Pharmacological Profile of Difamilast, a Novel Selective Phosphodiesterase 4 Inhibitor, for Topical Treatment of Atopic Dermatitis.

PDE4 inhibitors are expected to be anti-inflammatory agents based on their mechanism of action, but the application of this drug class is limited by a narrow therapeutic window due to adverse effects associated with gastrointestinal function. Difamilast, a novel selective phosphodiesterase 4 (PDE4) inhibitor, demonstrated significant efficacy without adverse reactions such as nausea and diarrhea in patients with atopic dermatitis (AD) and was recently approved in Japan. In this study, we investigated the pharmacological and pharmacokinetic properties of difamilast to provide nonclinical data to help understand the clinical effects. Difamilast selectively inhibited recombinant human PDE4 activity in assays. The IC50 of difamilast against PDE4B, a PDE4 subtype that plays an important role in the inflammatory response, was 0.0112 µM, representing a 6.6-fold decrease compared with the IC50 against PDE4D (0.0738 µM), a subtype that can trigger emesis. Difamilast inhibited TNF-α production in human (IC50 = 0.0109 µM) and mouse (IC50 = 0.0035 µM) peripheral blood mononuclear cells and improved skin inflammation in a mouse model of chronic allergic contact dermatitis. These effects of difamilast on TNF-α production and dermatitis were superior to those of other topical PDE4 inhibitors: CP-80633, cipamfylline, and crisaborole. In pharmacokinetic studies using miniature pigs and rats, the concentrations of difamilast in the blood and brain after topical application were not sufficient to support pharmacological activity. This nonclinical study contributes to explain the efficacy and safety of difamilast with a sufficient therapeutic window in the clinical trials. SIGNIFICANCE STATEMENT: This is the first report on the nonclinical pharmacological profile of difamilast ointment, a novel topical PDE4 inhibitor that demonstrated utility in clinical trials in patients with atopic dermatitis. Difamilast, which has high PDE4 selectivity (especially for the PDE4B subtype), ameliorated chronic allergic contact dermatitis in mice after topical application, with a pharmacokinetic profile in animals that suggests few systemic side effects; thus, difamilast is a promising new therapeutic treatment for atopic dermatitis.

Discovery of N-{2-Methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}-N'-[2-(propane-2-sulfonyl)phenyl]-1,3,5-triazine-2,4-diamine (ASP3026), a Potent and Selective Anaplastic Lymphoma Kinase (ALK) Inhibitor.

Anaplastic lymphoma kinase (ALK) is a validated therapeutic target for treating echinoderm microtubule-associated protein-like 4 (EML4)-ALK positive non-small cell lung cancer (NSCLC). We synthesized a series of 1,3,5-triazine derivatives and identified ASP3026 (14a) as a potent and selective ALK inhibitor. In mice xenografted with NCI-H2228 cells expressing EML4-ALK, once-daily oral administration of 14a demonstrated dose-dependent antitumor activity. Here, syntheses and structure-activity relationship (SAR) studies of 1,3,5-triazine derivatives are described.

Three-dimensional Navigation for Thoracoscopic Sublobar Resection Using a Novel Wireless Marking System.

We developed a novel localization technique for small intrapulmonary lesions using radiofrequency identification (RFID) technology. Micro-RFID markers with nickel-titanium coils were designed to be placed from subsegmental bronchi to the peripheral parenchyma. In this preclinical study, thoracoscopic subsegmentectomy of a canine pseudotumor model was performed to demonstrate the feasibility and three-dimensional positional accuracy of the system. To recover subcentimeter pseudotumors, markers were bronchoscopically placed to determine the resection line: (1) next to the pseudotumor; (2) in the responsible subsegmental bronchi as the central margin; and (3) on the intersubsegmental plane as the lateral margin. Specific marker positions were located by wireless communication using a wand-shaped probe with a 30-mm communication range, with the distance to the marker indicated by gradual changes in sound pitch. Thirty-four markers were placed for 10 pseudotumors (14.6 mm from the pleura) in 10 canines. Three markers were placed at a mean distance of 5.5 mm from the pseudotumors, and 11 central and 20 lateral markers were placed at mean distances of 17.2 and 20.7 mm from the pseudotumors, respectively. Central markers (20.5 mm from the pleura) were detected within 16.0 seconds in 2.9-mm-diameter bronchi. All resection stumps were within 5.4 mm (range 2-8 mm) from each marker, and pseudotumors were removed with adequate surgical margins toward the central (11.5 mm; range 7-16 mm) and lateral (12.4 mm; range 9-17 mm) directions. RFID wireless markers provided precise three-dimensional positional information and are a potential viable alternative to conventional markers.

