Effects of the prenatal and postnatal nurturing environment on the phenotype and gut microbiota of mice with polycystic ovary syndrome induced by prenatal androgen exposure: a cross-fostering study.

The gut microbiome is implicated in the pathogenesis of polycystic ovary syndrome (PCOS), and prenatal androgen exposure is involved in the development of PCOS in later life. Our previous study of a mouse model of PCOS induced by prenatal dihydrotestosterone (DHT) exposure showed that the reproductive phenotype of PCOS appears from puberty, followed by the appearance of the metabolic phenotype after young adulthood, while changes in the gut microbiota was already apparent before puberty. To determine whether the prenatal or postnatal nurturing environment primarily contributes to these changes that characterize prenatally androgenized (PNA) offspring, we used a cross-fostering model to evaluate the effects of changes in the postnatal early-life environment of PNA offspring on the development of PCOS-like phenotypes and alterations in the gut microbiota in later life. Female PNA offspring fostered by normal dams (exposed to an abnormal prenatal environment only, fostered PNA) exhibited less marked PCOS-like phenotypes than PNA offspring, especially with respect to the metabolic phenotype. The gut microbiota of the fostered PNA offspring was similar to that of controls before adolescence, but differences between the fostered PNA and control groups became apparent after young adulthood. In conclusion, both prenatal androgen exposure and the postnatal early-life environment created by the DHT injection of mothers contribute to the development of PCOS-like phenotypes and the alterations in the gut microbiota that characterize PNA offspring. Thus, both the pre- and postnatal environments represent targets for the prevention of PCOS and the associated alteration in the gut microbiota in later life.

Inhibitory effects of vaginal Lactobacilli on Candida albicans growth, hyphal formation, biofilm development, and epithelial cell adhesion.

Introduction: Antifungal agents are not always efficient in resolving vulvovaginal candidiasis (VVC), a common genital infection caused by the overgrowth of Candida spp., including Candida albicans, or in preventing recurrent infections. Although lactobacilli (which are dominant microorganisms constituting healthy human vaginal microbiota) are important barriers against VVC, the Lactobacillus metabolite concentration needed to suppress VVC is unknown. Methods: We quantitatively evaluated Lactobacillus metabolite concentrations to determine their effect on Candida spp., including 27 vaginal strains of Lactobacillus crispatus, L. jensenii, L. gasseri, Lacticaseibacillus rhamnosus, and Limosilactobacillus vaginalis, with inhibitory abilities against biofilms of C. albicans clinical isolates. Results: Lactobacillus culture supernatants suppressed viable fungi by approximately 24%-92% relative to preformed C. albicans biofilms; however, their suppression differed among strains and not species. A moderate negative correlation was found between Lactobacillus lactate production and biofilm formation, but no correlation was observed between hydrogen peroxide production and biofilm formation. Both lactate and hydrogen peroxide were required to suppress C. albicans planktonic cell growth. Lactobacillus strains that significantly inhibited biofilm formation in culture supernatant also inhibited C. albicans adhesion to epithelial cells in an actual live bacterial adhesion competition test. Discussion: Healthy human microflora and their metabolites may play important roles in the development of new antifungal agent against C. albicans-induced VVC.

Comparison of clinical severity, genotype and toxin gene expression of binary toxin-producing Clostridioides difficile clinical isolates in Japan.

The emerging Clostridioides difficile strain BI/NAP1/027 has been reported to be associated with more severe clinical symptoms and higher mortality rates, thought in part due to production of a novel binary toxin alongside conventional A and B toxins. However, recent studies suggest that this may not always be the case. Therefore, the purpose of this report was to investigate the correlation between clinical severity and microbiological characteristics of CDT-producing C. difficile isolates in Japan. Eight Japanese isolates of CDT producing C. difficile were investigated using genotyping, cytotoxic activity assays and toxin gene expression. Correlation with clinical severity was performed retrospectively using the patient record. Three of eight patients were assessed as having severe C. difficile infection (CDI). PCR ribotyping resolved six ribotypes including ribotype 027. No specific genes were identified determining severe compared with non-severe cases. Positive correlation of expression levels of tcdA, tcdB and cdtB were observed although these expression levels were not correlated with cytotoxicity. CDI severity index neither correlated with toxin gene expression level nor cytotoxicity. These data indicate that the possession of the CDT gene and toxin gene expression levels may not relate to C. difficile cytotoxicity or clinical severity.

