, so to speak, symptomatic treatment for acute thrombosis, which is a cardiovascular event derived from arteriosclerosis. An object of the present invention is to provide a novel pharmacotherapeutic means effective against arteriosclerosis itself.

As a result of extensive research, the inventors of the present application have surprisingly found that lubiprostone has an arteriosclerosis-suppressing effect, and completed the present invention.

That is, the present invention provides an antiarteriosclerotic agent containing lubiprostone or a pharmaceutically acceptable salt thereof as an active ingredient.

INDUSTRIAL APPLICABILITY The present invention provides a novel means capable of suppressing progression of arteriosclerosis. Unlike lifestyle-related disease (risk) control and endovascular treatment (angioplasty) using a catheter, the present invention is more effective by improving and eliminating pathological conditions that cause chronic inflammation in arteriosclerosis. can effectively suppress arteriosclerosis. Lubiprostone has already been marketed and used as a therapeutic agent for constipation, and its safety has been guaranteed. Therefore, if its anti-arteriosclerotic effect is confirmed in humans, it can be applied clinically at an early stage. In addition, from the study of the mechanism leading to arteriosclerosis, which was clarified by the present invention, it is expected that elimination and control of LGS will become a new therapeutic target for arteriosclerosis, leading to drug discovery of new drugs. By identifying lifestyle habits, including dietary habits, that cause abnormalities in intestinal bacteria and intestinal walls, it will be possible to apply efforts to improve the health and longevity of the people, not limited to medical care. Become.

FIG. 1 shows the experimental design. ND; standard diet, WD; western diet, Mg; magnesium hydroxide, Sen; sennosides, Lub; lubiprostone, FITC-Dx; fluorescein isothiocyanate-dextran. The first 5 weeks of ND feeding was an acclimatization period. Inhibition of progression of arteriosclerotic lesions by lubiprostone in longitudinally dissected aorta. (A) Representative front view of the aorta from the aortic arch to the common iliac artery visualized by Oil Red O staining. The percentage of the lesion area to the total area of the aorta (%) and the size of the aortic lesion (mm ² ) analyzed with the Image J program are shown in (B) and (C), respectively. The data were expressed as the mean±standard error of the mean (SEM) for 5 animals in each group. Scale bar = 200 µm. ND; standard diet, WD; western diet, Mg; magnesium hydroxide, Sen; sennosides, Lub; lubiprostone. # p<0.001, * p=0.001, * p<0.01 compared to the 25-week WD group. Effect of lubiprostone in inhibiting atherosclerotic lesion progression quantified by cross-sectional analysis of the aortic root (aortic valve level). Representative hematoxylin-eosin staining images of aortic root sections of mice in the 25-week WD group and mice in the 25-week WD+lubiprostone group are shown in (A) and (B), respectively. Plaque size (μm ² ) and percentage of plaque to vessel area (%) analyzed with Image J software are shown in (C) and (D), respectively. (E) is the result of evaluating macrophage infiltration as F4/80-positive area (μm ² ). The data are expressed as the mean±SEM of 5 animals in each group. ND; standard diet, WD; western diet, Mg; magnesium hydroxide, Sen; sennosides, Lub; lubiprostone. * p<0.01, # p<0.05 compared to the 25-week WD group. Lubiprostone ameliorated intestinal barrier dysfunction and suppressed immunoglobulin production through a pathway independent of lipid metabolism. (A) Results of measurement of intestinal permeability in the body based on serum FITC-dextran concentration 1 hour after oral gavage of FITC-dextran. (B-C) ZO-1 and occludin mRNA levels in the ileum were measured by quantitative real-time PCR. (D-E) Total IgG and IgG3 concentrations measured by ELISA. Data are expressed as mean±SEM. (D) and (E) n=7, otherwise n=5. * p<0.01, # p<0.05 compared with 25-week WD group. (Continued from Figure 4-1) (F-H) Serum levels of low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c) and triglycerides (TG). n=5. *: p<0.01, #: p<0.05 compared to the 25-week WD group. Relative mRNA expression level of each gene in PVAT measured by quantitative real-time PCR and normalized to GAPDH. It was expressed as a relative ratio to the mRNA expression level of the WD group (25-week WD-fed group). IL1β; interleukin-1 beta, TNFα; tumor necrosis factor α. ND; 25-week ND group, WD; 25-week WD group, WD+Mg; 25-week WD + magnesium hydroxide administration group, WD + Sen; 25-week WD + sennoside administration group, WD + Lub; 25-week WD + lubiprostone administration group. Data are expressed as the mean±SEM of n=5 for each group. * p<0.01, # p<0.05 compared to the 25-week WD group. (continuation of FIG. 5-1) IL1r1; interleukin-1 receptor type 1, TLR5; toll-like receptor 5; ND; 25-week ND group, WD; 25-week WD group, WD+Mg; 25-week WD + magnesium hydroxide administration group, WD + Sen; 25-week WD + sennoside administration group, WD + Lub; 25-week WD + lubiprostone administration group. Data are expressed as the mean±SEM of n=5 for each group. * p<0.01, # p<0.05 compared to the 25-week WD group. Relative mRNA expression level of each gene in spleen measured by quantitative real-time PCR and normalized to GAPDH. It was expressed as a relative ratio to the mRNA expression level of the WD group (25-week WD-fed group). L1β; interleukin-1 beta, TNFα; tumor necrosis factor α. ND; 25-week ND group, WD; 25-week WD group, WD+Mg; 25-week WD + magnesium hydroxide administration group, WD + Sen; 25-week WD + sennoside administration group, WD + Lub; 25-week WD + lubiprostone administration group. Data are expressed as the mean±SEM of n=5 for each group. * p<0.01, # p<0.05 compared to the 25-week WD group. (continuation of FIG. 6-1) IL1r1; interleukin-1 receptor type 1, TLR5; toll-like receptor 5; ND; 25-week ND group, WD; 25-week WD group, WD+Mg; 25-week WD + magnesium hydroxide administration group, WD + Sen; 25-week WD + sennoside administration group, WD + Lub; 25-week WD + lubiprostone administration group. Data are expressed as the mean±SEM of n=5 for each group. * p<0.01, # p<0.05 compared to the 25-week WD group.

