Genotoxicity evaluation of self-assembled-micelle inhibitory RNA-targeting amphiregulin (SAMiRNA-AREG), a novel siRNA nanoparticle for the treatment of fibrotic disease.

The self-assembled-micelle inhibitory RNA-targeting amphiregulin (SAMiRNA-AREG) is a novel small-interfering RNA (siRNA) nanoparticle that is used for treatment of pulmonary fibrosis. We investigated the potential genotoxicity of SAMiRNA-AREG based on the guidelines published by the Organization for Economic Cooperation and Development. In the bacterial reverse mutation assay (Ames test), SAMiRNA-AREG did not induce mutations in Salmonella typhimurium TA100, TA1535, TA98, and TA1537 and Escherichia coli WP2uvrA at concentrations of up to 3000 µg/plate with or without metabolic activation. The SAMiRNA-AREG (concentrations up to 500 µg/mL) did not induce chromosomal aberrations in cultured Chinese hamster lung cells with or without metabolic activation. In the in vivo mouse bone marrow micronucleus assay, the SAMiRNA-AREG (concentrations up to 300 mg/kg body weight) did not affect the proportions of polychromatic erythrocytes and total erythrocytes, nor did it increase the number of micronucleated polychromatic erythrocytes in ICR mice. Collectively, these results suggest that SAMiRNA-AREG is safe with regard to genotoxicity such as mutagenesis or clastogenesis under the present experimental conditions. These results might support the safety of SAMiRNA-AREG as a potential therapeutic agent for pharmaceutical development.

Four-Week Repeated Intravenous Dose Toxicity of Self-Assembled-Micelle Inhibitory RNA-Targeting Amphiregulin in Mice.

The present study investigated the potential subchronic toxicity of self-assembled-micelle inhibitory RNA-targeting amphiregulin (SAMiRNA-AREG) in mice. The test reagent was administered once-daily by intravenous injection for 4 weeks at 0, 100, 200, or 300 mg/kg/day doses. Additional recovery groups (vehicle control and high dose groups) were observed for a 2-week recovery period. During the test period, mortality, clinical signs, body weight, food consumption, ophthalmology, urinalysis, hematology, serum biochemistry, gross pathology, organ weight, and histopathology were examined. An increase in the percentages of basophil and large unstained cells was observed in the 200 and 300 mg/kg/day groups of both sexes. In addition, the absolute and relative weights of the spleen were higher in males given 300 mg/kg/day relative to the concurrent controls. However, these findings were considered of no toxicological significance because the changes were minimal, were not accompanied by other relevant results (eg, correlating microscopic changes), and were not observed at the end of the 2-week recovery period indicating recovery of the findings. Based on the results, SAMiRNA-AREG did not cause treatment-related adverse effects at dose levels of up to 300 mg/kg/day in mice after 4-week repeated intravenous doses. Under these conditions, the no-observed-adverse-effect level of the SAMiRNA-AREG was ≥300 mg/kg/day in both sexes and no target organs were identified.

Self-assembled Micelle Interfering RNA for Effective and Safe Targeting of Dysregulated Genes in Pulmonary Fibrosis.

The siRNA silencing approach has long been used as a method to regulate the expression of specific target genes in vitro and in vivo. However, the effectiveness of delivery and the nonspecific immune-stimulatory function of siRNA are the limiting factors for therapeutic applications of siRNAs. To overcome these limitations, we developed self-assembled micelle inhibitory RNA (SAMiRNA) nanoparticles made of individually biconjugated siRNAs with a hydrophilic polymer and lipid on their ends and characterized their stability, immune-stimulatory function, and in vivo silencing efficacy. SAMiRNAs form very stable nanoparticles with no significant degradation in size distribution and polydispersity index over 1 year. Overnight incubation of SAMiRNAs (3 µm) on murine peripheral blood mononuclear cells did not cause any significant elaboration of innate immune cytokines such as TNF-α, IL-12, or IL-6, whereas unmodified siRNAs or liposomes or liposome complexes significantly stimulated the expression of these cytokines. Last, the in vivo silencing efficacy of SAMiRNAs was evaluated by targeting amphiregulin and connective tissue growth factor in bleomycin or TGF-ß transgenic animal models of pulmonary fibrosis. Intratracheal or intravenous delivery two or three times of amphiregulin or connective tissue growth factor SAMiRNAs significantly reduced the bleomycin- or TGF-ß-stimulated collagen accumulation in the lung and substantially restored the lung function of TGF-ß transgenic mice. This study demonstrates that SAMiRNA nanoparticle is a less toxic, stable siRNA silencing platform for efficient in vivo targeting of genes implicated in the pathogenesis of pulmonary fibrosis.