Sustained suppression of enterohepatic circulation of mycophenolic acid by antimicrobial-associated diarrhea in a kidney transplant recipient with Crohn's disease: A case report.

Mycophenolic acid (MPA) undergoes enterohepatic circulation. A kidney transplant patient on mycophenolate mofetil was treated with tazobactam/piperacillin for pyelonephritis, and developed antimicrobial-associated diarrhea. Consequently, the MPA trough level decreased by approximately 90%. Furthermore, it took approximately a month for the MPA level to normalize even after diarrhea had resolved.

Utility of autologous fecal microbiota transplantation and elucidation of microbiota in diversion colitis.

Objectives: Diversion colitis (DC) is an inflammatory disorder caused by interruption of the fecal stream and subsequent nutrient deficiency from luminal bacteria. The utility of fecal microbiota transplantation (FMT) for DC was recently investigated; however, the precise pathogenesis of this condition remains unclear. This study aimed to evaluate the utility of autologous FMT in DC and to determine the related changes in the intestinal microbiota. Methods: Autologous FMT was performed to reestablish the intestinal microbiota in five patients (average age, 64.6 ± 8.3 years) with DC. They underwent double-ended colostomy. We assessed the diverted colon by endoscopy and evaluated the microbiota before and after FMT using the 16S rRNA gene sequencing method. Results: All five patients had mild inflammation (ulcerative colitis endoscopic index of severity [UCEIS] 2-3) in the diverted colon based on the colonoscopic findings. Three patients presented with symptoms, such as tenesmus, mucoid stool, and bloody stool. With FMT treatment, all patients achieved endoscopic remission (UCEIS score of 0 or 1) and symptomatic improvement. We observed a significantly decreased α-diversity in DC patients compared to healthy controls. The frequency of aerobic bacteria, such as Enterobacteriaceae, in the diverted colon decreased after autologous FMT. Conclusions: This study was the first to show that the microbiota in the diverted colon was significantly affected by autologous FMT. Since interruption of the fecal stream is central to the development of DC, FMT can be considered a promising treatment.

Hydrogen gas with extracorporeal cardiopulmonary resuscitation improves survival after prolonged cardiac arrest in rats.

BACKGROUND: Despite the benefits of extracorporeal cardiopulmonary resuscitation (ECPR) in cohorts of selected patients with cardiac arrest (CA), extracorporeal membrane oxygenation (ECMO) includes an artificial oxygenation membrane and circuits that contact the circulating blood and induce excessive oxidative stress and inflammatory responses, resulting in coagulopathy and endothelial cell damage. There is currently no pharmacological treatment that has been proven to improve outcomes after CA/ECPR. We aimed to test the hypothesis that administration of hydrogen gas (H2) combined with ECPR could improve outcomes after CA/ECPR in rats. METHODS: Rats were subjected to 20 min of asphyxial CA and were resuscitated by ECPR. Mechanical ventilation (MV) was initiated at the beginning of ECPR. Animals were randomly assigned to the placebo or H2 gas treatment groups. The supplement gas was administered with O2 through the ECMO membrane and MV. Survival time, electroencephalography (EEG), brain functional status, and brain tissue oxygenation were measured. Changes in the plasma levels of syndecan-1 (a marker of endothelial damage), multiple cytokines, chemokines, and metabolites were also evaluated. RESULTS: The survival rate at 4 h was 77.8% (7 out of 9) in the H2 group and 22.2% (2 out of 9) in the placebo group. The Kaplan-Meier analysis showed that H2 significantly improved the 4 h-survival endpoint (log-rank P = 0.025 vs. placebo). All animals treated with H2 regained EEG activity, whereas no recovery was observed in animals treated with placebo. H2 therapy markedly improved intra-resuscitation brain tissue oxygenation and prevented an increase in central venous pressure after ECPR. H2 attenuated an increase in syndecan-1 levels and enhanced an increase in interleukin-10, vascular endothelial growth factor, and leptin levels after ECPR. Metabolomics analysis identified significant changes at 2 h after CA/ECPR between the two groups, particularly in D-glutamine and D-glutamate metabolism. CONCLUSIONS: H2 therapy improved mortality in highly lethal CA rats rescued by ECPR and helped recover brain electrical activity. The underlying mechanism might be linked to protective effects against endothelial damage. Further studies are warranted to elucidate the mechanisms responsible for the beneficial effects of H2 on ischemia-reperfusion injury in critically ill patients who require ECMO support.