In the present invention, arteriosclerosis is typically atherosclerosis. The term atherosclerotic lesions includes atherosclerotic lesions as well as stenosis and infarction of blood vessels.

The term anti-arteriosclerosis includes treatment and prevention of arteriosclerosis, specifically, suppression of arteriosclerosis development, reduction of arteriosclerotic lesions, suppression of progression of arteriosclerosis, arteriosclerosis amelioration of symptoms is included. The antiarteriosclerotic agent of the present invention can be, for example, an agent that reduces the occurrence of arteriosclerotic lesions or suppresses the progression of arteriosclerosis. In addition, the arteriosclerotic agent of the present invention can be an agent that suppresses progression of arteriosclerosis.

The subject of administration of the antiarteriosclerotic agent of the present invention includes various mammals, typically humans. The anti-arteriosclerotic agent of the present invention is preferable for patients with arteriosclerosis, patients who desire reduction in the development of arteriosclerotic lesions or suppression of progression of arteriosclerosis, patients who desire improvement of arteriosclerosis, and the like. can be administered. For example, ischemic heart disease (angina pectoris, myocardial infarction), aortic aneurysm (thoracic/abdominal aortic aneurysm, iliac aneurysm, etc.), arteriosclerosis obliterans, aortic valve stenosis, cerebrovascular disease (cerebral infarction, stroke) ) are typical examples of patients for whom it is desirable to reduce the occurrence of arteriosclerotic lesions, inhibit the progression of arteriosclerosis, and improve arteriosclerosis. A patient is typically a human patient.

Lubiprostone ((-)-7-[(2R,4aR,5R,7aR)-2-(1,1-difluoropentyl)-2-hydroxy-6-oxooctahydrocyclopenta[b]pyran-) used in the present invention 5-yl]heptanoic acid) is a compound having the following structure.

Lubiprostone usually exists in the following tautomeric forms, and it is known that bicyclic lubiprostone is dominant over monocyclic forms (Patent No. 4786866, JP 2014-501698, etc.) ). Therefore, the antiarteriosclerotic agent of the present invention may be an agent containing an active ingredient in a form in which monocyclic tautomers coexist.