SAMiRNA Targeting Amphiregulin Alleviate Total-Body-Irradiation-Induced Renal Fibrosis.

Fibrosis is a serious unintended side effect of radiation therapy. In this study, we aimed to investigate whether amphiregulin (AREG) plays a critical role in fibrosis development after total-body irradiation (TBI). We found that the expression of AREG and fibrotic markers, such as α-smooth muscle actin (α-SMA) and collagen type I alpha 1 (COL1α1), was elevated in the kidneys of 6 Gy TBI mice. Expression of AREG and α-SMA was mainly elevated in the proximal and distal tubules of the kidney in response to TBI, which was confirmed by immunofluorescence staining. Knockdown of Areg mRNA using self-assembled-micelle inhibitory RNA (SAMiRNA) significantly reduced the expression of fibrotic markers, including α-SMA and COL1α1, and inflammatory regulators. Finally, intravenous injections of SAMiRNA targeting mouse Areg mRNA (SAMiRNA-mAREG) diminished radiation-induced collagen accumulation in the renal cortex and medulla. Taken together, the results of the present study suggest that blocking of AREG signaling via SAMiRNA-mAREG treatment could be a promising therapeutic approach to alleviate radiation-induced kidney fibrosis.

Weekly treatment with SAMiRNA targeting the androgen receptor ameliorates androgenetic alopecia.

Androgenetic alopecia (AGA) is the most common type of hair loss in men and women. Dihydrotestosterone (DHT) and androgen receptor (AR) levels are increased in patients with AGA, and DHT-AR signaling correlates strongly with AGA pathogenesis. In this study, treatment with self-assembled micelle inhibitory RNA (SAMiRNA) nanoparticle-type siRNA selectively suppressed AR expression in vitro. Clinical studies with application of SAMiRNA to the scalp and massaging to deliver it to the hair follicle confirmed its efficacy in AGA. For identification of a potent SAMiRNA for AR silencing, 547 SAMiRNA candidates were synthesized and screened. SAMiRNA-AR68 (AR68) was the most potent and could be efficiently delivered to human follicle dermal papilla cells (HFDPCs) and hair follicles, and this treatment decreased the AR mRNA and protein levels. We confirmed that 10 µM AR68 elicits no innate immune response in human PBMCs and no cytotoxicity up to 20 µM with HFDP and HaCaT cells. Clinical studies were performed in a randomized and double-blind manner with two different doses and frequencies. In the low-dose (0.5 mg/ml) clinical study, AR68 was applied three times per week for 24 weeks, and through quantitative analysis using a phototrichogram, we confirmed increases in total hair counts. In the high-dose (5 mg/ml) clinical study, AR68 was given once per week for 24 weeks and showed 83% efficacy in increasing hair counts compared with finasteride. No side effects were observed. Therefore, SAMiRNA targeting AR mRNA is a potential novel topical treatment for AGA.

The Effect of Lactobacillus gasseri BNR17 on Postmenopausal Symptoms in Ovariectomized Rats.

Clinical and preclinical studies have reported that Lactobacillus gasseri BNR17, a probiotic bacterial strain isolated from human breast milk, reduces body weight and white adipose tissue volume. In order to further explore the actions of L. gasseri BNR17, we investigated the anti-menopausal effects of L. gasseri BNR17 in an ovariectomized (OVX) rat model. The serum alanine aminotransferase levels of the rats in the OVX-BNR17 group were lower than those of the rats in the OVX-vehicle only (OVX-Veh) group. Upon administration of L. gasseri BNR17 after ovariectomy, calcitonin and Serotonin 2A levels increased significantly, whereas serum osteocalcin levels showed a decreasing tendency. Compared to the rats in the OVX-Veh group, those in the OVX-BNR17 group showed lower urine deoxypyridinoline levels, lower pain sensitivity, and improved vaginal cornification. Furthermore, L. gasseri BNR17 administration increased bone mineral density in the rats with OVX-induced femoral bone loss. These results suggest that L. gasseri BNR17 administration could alleviate menopausal symptoms, indicating that this bacterium could be a good functional probiotic for managing the health of older women.