Sensitive quantification of free pazopanib using ultra-high performance liquid chromatography coupled to tandem mass spectrometry and assessment of clinical application.

Pazopanib is widely used to treat renal cell carcinomas and soft tissue tumors in Japan. Although several reports demonstrated the usefulness of therapeutic drug monitoring (TDM) of pazopanib, those studies measured only total pazopanib concentration. For drugs with high protein binding rates such as pazopanib, measuring free concentrations may be clinically more useful than measuring total concentrations. In this study, we aimed to develop a high-throughput method for quantification of free pazopanib in human plasma using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). Free pazopanib was separated by ultrafiltration. After a simple solid-phase extraction step using a 96-well plate, pazopanib was analyzed by UHPLC-MS/MS in positive electrospray ionization mode. The novel method fulfilled the requirements of the US Food and Drug Administration guidelines for assay validation, and the lower limit of quantification was 0.05 ng/mL. The calibration curve was linear over the concentration range of 0.05-50 ng/mL. The average recovery rate was 66.9 ± 2.1% (mean ± SD). The precision was below 7.02%, and accuracy was within 10.60% across all quality control levels. Matrix effect varied between 44.4% and 60.4%. This assay was successfully applied to measure trough free pazopanib concentrations in three patients treated with pazopanib for soft tissue tumors. We succeeded to develop a novel high-throughput UHPLC-MS/MS method for quantification of free pazopanib in human plasma. This method can be applied to TDM for patients receiving pazopanib in the clinical setting.

Pathogenic Characterization of Clostridium perfringens Strains Isolated From Patients With Massive Intravascular Hemolysis.

Sepsis caused by Clostridium perfringens infection is rare but often fatal. The most serious complication leading to poor prognosis is massive intravascular hemolysis (MIH). However, the molecular mechanism underlying this fulminant form of hemolysis is unclear. In the present study, we employed 11 clinical strains isolated from patients with C. perfringens septicemia and subdivided these isolates into groups H and NH: septicemia with (n = 5) or without (n = 6) MIH, respectively. To elucidate the major pathogenic factors of MIH, biological features were compared between these groups. The isolates of two groups did not differ in growth rate, virulence-related gene expression, or phospholipase C (CPA) production. Erythrocyte hemolysis was predominantly observed in culture supernatants of the strains in group H, and the human erythrocyte hemolysis rate was significantly correlated with perfringolysin O (PFO) production. Correlations were also found among PFO production, human peripheral blood mononuclear cell (PBMC) cytotoxicity, and production of interleukin-6 (IL-6) and interleukin-8 (IL-8) by human PBMCs. Analysis of proinflammatory cytokines showed that PFO induced tumor necrosis factor-α (TNF-α), IL-5, IL-6, and IL-8 production more strongly than did CPA. PFO exerted potent cytotoxic and proinflammatory cytokine induction effects on human blood cells. PFO may be a major virulence factor of sepsis with MIH, and potent proinflammatory cytokine production induced by PFO may influence the rapid progression of this fatal disease caused by C. perfringens.

Increase in muscle mass associated with the prebiotic effects of 1-kestose in super-elderly patients with sarcopenia.

Sarcopenia causes functional disorders and decreases the quality of life. Thus, it has attracted substantial attention in the aging modern world. Dysbiosis of the intestinal microbiota is associated with sarcopenia; however, it remains unclear whether prebiotics change the microbiota composition and result in the subsequent recovery of muscle atrophy in elderly patients with sarcopenia. This study aimed to assess the effects of prebiotics in super-elderly patients with sarcopenia. We analyzed the effects of 1-kestose on the changes in the intestinal microbiota and body composition using a next-generation sequencer and a multi-frequency bioimpedance analysis device. The Bifidobacterium longum population was significantly increased in the intestine after 1-kestose administration. In addition, in all six patients after 12 weeks of 1-kestose administration, the skeletal muscle mass index was greater, and the body fat percentage was lower. This is the first study to show that administration of a prebiotic increased the population of B. longum in the intestinal microbiota and caused recovery of muscle atrophy in super-elderly patients with sarcopenia.