Pterosin B prevents chondrocyte hypertrophy and osteoarthritis in mice by inhibiting Sik3.

Osteoarthritis is a common debilitating joint disorder. Risk factors for osteoarthritis include age, which is associated with thinning of articular cartilage. Here we generate chondrocyte-specific salt-inducible kinase 3 (Sik3) conditional knockout mice that are resistant to osteoarthritis with thickened articular cartilage owing to a larger chondrocyte population. We also identify an edible Pteridium aquilinum compound, pterosin B, as a Sik3 pathway inhibitor. We show that either Sik3 deletion or intraarticular injection of mice with pterosin B inhibits chondrocyte hypertrophy and protects cartilage from osteoarthritis. Collectively, our results suggest Sik3 regulates the homeostasis of articular cartilage and is a target for the treatment of osteoarthritis, with pterosin B as a candidate therapeutic.

Characterization and Thermodynamic Stability of Polymorphs of Di(arylamino) Aryl Compound ASP3026.

ASP3026 (N-{2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}-N'-[2-(propane-2-sulfonyl)phenyl]-1,3,5-triazine-2,4-diamine) was developed in Astellas Pharma Inc. as a novel and selective inhibitor of the fusion protein EML4-ALK. We investigated the thermodynamic stability of five polymorphs of ASP3026 (A01, A02, A03, A04, and A05) in detail. To determine the most stable form at ambient temperature, powder X-ray diffraction, differential scanning calorimetry, and solubility measurements were conducted. Of the five polymorphs, A04 was the most stable and A05 was the least stable. The relationship between A04 and A03 and A04 and A01 were mutually monotropic, while that between A01 and A02 was enantiotropic. The transition temperature from A02 to A01 was estimated as 325 K. A02 was more thermodynamically stable at ambient temperature than A01. Furthermore, the method to estimate polymorphic transition temperatures using solution calorimetry was found to be effective. The systematic characterization of ASP3026 polymorphs presented in this study enables the selective crystallization of the most stable form and design of solid formulations.

Generation of scaffoldless hyaline cartilaginous tissue from human iPSCs.

Defects in articular cartilage ultimately result in loss of joint function. Repairing cartilage defects requires cell sources. We developed an approach to generate scaffoldless hyaline cartilage from human induced pluripotent stem cells (hiPSCs). We initially generated an hiPSC line that specifically expressed GFP in cartilage when teratoma was formed. We optimized the culture conditions and found BMP2, transforming growth factor ß1 (TGF-ß1), and GDF5 critical for GFP expression and thus chondrogenic differentiation of the hiPSCs. The subsequent use of scaffoldless suspension culture contributed to purification, producing homogenous cartilaginous particles. Subcutaneous transplantation of the hiPSC-derived particles generated hyaline cartilage that expressed type II collagen, but not type I collagen, in immunodeficiency mice. Transplantation of the particles into joint surface defects in immunodeficiency rats and immunosuppressed mini-pigs indicated that neocartilage survived and had potential for integration into native cartilage. The immunodeficiency mice and rats suffered from neither tumors nor ectopic tissue formation. The hiPSC-derived cartilaginous particles constitute a viable cell source for regenerating cartilage defects.

iPS cell technologies and cartilage regeneration.

Articular cartilage covers the ends of bone and provides shock absorption and lubrication to the diarthrodial joints. Cartilage has a limited capacity for repair when injured, and there is a need for cell sources for chondrocytes that can be transplanted as part of a regenerative medicine approach. Induced pluripotent stem cells (iPSCs) have pluripotency and the potential for self-renewal similar to embryonic stem cells (ESCs), but are not associated with the ethical issues that have plagued ESCs. Recent progress has made it possible to generate integration-free iPSCs and to differentiate iPSCs toward chondrocytes. An iPSC library prepared from donors homozygous for common HLA types is being developed, and will be able to provide allogeneic iPSC-derived chondrocytes at low cost that can cover the majority of the population. As an alternative approach, chondrocytic cells can be induced directly from dermal fibroblasts without going through the iPSC stage. Another important application of the iPSC technology is modeling cartilage diseases, such as skeletal dysplasia. Chondrogenically differentiated iPSCs generated from patients would recapitulate the pathology, and may serve as a useful platform both for exploring the disease mechanisms and for drug screening. This article is part of a Special Issue entitled "Stem Cells and Bone".