Pharmaceutically acceptable salts of lubiprostone include, for example, alkali metal salts (e.g. sodium and potassium salts), alkaline earth metal salts (e.g. calcium and magnesium salts), ammonium salts, with inorganic bases. salts; and amine salts such as methylamine salts, dimethylamine salts, cyclohexylamine salts, benzylamine salts, piperidine salts, ethylenediamine salts, ethanolamine salts, diethanolamine salts, triethanolamine salts, tris(hydroxymethylamino)ethane salts. , monomethyl-monoethanolamine salts, procaine and caffeine salts), basic amino acid salts (eg, arginine and lysine salts), tetraalkylammonium salts, and salts with organic bases. These salts can be prepared, for example, from the corresponding acids and bases by conventional reactions or by salt exchange.

Examples of ethers include alkyl ethers, for example lower alkyl ethers such as methyl ether, ethyl ether, propyl ether, isopropyl ether, butyl ether, isobutyl ether, t-butyl ether, pentyl ether and 1-cyclopropylethyl ether; and octyl middle or higher alkyl ethers such as ether, diethylhexyl ether, lauryl ether and cetyl ether; unsaturated ethers such as oleyl ether and linolenyl ether; lower alkenyl ethers such as vinyl ether, allyl ether; hydroxy (lower) alkyl ethers such as hydroxyethyl ether and hydroxyisopropyl ether; lower alkoxy (lower) alkyl ethers such as methoxymethyl ether and 1-methoxyethyl ether; phenyl ether, tosyl ether, t - optionally substituted aryl ethers such as butylphenyl ether, salicyl ether, 3,4-dimethoxyphenyl ether and benzamidophenyl ether; and aryl (lower) alkyl ethers such as benzyl ether, trityl ether and benzhydryl ether. mentioned.

Examples of esters include aliphatic esters, lower alkyl esters such as methyl ester, ethyl ester, propyl ester, isopropyl ester, butyl ester, isobutyl ester, t-butyl ester, pentyl ester and 1-cyclopropylethyl ester. lower alkenyl esters such as vinyl and allyl esters; lower alkynyl esters such as ethynyl and propynyl esters; hydroxy (lower) alkyl esters such as hydroxyethyl ester; methoxymethyl and 1-methoxyethyl esters. lower alkoxy (lower) alkyl esters; and optionally substituted aryl esters such as, for example, phenyl esters, tolyl esters, t-butylphenyl esters, salicyl esters, 3,4-dimethoxyphenyl esters and benzamidophenyl esters; and benzyl. Aryl (lower) alkyl esters such as esters, trityl esters and benzhydryl esters are included.

Amides of lubiprostone are such that the -COOH of lubiprostone is -CONR'R'' (wherein R' and R'' are each hydrogen, lower alkyl, aryl, alkyl- or aryl-sulfonyl, lower alkenyl and lower alkynyl, etc.). amide compounds represented by, for example, lower alkylamides such as methylamide, ethylamide, dimethylamide and diethylamide; arylamides such as anilides and toluidide; and methylsulfonylamides, ethylsulfonylamides and tolylsulfonylamides such as Alkyl or arylsulfonyl amides may be mentioned.

Lubiprostone itself is a well-known compound, and its synthesis method is also widely known (for example, JP-A-2007-211011, JP-A-2014-501698, etc.). In addition, soft capsules of lubiprostone under the trade name of AMITIZA are sold by Sucampo Pharmaceuticals, Inc. and Mylan EPD LLC. Lubiprostone or a pharmaceutically acceptable salt thereof used in the present invention can be produced by a known method, or a commercially available product may be used.

The administration route of the agent of the present invention is not particularly limited, and may be systemic administration or local administration, oral administration or parenteral administration (e.g., intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, transdermal administration, transdermal administration, Intestinal administration, nasal administration, etc.) may be used, but oral administration is preferred.

The dosage form of the agent of the present invention is also not particularly limited. , diluents, excipients, binders, lubricants, disintegrants, sweeteners, suspending agents, emulsifiers, coloring agents, flavoring agents, stabilizers, etc. . Formulations include oral agents such as tablets, hard capsules, soft capsules, granules, powders and syrups, and parenteral agents such as inhalants, injections, suppositories and liquid agents. Formulation methods and usable excipients are well known in the field of pharmaceutical formulations, and any method and excipients can be used.