Evaluation of intestinal microbiota, short-chain fatty acids, and immunoglobulin a in diversion colitis.

It is reported that an increase in aerobic bacteria, a lack of short-chain fatty acids (SCFAs), and immune disorders in the diverted colon are major causes of diversion colitis. However, the precise pathogenesis of this condition remains unclear. The aim of the present study was to examine the microbiota, intestinal SCFAs, and immunoglobulin A (IgA) in the diverted colon. Eight patients underwent operative procedures for colostomies. We assessed the diverted colon using endoscopy and obtained intestinal samples from the diverted colon and oral colon in these patients. We analyzed the microbiota and SCFAs of the intestinal samples. The bacterial communities were investigated using a 16S rRNA gene sequencing method. The microbiota demonstrated a change in the proportion of some species, especially Lactobacillus, which significantly decreased in the diverted colon at the genus level. We also showed that intestinal SCFA values were significantly decreased in the diverted colon. Furthermore, intestinal IgA levels were significantly increased in the diverted colon. This study was the first to show that intestinal SCFAs were significantly decreased and intestinal IgA was significantly increased in the diverted colon. Our data suggest that SCFAs affect the microbiota and may play an immunological role in diversion colitis.

Development of a High-Throughput Quantification Method for Pazopanib Using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry and Its Clinical Application in Patients With Soft Tissue Tumors.

BACKGROUND: Pazopanib is widely used to treat renal cell carcinomas and soft tissue tumors in Japan. Pazopanib has significant therapeutic efficacy but it is associated with frequent severe adverse effects. Therapeutic drug monitoring (TDM) may help to prevent adverse effects. A more convenient and rapid pazopanib assay is desirable for the application of TDM in clinical settings. In this study, the authors developed a high-throughput method for quantifying pazopanib in human plasma using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). METHODS: After a simple solid-phase extraction step using a 96-well plate, pazopanib was analyzed by UHPLC-MS/MS in the positive electrospray ionization mode. RESULTS: The novel method fulfilled the requirements of the US Food and Drug Administration and the European Medicines Agency guidelines for assay validation, and the lower limit of quantification was 0.5 mcg/mL. The calibration curves were linear over the concentration range of 0.5-100 mcg/mL. The average recovery rate was 102.0% ± 3.9% (mean ± SD). The precision was below 5.0%, and the accuracy was within 12.0% for all quality control levels. Matrix effect varied between 90.9% and 97.1%. This assay was successfully applied to TDM of pazopanib trough concentrations in 3 patients treated with the drug for soft tissue tumors. CONCLUSIONS: The authors succeeded in developing a novel high-throughput UHPLC-MS/MS method for quantifying pazopanib in human plasma. This method can be applied to TDM of patients receiving pazopanib in clinical settings.

Clostridium butyricum Modulates the Microbiome to Protect Intestinal Barrier Function in Mice with Antibiotic-Induced Dysbiosis.

Clostridium butyricum MIYAIRI 588 (CBM 588) is a probiotic bacterium that has previously been used to prevent antibiotic-associated diarrhea. However, the underlying mechanism by which CBM 588 protects the gut epithelial barrier remains unclear. Here, we show that CBM 588 increased the abundance of Bifidobacterium, Lactobacillus, and Lactococcus species in the gut microbiome and also enhanced the intestinal barrier function of mice with antibiotic-induced dysbiosis. Additionally, CBM 588 significantly promoted the expansion of IL-17A-producing γδT cells and IL-17A-producing CD4 cells in the colonic lamina propria (cLP), which was closely associated with changes in the intestinal microbial composition. Additionally, CBM 588 plays an important role in controlling antibiotic-induced gut inflammation through upregulation of anti-inflammatory lipid metabolites such as palmitoleic acid, 15d-prostaglandin J2, and protectin D1. This study reveals a previously unrecognized mechanism of CBM 588 and provides new insights into gut epithelial barrier protection with probiotics under conditions of antibiotic-induced dysbiosis.