Modeling type II collagenopathy skeletal dysplasia by directed conversion and induced pluripotent stem cells.

Type II collagen is a major component of cartilage. Heterozygous mutations in the type II collagen gene (COL2A1) result in a group of skeletal dysplasias known as Type II collagenopathy (COL2pathy). The understanding of COL2pathy is limited by difficulties in obtaining live chondrocytes. In the present study, we converted COL2pathy patients' fibroblasts directly into induced chondrogenic (iChon) cells. The COL2pathy-iChon cells showed suppressed expression of COL2A1 and significant apoptosis. A distended endoplasmic reticulum (ER) was detected, thus suggesting the adaptation of gene expression and cell death caused by excess ER stress. Chondrogenic supplementation adversely affected the chondrogenesis due to forced elevation of COL2A1 expression, suggesting that the application of chondrogenic drugs would worsen the disease condition. The application of a chemical chaperone increased the secretion of type II collagen, and partially rescued COL2pathy-iChon cells from apoptosis, suggesting that molecular chaperons serve as therapeutic drug candidates. We next generated induced pluripotent stem cells from COL2pathy fibroblasts. Chondrogenically differentiated COL2pathy-iPS cells showed apoptosis and increased expression of ER stress-markers. Finally, we generated teratomas by transplanting COL2pathy iPS cells into immunodeficient mice. The cartilage in the teratomas showed accumulation of type II collagen within cells, a distended ER, and sparse matrix, recapitulating the patient's cartilage. These COL2pathy models will be useful for pathophysiological studies and drug screening.

Synthesis and pharmacological evaluation of optically pure, novel carbonyl guanidine derivatives as dual 5-HT2B and 5-HT7 receptor antagonists.

A series of 9-disubstituted N-(9H-fluorene-2-carbonyl)guanidine derivatives have been discovered as potent and orally active dual 5-HT(2B) and 5-HT(7) receptor antagonists. Upon screening several compounds, N-(diaminomethylene)-4',5'-dihydro-3'H-spiro[fluorene-9,2'-furan]-2-carboxamide (17) exhibited potent affinity for both 5-HT(2B) (Ki = 5.1 nM) and 5-HT(7) (K(i) = 1.7 nM) receptors with high selectivity over 5-HT(2A), 5-HT(2C), α(1), D(2) and M(1) receptors. Optical resolution of the intermediate carboxylic acid 16 via the formation of diastereomeric salts using chiral alkaloids gave the optically pure compounds (R)-17 and (S)-17. Both enantiomers suppressed 5-HT-induced dural protein extravasation in guinea pigs in a dose-dependent manner and the amount of leaked protein was suppressed to near normal levels when orally administrated at 10 mg/kg. (R)-17 and (S)-17 were therefore selected as candidates for human clinical trials.

Statin treatment rescues FGFR3 skeletal dysplasia phenotypes.

Gain-of-function mutations in the fibroblast growth factor receptor 3 gene (FGFR3) result in skeletal dysplasias, such as thanatophoric dysplasia and achondroplasia (ACH). The lack of disease models using human cells has hampered the identification of a clinically effective treatment for these diseases. Here we show that statin treatment can rescue patient-specific induced pluripotent stem cell (iPSC) models and a mouse model of FGFR3 skeletal dysplasia. We converted fibroblasts from thanatophoric dysplasia type I (TD1) and ACH patients into iPSCs. The chondrogenic differentiation of TD1 iPSCs and ACH iPSCs resulted in the formation of degraded cartilage. We found that statins could correct the degraded cartilage in both chondrogenically differentiated TD1 and ACH iPSCs. Treatment of ACH model mice with statin led to a significant recovery of bone growth. These results suggest that statins could represent a medical treatment for infants and children with TD1 and ACH.

Synthesis and structure-activity relationships of new carbonyl guanidine derivatives as novel dual 5-HT2B and 5-HT7 receptor antagonists. Part 2.