Since lubiprostone is extremely soluble in ethanol and hardly soluble in water (Non-Patent Document 5), for example, when lubiprostone is prepared as a soft capsule, it is preferable to prepare a capsule-filling liquid using alcohol as a solvent. In addition, since it is known that the stability of lubiprostone is improved by glycerides (Japanese Patent No. 4332316), the capsule-filling liquid may contain suitable glycerides such as medium-chain fatty acid triglycerides.

The dosage of the agent of the present invention may be an amount that can treat or prevent arteriosclerosis, and can be appropriately selected according to the age, body weight, severity, etc. of the subject to be administered. Although not particularly limited, the dosage may be about 0.24 μg/kg body weight to 2.88 μg/kg body weight, for example, about 0.48 μg/kg body weight to 1.44 μg/kg body weight as the active ingredient amount per day to the subject. The daily dose may be administered once or divided into several doses. It can be administered daily or every few days or weeks.

EXAMPLES The present invention will be described in more detail below based on examples. However, the present invention is not limited to the following examples.

Methods Animals, Diet and Treatment The experimental design is shown in FIG. Five-week-old ApoE-/- mice were cloned from known mice on the C57BL/6J strain background (Lo Sasso et al., J Transl Med 2016, 14:146, DOI 10.1186/s12967-016-0901-1; Kapourchali et al., World J Clin Cases 2014 May 16, vol. 2, issue 5, p.126-132; Li et al., Protein Cell 2011, vol. 2, issue 3, p.189-201). Received from Ph.D. (Graduate School of Medicine, Yokohama City University). All mice used in the experiment survived the 30-week study period (5 weeks of acclimation, 25 weeks of experimental observation period). All mice were fed a standard chow diet (Normal diet; ND) and tap water ad libitum for 5 weeks before being randomly assigned to experimental and control groups. After this acclimation period the mice were grouped as follows. ND groups continued to feed ND for 15 or 25 weeks. After the acclimation period, the WD group was switched to a high-fat, high-cholesterol western diet (Oriental Yeast, Tokyo, Japan) containing 21.22% lipid (g/100 g), 17.01% protein, 48.48% carbohydrate, and 0.15% cholesterol. . After 15 weeks, mice in the WD group were further subdivided into a control group (continued on WD for 10 weeks until week 30, for a total of 25 weeks of WD) and a group that received WD feeding plus laxative treatment (lubiprostone (500 μg/ml)). kg), sennoside (25 mg/kg) or magnesium hydroxide (Mg(OH) ₂ ) (60 mg/kg), daily gavage for 10 weeks up to week 30). Mice in the ND and WD groups were sacrificed at week 20 for baseline measurements of atherosclerotic lesion progression and serum levels of lipid profile.

Measurement of intestinal permeability in vivo The in vivo permeability of the intestinal wall was quantified as the permeability of 4 kDa fluorescein isothiocyanate (FITC)-dextran (Sigma-Aldrich, St. Louis, MO, USA) as previously reported [1]. turned into Briefly, 27-week-old mice (with or without laxative treatment for 7 weeks) were fasted for 6 hours and given FITC-dextran (500 mg/kg) by oral gavage, followed by blood collection from the facial vein for serum samples. was prepared. Serum concentrations of FITC-dextran were measured with a fluorescence spectrophotometer (ARVO MX, PerkinElmer, Boston, Mass., USA) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. This enabled direct evaluation of changes in intestinal barrier function.

Quantification and histological analysis of arteriosclerosis
After 15 or 25 weeks of feeding with ND or WD, mice were sacrificed and hearts and aortas were harvested and analyzed by cross-sectional analysis of the aortic root and frontal analysis of the entire aorta (ascending aorta to common iliac artery). Lesion was quantified. After collection of perivascular adipose tissue (PVAT), aortas were fixed in 10% formalin, longitudinally dissected and stained for lipid deposits with Oil Red O solution (Sigma-Aldrich). Areas of aortic lesions were calculated using the Image J program (National Institutes of Health, Baltimore, MD, USA). The areas of all observed plaques were summed and expressed as a percentage of the total vessel area. Hearts were embedded in paraffin and serial sections of 5 μm thickness were cut from the beginning of the three aortic valve cusps to the ascending aorta and stained with hematoxylin-eosin. For immunohistochemistry, serial sections were stained with anti-F4/80 antibody (Abcam, Cambridge, UK), a specific marker for mature macrophages. The intensity of positively stained areas (F4/80 positive areas) was analyzed with Image J software.