The impact of probiotic Clostridium butyricum MIYAIRI 588 on murine gut metabolic alterations.

INTRODUCTION: Clostridium butyricum MIYAIRI 588 (CBM 588) is a probiotic bacterium used in antidiarrheal medicine in Japan. A few studies analyzed the changes in gut microbiome in patients treated with antimicrobials based on metagenomics sequencing. However, the impact of CBM 588 on gut metabolic alterations has not been fully elucidated. This study was to reveal the impact of CBM 588 on gut metabolic alterations. MATERIAL AND METHODS: In this in vivo study, mice were divided into four groups and CBM 588, clindamycin (CLDM), and normal saline (control) was orally administered (1. CLDM, 2. CBM 588, 3. CBM 588 + CLDM, 4. water) for 4 days. Fecal samples were collected to extract DNA for metagenomics analysis. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) was used to obtain relative Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway abundance information derived from metagenomics data. RESULTS: CLDM treatment resulted in a dramatic increase in Firmicutes phylum compared to non-CLDM-treated groups (control and CBM 588-treated group). Then, the CBM 588 + CLDM-treated group showed a trend similar in many metabolic pathways to the CLDM-treated group. On the other hand, the CBM 588 + CLDM-treated group showed higher relative abundance compared to the CLDM-treated group especially in starch and sucrose metabolism. DISCUSSION: We concluded that CBM 588 caused a gut microbiome functional shift toward increased carbohydrate metabolism. These results support the hypothesis that CBM 588 treatment modulates gut microbiome under dysbiosis conditions due to antimicrobials.

Characterization of the vaginal microbiota of Japanese women.

The composition of vaginal microbiota changes throughout life in response to health status, sexual activity, and pregnancy. Here the constitution of the vaginal microbiota among non-pregnant women, pregnant woman, and commercial sex workers (CSWs) in Japan were compared. Vaginal samples were obtained from 54 women between January 2014 and February 2015 and the microbiota of each was analyzed by 16S metagenomics as well as cluster and diversity analyses to identify differences. In addition, vaginal Lactobacillus spp. were isolated for comparison. Furthermore, data regarding the use of ritodrine hydrochloride by pregnant women was collected from medical charts. The vaginal microbiota were clustered into three groups. Group 1 was most often dominated by Lactobacillus spp., whereas groups 2 and 3 included not only Lactobacillus spp. but also Bifidobacterium, Atopobium, Prevotella, and Gardnerella spp., in addition to a few other taxa. In non-pregnant women, the proportions of microbes in groups 1, 2, and 3 were 31.8%, 36.4%, and 31.8%, respectively. In pregnant women, the abundance of group 1 microbes was notably greater than that of groups 2 and 3 (66.7% vs. 12.5% and 20.8%, respectively). In CSWs, the prevalence of group 3 microbes was far greater than that of group 1 (87.5% vs. 12.5%, respectively). The alpha diversity of non-pregnant women was significantly greater than that of pregnant women. The detection rate of live Lactobacillus spp. in CSWs was lower than in pregnant and non-pregnant women (25% vs. 50% and 68.2%, respectively). The vaginal microbiota of most pregnant women (60%) who received ritodrine hydrochloride was not dominated by Lactobacillus spp. These results suggest that there were clear differences in the colonization rate of Lactobacillus spp. among non-pregnant, pregnant, and CSW women groups. In addition, the dominance of Lactobacillus may influence the risk of preterm birth among women who received ritodrine hydrochloride during pregnancy.

Association between Clostridioides difficile ribotypes, restriction endonuclease analysis types, and toxin gene expression.