In vivo silencing of amphiregulin by a novel effective Self-Assembled-Micelle inhibitory RNA ameliorates renal fibrosis via inhibition of EGFR signals.

Amphiregulin (AREG) is a transmembrane glycoprotein recently implicated in kidney fibrosis. Previously, we reported that the AREG-targeting Self-Assembled-Micelle inhibitory RNA (SAMiRNA-AREG) alleviated fibrosis by stably silencing the AREG gene, and reduced the side effects of conventional siRNA treatment of pulmonary fibrosis. However, the therapeutic effect of SAMiRNA-AREG in renal fibrosis has not been studied until now. We used two animal models of renal fibrosis generated by a unilateral ureteral obstruction (UUO) and an adenine diet (AD) to investigate whether SAMiRNA-AREG inhibited renal fibrosis. To investigate the delivery of SAMiRNA-AREG to the kidney, Cy5-labeled SAMiRNA-AREG was injected into UUO- and AD-induced renal fibrosis models. In both kidney disease models, SAMiRNA-AREG was delivered primarily to the damaged kidney. We also confirmed the protective effect of SAMiRNA-AREG in renal fibrosis models. SAMiRNA-AREG markedly decreased the UUO- and AD-induced AREG mRNA expression. Furthermore, the mRNA expression of fibrosis markers, including α-smooth muscle actin, fibronectin, α1(I) collagen, and α1(III) collagen in the UUO and AD-induced kidneys, was diminished in the SAMiRNA-AREG-treated mice. The transcription of inflammatory markers (tumor necrosis factor-α and monocyte chemoattractant protein-1) and adhesion markers (vascular cell adhesion molecule 1 and intercellular adhesion molecule 1) was attenuated. The hematoxylin and eosin, Masson's trichrome, and immunohistochemical staining results showed that SAMiRNA-AREG decreased renal fibrosis, AREG expression, and epidermal growth factor receptor (EGFR) phosphorylation in the UUO- and AD-induced models. Moreover, we studied the effects of SAMiRNA-AREG in response to TGF-ß1 in mouse and human proximal tubule cells, and mouse fibroblasts. TGF-ß1-induced extracellular matrix production and myofibroblast differentiation were attenuated by SAMiRNA-AREG. Finally, we confirmed that upregulated AREG in the UUO or AD models was mainly localized in the distal tubules. In conclusion, SAMiRNA-AREG represents a novel siRNA therapeutic for renal fibrosis by suppressing EGFR signals.

Safety pharmacology of self-assembled-micelle inhibitory RNA-targeting amphiregulin (SAMiRNA-AREG), a novel siRNA nanoparticle platform.

The present safety pharmacology core battery studies (neurobehavior, respiratory, cardiovascular system, and human ether a-go-go (hERG) channel current) investigated the potential harmful effects of self-assembled-micelle inhibitory RNA-targeting amphiregulin (SAMiRNA-AREG). The SAMiRNA-AREG was administered by single intravenous injection at up to 300 mg/kg and 100 mg/kg in mice and monkeys, respectively. The hERG assay was performed in Chinese hamster ovary (CHO) cells at SAMiRNA-AREG concentrations of up to 200 µg/mL. In the evaluation on neurobehavior, a transient decrease in body temperature was found at 0.5 h (30 min) post-dose at both sexes in mice, with a single 300 mg/kg dose of SAMiRNA-AREG. However, these effects had returned to normal at 1 h post-dose. In the evaluation on hERG channel current, there were statistically significant differences in the inhibition of peak hERG potassium channel current between the 20, 100, and 200 µg/mL SAMiRNA-AREG treatment groups and the vehicle control group. However, these effects were less potent than that of E-4031, a positive control article. For the respiratory and cardiovascular systems, no treatment-related changes were observed in mice or monkeys. Thus, under these experimental conditions, these studies suggest that SAMiRNA-AREG showed no adverse effects on the neurobehavior, respiratory, and cardiovascular function.

Anti-obesity effect of Lactobacillus gasseri BNR17 in high-sucrose diet-induced obese mice.