Quantitative real-time PCR analysis Whole spleen, PVAT, and ileum were each homogenized in TRIzol Reagent (Cat.#00571510, Thermo Fisher Scientific) and total RNA was extracted using the RiboPure Kit (Cat.# AM1924, Life Technologies) according to the manufacturer's protocol. bottom. A total of 500 ng of RNA was reverse transcribed into cDNA using the High Capacity RNA-to-cDNA Kit (catalog number 4387406, Applied Biosystems) according to the manufacturer's protocol. The cDNA was then mixed with RT2 SYBR Green ROX qPCR Master mix (catalog number 1712516, Thermo Fisher Scientific) and real-time PCR was performed according to the manufacturer's instructions. Temperature cycling and fluorescence detection were performed using an ABI7500 real-time PCR machine (Applied Biosystems) according to the manufacturer's recommended conditions. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene, relative mRNA expression levels were determined by the 2[-ΔΔC(T)] method. Table 1 shows the sequences of the primers used for real-time PCR.

Quantitation of Plasma Parameters Whole blood samples were obtained via right ventricular puncture at the time of mouse sacrifice. Low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c) and triglyceride (TG) levels were measured by SRL Inc. (Tokyo, Japan). Total serum IgG was measured on diluted sera using the Mouse IgG ELISA Kit (Bethyl Laboratories, E99-131) according to the manufacturer's instructions. Serum IgG3 was measured using the Mouse IgG3 ELISA Kit (Bethyl Laboratories, E99-111). Data are expressed in micrograms per milliliter (μg/ml) based on a standard curve of isotype standards.

Ethics Statement All experiments in this study were performed in accordance with the animal care guidelines of Yokohama City University Graduate School of Medicine.

Statistical Analysis Continuous data were expressed as the mean±standard error of the mean (SEM). Differences between groups were analyzed A priori, analysis of variance (ANOVA) was performed, and tested post hoc. A p-value <0.05 was considered significant. All analyzes were performed using JMP 9.1 software (SAS Institute Inc., Cary, NC, USA).

Results 1. Oral administration of lubiprostone attenuates atherosclerotic progression As shown by oil red O staining of longitudinally dissected aortas, 15-week feeding of WD induced greater atherosclerotic lesion formation compared to ND. , this lesion formation increased with continued feeding of WD for an additional 10 weeks (Fig. 2). A 10-week administration of lubiprostone after a 15-week WD diet significantly inhibited atherosclerotic lesion progression by 69% (p<0.01) compared to the 25-week WD group. Histological examination of the aortic root confirmed a 26% reduction in atherosclerotic lesions (p<0.05, Figure 3). However, the area of aortic lesions in both longitudinally dissected aorta and aortic root areas was still greater in the lubiprostone-treated mice group than at baseline (15-week WD group). These results indicate that oral administration of lubiprostone attenuated WD-induced exacerbation of atherosclerotic lesion formation, but was insufficient to induce atherosclerotic plaque regression. Immunohistochemical analysis of aortic roots showed that lubiprostone significantly reduced macrophage infiltration into plaques examined by F4/80 staining (P<0.05, FIG. 3E).

2. Lubiprostone reverses WD-induced intestinal hyperpermeability In vivo intestinal permeability, determined by translocation of orally administered FITC-dextran to plasma components, was significantly higher in the WD group than in the ND group (Fig. 4A). . However, despite continued WD feeding, FITC-dextran translocation into plasma components was significantly reduced by lubiprostone administration.

We further examined the expression of tight junctions in the intestinal epithelium, which inhibit pathogen entry into the body by controlling intestinal permeability. Expression of tight junction protein-1 (zona occludens protein-1; ZO-1), an epithelial tight junction protein, was significantly decreased by WD feeding, but was upregulated by lubiprostone administration (Fig. 4B). Expression of another tight junction protein, occludin, was also elevated by lubiprostone administration (Fig. 4C). These results suggest that oral administration of lubiprostone may improve intestinal barrier function and attenuate the release of pathogens into circulation to prevent LGS-induced systemic inflammation.