We previously reported that the novel dual 5-HT2B and 5-HT7 receptor antagonist N-(9-hydroxy-9H-fluorene-2-carbonyl)guanidine (4) exerted a suppressing effect on 5-HT-induced dural protein extravasation in guinea pigs. To develop a synthetic strategy, we performed docking studies of lead compound 4 bound to 5-HT2B and 5-HT7 receptors, and observed that the carbonyl guanidine group forms a tight interaction network with an active center Asp (D135:5-HT2B, D162:5-HT7), Tyr (Y370:5-HT2B, Y374:5-HT7) and aromatic residue (W131:5-HT2B, F158:5-HT7). Based on molecular modeling results, we optimized the substituents at the 5- to 8-position and 9-position of the fluorene ring and identified N-(diaminomethylene)-9-hydroxy-9-methyl-9H-fluorene-2-carboxamide (24a) exhibits potent affinity for 5-HT2B (Ki=4.3 nM) and 5-HT7 receptor (Ki=4.3 nM) with high selectivity over 5-HT2A, 5-HT2C, α1, D2 and M1 receptors. Compound 24a reversed the hypothermic effect of 5-carboxamidotryptamine (5-CT) in mice and also showed a suppressing effect on 5-HT-induced dural protein extravasation in guinea pigs when orally administered at 30 mg/kg. Compound 24a is therefore a promising candidate for a novel class of anti-migraine agent without any adverse effects.

Solid-state emission enhancement in vaulted trans-bis(salicylaldiminato)platinum(II) crystals with halogen functionality.

The synthesis, structure, and solid-state emission of vaulted trans-bis(salicylaldiminato)platinum(ii) complexes 1-3 with halogen functionalities are described and compared with the non-substituted analogues. Chloro-substitution provided an improvement of the low emission properties of short-vaulted, non-substituted complexes 1 and 2 in the crystalline state at ambient temperature, while the intense emission of long-vaulted analogues 3 remained unchanged. Bromo-substituted crystals also emit intensively, while the fluoro analogue is non-emissive under the same conditions. Temperature-dependent emission spectra indicate that all chloro- and bromo-substituted crystals with enhanced emission properties at ambient temperature exhibit improved heat resistance properties towards emission decay with the halogen functionalities. X-ray diffraction studies revealed that such a positive effect of halogenation for the enhancement of solid-state emission is due to significant molecular constraints in the crystals by a combination of the vaulted structure and three-dimensional HX hydrogen bonding interactions.

Development of a novel marking system for laparoscopic gastrectomy using endoclips with radio frequency identification tags: feasibility study in a canine model.

BACKGROUND: Intraoperative identification of early gastric cancer is difficult to conduct during laparoscopic procedures. In this study, we investigated the feasibility and accuracy of a newly developed marking system using endoclips with radio frequency identification (RFID) tags in a canine model. METHODS: RFID is a wireless near field communication technology. Among the open frequency bands available for medical use, 13.56 MHz is suitable for a surgical marking system because of the similar and linear signal decay both in air and in biological tissues. The proposed system consists of four parts: (a) endoclips with RFID tags, (b) endo-clip applier equipment, (c) laparoscopic locating probe, and (d) signal processing units with audio interface. In the experimental setting using canine models, RFID-tagged endoclips were applied to the mucosa of each dog's stomach. During the subsequent operation, the clips with RFID tags placed in five dogs were located by the detection of the RFID signal from the tag (RFID group), and the conventional clips in the other six dogs were located by finger palpation (FP group). The detected sites were marked by ablation on the serosal surface. Distance between the clips and the metal pin needles indicating ablated sites were measured with X-ray radiographs of the resected specimen. RESULTS: All clips were successfully detected by the marking system in the RFID group (10/10) and by finger palpation in the FP group (17/17). The medians of detection times were 31.5 and 25.0 s, respectively; the distances were 5.63 and 7.62 mm, respectively. The differences were not statistically significant. No adverse event related to the procedures was observed. CONCLUSIONS: Endoclips with RFID tags were located by our novel marking system in an experimental laparoscopic setting using canine stomachs with substantial accuracy comparable to conventional endoclips located by finger palpation through an open approach.

A novel surgical marking system for small peripheral lung nodules based on radio frequency identification technology: Feasibility study in a canine model.