Previously, we reported that Lactobacillus gasseri BNR17 (BNR17), a probiotic strain isolated from human breast milk, inhibited increases in body weight and adipocyte tissue weight in high-sucrose diet-fed Sprague-Dawley (SD) rats and reduced glucose levels in type 2 diabetes mice. In the current study, we conducted further experiments to extend these observations and elucidate the mechanism involved. C57BL/6J mice received a normal diet, high-sucrose diet or high-sucrose diet containing L. gasseri BNR17 (10(9) or 10(10) CFU) for 10 weeks. The administration of L. gasseri BNR17 significantly reduced the body weight and white adipose tissue weight regardless of the dose administered. In BNR17-fed groups, mRNA levels of fatty acid oxidation-related genes (ACO, CPT1, PPARα, PPARδ) were significantly higher and those of fatty acid synthesis-related genes (SREBP-1c, ACC) were lower compared to the high-sucrose-diet group. The expression of GLUT4, main glucose transporter-4, was elevated in BNR17-fed groups. L. gasseri BNR17 also reduced the levels of leptin and insulin in serum. These results suggest that the anti-obesity actions of L. gasseri BNR17 can be attributed to elevated expression of fatty acid oxidation-related genes and reduced levels of leptin. Additionally, data suggested the anti-diabetes activity of L. gasseri BNR17 may be to due elevated GLUT4 and reduced insulin levels.

Effect of Lactobacillus gasseri BNR17 on Overweight and Obese Adults: A Randomized, Double-Blind Clinical Trial.

BACKGROUND: Lactobacillus gasseri BNR17 is a type of probiotic strain isolated from human breast milk. A study was reported regarding the fact that BNR17 was an inhibitor of obesity and diabetic activities in the human body through previous animal experiments. This study was furthered to investigate the effect of BNR17, a probiotic strain isolated from human breast milk, on obese and overweight adults. METHODS: Sixty-two obese volunteers aged 19 to 60 with body mass index ≥ 23 kg/m(2) and fasting blood sugar ≥ 100 mg/dL participated in a placebo controlled, randomized, and double-blind trial. For 12 weeks, 57 participants were given either placebo or BNR17 and were tested by measuring body fat, body weight, various biochemical parameters, vital signs, and computed tomography at the start of the study and at weeks 4, 8, and 12. The subjects assumed usual daily activities without having to make behavioral or dietary modifications during the course of the study. RESULTS: At the 12th week, a slight reduction in body weight was noted in the BNR17 group, but there were no significant weight changes between groups. Decrease of waist and hip circumferences in the BNR17 group was more pronounced than those in the placebo group. The two groups had no special or severe adverse reactions. CONCLUSION: Despite there being no change in behavior or diet, administration of only the supplement of BNR17 reduced weight and waist and hip circumference. However, there were no significant differences between the two groups. These findings warrant a subsequent longer-term prospective clinical investigation with a large population.

Effects of Lactobacillus gasseri BNR17 on body weight and adipose tissue mass in diet-induced overweight rats.

We investigated the weight-gain suppressive effect of Lactobacillus gasseri BNR17 isolated from human breast milk. Rats were fed a high-carbohydrate diet and administered BNR17 (BNR17 group) twice daily for twelve weeks. Changes were observed in body weight and white adipose tissue mass. The percent increase in body weight (P=0.0331) and fat pad mass (P<0.01) was significantly lower in the BNR17 group, and the FER was moderately lower (P=0.0769). These data suggest that BNR17 can prevent diet-induced overweight and may become an alternative method for treating weight problems and obesity.

Use of microsatellite markers to detect quantitative trait loci in Yorkshire pigs.

To identify genetic markers associated with economic traits in pigs, 157 microsatellite markers were examined in Yorkshire pigs. Thirty eight female Yorkshire pigs were initially examined and six of them were selected as progenitors; half were more than 1.5 standard deviations (SD) above the mean for average daily gain (ADG) and backfat thickness (BFT), and the remaining half were more than 1.5 SD below the mean. These pigs were then mated to male Duroc pigs, and 200 F2 pig offspring were examined for the association of specific alleles with ADG and BFT. To confirm the specific markers identified in the initial analysis, associations of significant markers with economic traits were further examined in 228 additional performance-tested purebred pigs. Twenty-five microsatellite markers were significantly associated with either ADG or BFT, and among these, 17 were associated with both traits. The markers with the highest association to ADG were also associated with BFT. Our study reveals that specific markers could be used to predict economic significance, and confirms several quantitative trait loci (QTL) identified in previous studies. However, further analysis with more closely-spaced microsatellite markers is required to refine predictive values for economic traits and positions of QTL that are reliable for actual phenotypic prediction.