3. Improving intestinal barrier function attenuates WD-induced FOB cell activation
B cells are known to exist in the spleen as well as in the PVAT and adventitial layers [2,3]. We previously reported that PVAT of WD-fed mice increased overt B2 cell infiltration and that B2 cell infiltration was an essential contributor to the induction of atherosclerosis (Non-Patent Document 3). To test the hypothesis that the effect of lubiprostone is mediated by inactivation of B2 cells, we analyzed B2 cell aggregation in PVAT.

The expression of B220 and CD23, surface markers of follicular B cells (FOB), was significantly reduced in the WD + laxative mice group compared to the WD alone group (p < 0.001, Fig. 5A, 5B). In the most significant difference between the laxative-treated groups, transcription of CD14, which encodes a surface antigen molecule required for B-cell function, was significantly down-regulated in lubiprostone-treated PVAT (Fig. 5C). Furthermore, genes encoding inflammation-activation-related molecules such as interleukin-1β (IL1β) and tumor necrosis factor-α (TNFα) were also significantly down-regulated only in the lubiprostone-treated group (Figs. 5D, 5E). These data suggest that the effects of PVAT on reducing the inflammatory phenotype and suppressing the progression of arteriosclerosis observed by lubiprostone administration are mediated by inactivation of the B2 cell Toll-like receptor (TLR) signaling pathway. strongly indicates The expression of macrophage surface marker F4/80, another factor contributing to inflammation, was also decreased in the laxative-treated group (Fig. 5F), but the rate of decrease was not as large as that of the FOB surface marker.

B2 cells in the spleen, the major B-cell reservoir, [4] were also examined, and there was no significant difference in B220 expression, but CD23 expression was significantly reduced in the laxative-treated spleen ( 6A, 6B). Although the presence of marginal zone B (MZB) cells in the splenic marginal zone enables rapid responses to blood-borne antigens [5], the presence of CD21, a surface marker for MZB cells, is The expression was most strongly suppressed in the lubiprostone-administered group (Fig. 6I).

A major function of activated B cells is antibody production. Therefore, we also examined blood IgG titers (Figs. 4D, 4E). Both the 15-week and 25-week WD-fed groups had higher IgG levels compared to their respective ND groups. The WD-fed mice group gavaged with Mg(OH) ₂ and sennosides had similar IgG levels to the WD-only group, but the lubiprostone-treated group had significantly lower total IgG titers than the WD-only group. (Fig. 4D). Moreover, blood levels of IgG3 [6,7], a subclass of IgG and produced primarily by MZB cells in a T cell-independent pathway in pre- or post-immune situations, were significantly reduced by lubiprostone administration. group and Mg(OH) ₂ administration group were significantly lower than the WD only group (p<0.05; FIG. 4E). These data also suggest that LGS-induced atherosclerosis is characterized by increased antibody production resulting from B2 cell activation, and are consistent with the real-time PCR analysis data.

4. The antiatherogenic effects of lubiprostone are mediated by a lipid-independent pathway
Serum levels of LDL-c and TG were significantly lower in the Mg(OH) ₂ -treated and sennoside-treated groups than in the WD group (FIGS. 4F, 4H). Serum levels of LDL-c and TG decreased slightly in the lubiprostone-treated group, and there was no significant difference. However, among the 3 groups (Mg(OH) ₂ , sennoside, lubiprostone) administered with laxatives, although lubiprostone was superior in its antiatherogenic effect, only the lubiprostone group showed a significant decrease in serum HDL-c level. seen (Fig. 4G). These data suggest that lipid metabolism may at least partially confer moderate antiatherogenic effects in mice treated with Mg(OH) ₂ or sennosides, whereas lubiprostone is independent of lipid metabolism. It suggests that the mechanism can suppress the progression of arteriosclerosis.

References
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Claims (2)

An antiarteriosclerotic agent containing lubiprostone or a pharmaceutically acceptable salt thereof as an active ingredient. 2. The anti-arteriosclerotic agent according to claim 1, which is an agent that reduces the occurrence of arteriosclerotic lesions or suppresses the progression of arteriosclerosis.

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