OBJECTIVES: Clostridioides difficile strains cause severe infection. Previous studies suggested that the virulence of C. difficile is dependent on ribotype; however, this hypothesis is still controversial. We aim to investigate the relationship between ribotypes, restriction endonuclease analysis (REA) types, and toxin gene expression in C. difficile strains. METHODS: We utilized 53 clinical C. difficile strains. All strains were assigned a molecular strain type using PCR ribotyping and REA typing and classified into 17 ribotypes and six REA types. The expression of toxin genes (tcdA, tcdB, and cdtB) in C. difficile strains were quantified by real-time PCR using each specific primer set, and expression was normalized to that of the housekeeping gene rpoA. RESULTS: All 53 strains expressed tcdB and four strains expressed cdtB. Five strains did not express tcdA. Most ribotype and REA type strains expressed tcdA and tcdB similar to the BAA-1870 strain. In cdtB-positive strains, the cdtB expression levels were similar to those in the BAA-1870 strain. tcdA and tcdB expression levels were similar in the cdtB-positive and cdtB-negative strains. CONCLUSION: Toxin gene expression was not associated with the ribotype. Production of binary toxin C. difficile transferase was not related to tcdA and tcdB expression levels.

The impact of Clostridium butyricum MIYAIRI 588 on the murine gut microbiome and colonic tissue.

BACKGROUND: Clostridium butyricum MIYAIRI 588 (CBM 588) is a probiotic bacterium that is used as an anti-diarrheal medicine in Japan. However, the impact of this probiotic on the gut microbiome has not been fully elucidated, especially, when used with antimicrobials. MATERIAL AND METHODS: In an in vivo study, CBM 588 monotherapy, clindamycin monotherapy, CBM 588 and clindamycin (combination therapy), or normal saline (control) was orally administered to mice for 4 days, and fecal samples were collected for 18 days to enumerate C. butyricum. We also extracted DNA from these fecal samples for metagenomics analysis by amplification of the V3-V4 region of the bacterial 16S rRNA gene and MiSeq Illumina sequencing. In addition, the concentrations of some short chain fatty acids were assessed in the fecal samples. A histological analysis was also conducted. RESULTS: On day 4 (the last treatment day), there was no difference in the total counts of C. butyricum between the CBM 588 monotherapy and combination therapy groups (5.21 ±â€¯0.78 vs. 5.13 ±â€¯0.45 log10 cfu/g, p = 0.86). Clindamycin treatment resulted in dramatic increases in the phylum Firmicutes, especially Enterobacteriaceae, Clostridiaceae, Lactobacillus, and Enterococcus, compared with the other groups during the treatment period. CBM 588 treatment modified the bacterial community composition at lower phylogenetic levels. Some bacterial taxa, such as Bifidobacterium, Coprococcus, and Bacteroides, were significantly increased in the combination therapy group when compared with the other groups. In the metabolic analysis, CBM 588 enhanced lactic acid production. It also enhanced the efficiency of lactic acid use for the production of butyric acid. Only the clindamycin monotherapy group showed abnormal colon tissue, with superficial epithelial necrosis and the presence of inflammatory cells. CONCLUSION: CBM 588 treatment modulated the gut microbiota composition under dysbiosis due to the use of an antimicrobial with strong activity against anaerobes and significantly reduced epithelial damage.

Coordination-driven folding and assembly of a short peptide into a protein-like two-nanometer-sized channel.

Short peptide helices have attracted attention as suitable building blocks for soft functional materials, but they are rarely seen in crystalline materials. A new artificial nanoassembly of short peptide helices in the crystalline state is presented in which peptide helices are arranged three-dimensionally by metal coordination. The folding and assembly processes of a short peptide ligand containing the Gly-Pro-Pro sequence were induced by silver(I) coordination in aqueous alcohol, and gave rise to a single crystal composed of polyproline II helices. Crystallographic studies revealed that this material possesses two types of unique helical nanochannel; the larger channel measures more than 2 nm in diameter. Guest uptake properties were investigated by soaking the crystals in polar solutions of guest molecules; anions, organic chiral molecules, and bio-oligomers are effectively encapsulated by this peptide-folded porous crystal, with moderate to high chiral recognition for chiral molecules.

Effects of anthocyanin-rich purple potato flakes on antioxidant status in F344 rats fed a cholesterol-rich diet.