OBJECTIVE: We investigated the feasibility and accuracy of a novel surgical marking system based on radiofrequency identification (RFID) technology for the localization of small peripheral lung nodules (SPLNs) in a canine model. METHODS: The system consists of 4 components: (1) micro RFID tags (13.56 MHz, 1.0 × 1.0 × 0.8 mm), (2) a tag delivery system with a bronchoscope, (3) a wand-shaped locating probe (10-mm diameter), and (4) a signal processing unit with audio interface. Before the operation, pseudolesions mimicking SPLNs were prepared in 7 dogs by injecting colored collagen. By use of a computed tomographic (CT) guide, an RFID tag was placed via a bronchoscope close to each target lesion. This was then followed by scanning with the locating probe, and wedge resection was performed when possible. Operators can locate the tag by following the sound emitted by the system, which exhibits tone changes according to the tag-probe distance. The primary outcome measure was the rate of wedge resection with good margins. RESULTS: A total of 10 pseudolesions imitating SPLNs were selected as targets. During thoracoscopic procedures, 9 of 10 tags were detected by the system within a median of 27 seconds. Wedge resections were performed for these 9 lesions with a median margin of 11 mm. The single failure was caused by tag dislocation to the central airway. CONCLUSIONS: Successful localization and wedge resection of pseudolesions with appropriate margins were accomplished in an experimental setting. Our RFID marking system has future applications for accurately locating SPLNs in a clinical setting.

Synthesis and structure-activity relationships of new carbonyl guanidine derivatives as novel dual 5-HT2B and 5-HT7 receptor antagonists.

To identify potent dual 5-HT2B and 5-HT7 receptor antagonists, we synthesized a series of novel carbonyl guanidine derivatives and examined their structure-activity relationships. Among these compounds, N-(9-hydroxy-9H-fluorene-2-carbonyl)guanidine (10) had a good in vitro profile, that is, potent affinity for human 5-HT2B and 5-HT7 receptor subtypes (Ki=1.8 nM and Ki=17.6 nM, respectively) and high selectivity over 5-HT2A, 5-HT2C, α1, D2 and M1 receptors. Compound 10 also showed a suppressing effect on 5-HT-induced dural protein extravasation in guinea pigs when orally administered.

Direct induction of chondrogenic cells from human dermal fibroblast culture by defined factors.

The repair of large cartilage defects with hyaline cartilage continues to be a challenging clinical issue. We recently reported that the forced expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) can induce chondrogenic cells from mouse dermal fibroblast culture without going through a pluripotent state. We here generated induced chondrogenic (iChon) cells from human dermal fibroblast (HDF) culture with the same factors. We developed a chondrocyte-specific COL11A2 promoter/enhancer lentiviral reporter vector to select iChon cells. The human iChon cells expressed marker genes for chondrocytes but not fibroblasts, and were derived from non-chondrogenic COL11A2-negative cells. The human iChon cells formed cartilage but not tumors in nude mice. This approach could lead to the preparation of cartilage directly from skin in human, without going through pluripotent stem cells.

Discovery of 2-methyl-1-{1-[(5-methyl-1H-indol-2-yl)carbonyl]piperidin-4-yl}propan-2-ol: a novel, potent and selective type 5 17ß-hydroxysteroid dehydrogenase inhibitor.

Type 5 17ß-hydroxysteroid dehydrogenase (17ß-HSD5), also known as aldo-keto reductase 1C3 (AKR1C3), is a member of the aldo-keto reductase superfamily of enzymes and is expressed in the human prostate. One of the main functions of 17ß-HSD5 is to catalyze the conversion of the weak androgen, androstenedione, to the potent androgen, testosterone. The concentration of intraprostatic 5α-dihydrotestosterone (DHT) in patients following chemical or surgical castration has been reported to remain as high as 39% of that of healthy men, with 17ß-HSD5 shown to be involved in this androgen synthesis. Inhibition of 17ß-HSD5 therefore represents a promising target for the treatment of castration-resistant prostate cancer (CRPC). To investigate this, we conducted high-throughput screening (HTS) and identified compound 2, which displayed a structure distinct from known 17ß-HSD5 inhibitors. To optimize the inhibitory activity of compound 2, we first introduced a primary alcohol group. We then converted the primary alcohol group to a tertiary alcohol, which further enhanced the inhibitory activity, improved metabolic stability, and led to the identification of compound 17. Oral administration of compound 17 to castrated nude mice bearing the CWR22R xenograft resulted in the suppression of androstenedione (AD)-induced intratumoral testosterone production. Compound 17 also demonstrated good isoform selectivity, minimal inhibitory activity against either CYP or hERG, and enhanced pharmacokinetic and physicochemical properties.

Vaulted trans-bis(salicylaldiminato)platinum(II) crystals: heat-resistant, chromatically sensitive platforms for solid-state phosphorescence at ambient temperature.