We examined the antioxidant effects of polyphenol/anthocyanin-rich potato (Solanum tuberosum cv. Shadow-Queen) flakes in male rats fed a high-cholesterol diet. The rats were served either a high-cholesterol (0.5% cholesterol plus 0.125% sodium cholate) diet, or a high-cholesterol diet containing a mixture of 243 g alpha-maize starch/kg supplemented with one of the following (per kg diet): 300 g medium purple potato (Shadow-Queen), 300 g white potato (Solanum tuberosum cv. Toyoshiro) or 300 g dark purple sweet potato (Ipomoea batatas cv. Ayamurasaki) flakes for 28 d. We analysed thiobarbituric acid reactive substance (TBARS) levels in the serum and liver, and antioxidant enzyme activities in the liver. At this dosage, TBARS levels in the serum and liver of the Shadow-Queen and Ayamurasaki groups were significantly lower than those in the control and Toyoshiro groups. The serum urate levels in all the flake groups were significantly lower than that in the control group. The hepatic glutathione levels in the Shadow-Queen and Ayamurasaki groups were significantly higher than in the control and Toyoshiro groups. The activities of hepatic glutathione reductase and glutathione S-transferase in the Shadow-Queen and Ayamurasaki groups were significantly greater than those in the control group. These results show that modulation of antioxidant enzymes and oxidative status in the serum and liver by the purple potato flake diet (Shadow-Queen) containing polyphenols/anthocyanins may play an important role in the protection against adverse effects related to oxidative damage in rats fed a high-cholesterol diet.

Development of dry powder inhalation system of novel vasoactive intestinal peptide (VIP) analogue for pulmonary administration.

Vasoactive intestinal peptide (VIP) exerts a relaxing action on tracheal smooth muscle which is mediated through interaction with VIP receptors. The deficiency of VIP in the airways has been implicated in the pathogenesis of asthma. Thus, the administration of VIP may be useful for the therapy of pulmonary diseases. However, the therapeutic application of VIP is largely limited by its rapid degradation in addition to the systemic adverse effects due to the wide distribution of VIP receptors. To overcome these problems, we succeeded to synthesize a novel VIP derivative of VIP, [R15, 20, 21, L17]-VIP-GRR (IK312532), and to prepare its dry powder for the topical administration to the lung. The physicochemical properties of dry powder were evaluated by laser diffraction and cascade impactor. The laser diffraction analysis indicated that the carrier and fine particles had median diameter of 65.6 and 4.5 microm, respectively, and the air flow at the pressure of 0.15 MPa or higher resulted in the high dispersion and significant separation of fine particle containing peptide from the carrier molecule. The cascade impactor analysis clearly showed the high emission of dry powder from capsule and the deposition of peptide on stages 3 of the cascade impactor. The intratracheal administration of dry powder inhaler (DPI) of VIP or IK312532 brought about a significant decrease of maximal number of binding sites (Bmax) for [125I]VIP in anterior and posterior lobes of rat right lung, suggesting a significant occupancy of lung VIP receptors. This effect by IK312532-DPI compared with VIP-DPI lasted for a longer period. Thus, IK312532-DPI may be a pharmacologically useful drug delivery system for the VIP therapy of pulmonary diseases such as asthma.

Erythritol-based dry powder of glucagon for pulmonary administration.

Glucagon, a key regulatory element of glycogen metabolism, is known to be effective in the clinical treatment of hypoglycemia and the maintenance of normal circulating glucose levels in patients with total pancreatectomy, however the clinical use of this gut hormone has been restricted to parenteral administration. In this investigation, we prepared dry powder dosage forms of glucagon, which were formulated by mixing micronized glucagon particles and excipients with larger carrier particles. To achieve alveolar deposition for subsequent systemic absorption, a dry powder inhalant (DPI) of glucagon was size-reduced to a mass median diameter between 1 and 6 microm, as measured by laser diffraction analysis. The use of erythritol as both excipient and carrier in DPI of glucagon resulted in high and reproducible flowability and dispersibility of the powder mixtures, and therefore it provided a low dosing of the active substances. Distinct transpulmonary absorption of glucagon was confirmed after intratracheal administration of the glucagon dry powder to anesthetized rats, as evidenced by the increase in the blood glucagon and blood sugar levels. These results suggested the usefulness of an erythritol-based powder form of glucagon for systemic administration.