The synthesis, structure, and solid-state emission of vaulted trans-bis(salicylaldiminato)platinum(II) complexes are described. A series of polymethylene (1: n=8; 2: n=9; 3: n=10; 4: n=11; 5: n=12; 6: n=13) and polyoxyethylene (7: m=2; 8: m=3; 9: m=4) vaulted complexes (R=H (a), 3-MeO (b), 4-MeO (c), 5-MeO (d), 6-MeO (e), 4-CF3O (f), 5-CF3O (g)) was prepared by treating [PtCl2(CH3CN)2] with the corresponding N,N'-bis(salicylidene)-1,ω-alkanediamines. The trans coordination, vaulted structures, and the crystal packing of 1-9 have been unequivocally established from X-ray diffraction studies. Unpredictable, structure-dependent phosphorescent emission has been observed for crystals of the complexes under UV excitation at ambient temperature, whereas these complexes are entirely nonemissive in the solution state under the same conditions. The long-linked complex crystals 4-6, 8, and 9 exhibit intense emission (Φ77K =0.22-0.88) at 77 K, whereas short-linked complexes 1-3 and 7 are non- or slightly emissive at the same temperature (Φ77K <0.01-0.18). At 298 K, some of the long-linked crystals, 4a, 4b, 5c, 5e, 6c, 6e, and 9b, completely lose their high-emission properties with elevation of the temperature (Φ298K <0.01-0.02), whereas the other long-linked crystals, 5a, 6a, 9a, and 9d, exhibit high heat resistance towards emission decay with increasing temperature (Φ298K =0.21-0.38). Chromogenic control of solid-state emission over the range of 98 nm can be performed simply by introducing MeO groups at different positions on the aromatic rings. Orange, yellow-green, red, and yellow emissions are observed in the glass and crystalline state upon 3-, 4-, 5-, and 6-MeO substitution, respectively, whereas those with CF3 O substituents have orange emission, irrespective of the substitution position. DFT calculations (B3LYP/6-31G*, LanL2DZ) showed that such chromatic variation is ascribed to the position-specific influence of the substituents on the highest-occupied molecular orbital (HOMO) and lowest-unoccupied molecular orbital (LUMO) levels of the trans-bis(salicylaldiminato)platinum(II) platform. The solid-state emission and its heat resistance have been discussed on the basis of X-ray diffraction studies. The planarity of the trans-coordination sites is strongly correlated to the solid-state emission intensities of crystals 1-9 at lower temperatures. The specific heat-resistance properties shown exclusively by the 5a, 6a, 9a, and 9d crystals are due to their strong three-dimensional hydrogen-bonding interactions and/or Pt···Pt contacts, whereas heat-quenchable crystals 4a, 4b, 5c, 5e, 6c, 6e, and 9b are poorly bound with limited interactions, such as non-, one-, or two-dimensional hydrogen-bonding networks. These results lead to the conclusion that Pt···Pt contacts are an important factor in the heat resistance of solid-state phosphorescence at ambient temperature, although the role of Pt···Pt contacts can be substituted by only higher-ordered hydrogen-bonding fixation.

Reduction of visceral fat correlates with the decrease in the number of obesity-related cardiovascular risk factors in Japanese with Abdominal Obesity (VACATION-J Study).

AIM: Visceral fat accumulation is associated with obesity-related cardiovascular risk factor accumulation and atherosclerosis. The present study investigated whether one-year reduction of the visceral fat area (VFA) correlates with a decrease in the number of such factors in Japanese with or without visceral fat accumulation. METHODS: The study subjects comprised 5,347 Japanese, who underwent health check-ups in 2007 and 2008, including measurements of VFA and subcutaneous fat area (SFA) by computed tomography at 9 centers in Japan. Subjects with one or more such factor(s) were categorized into tertiles based on the one-year change in VFA. We investigated the multivariate age, sex, and one-year change in SFA-adjusted odds ratios (ORs) and 95% confidence intervals (CI) for reductions in the number of risk factors in each of the three categories based on the one-year change in VFA, in subjects with one or more such factors (n= 3,648). RESULTS: In the entire group (n=3,648), the OR and 95%CI for reductions in the number of risk factors in the first tertile were 0.804 (0.673-0.962, p=0.0172), compared with the second tertile set at 1.0. Subjects with VFA <100cm(2) showed no reduction in the number of risk factors. In subjects with VFA≥100 cm(2), OR in the first tertile was 0.788 (0.639-0.972, p=0.0257) relative to the second tertile set at 1.0. CONCLUSIONS: In subjects with multiple cardiovascular risk factors, visceral fat reduction correlated with a decrease in the number of such factors in subjects with VFA≥100cm(2), but not in those with VFA<100cm(2).