One-step construction of ferritin encapsulation drugs for cancer chemotherapy.

Conventionally, a disassembly and reassembly method has been used for encapsulation of drug molecules in ferritin protein nano-cages. However, clinical applications of ferritin have been greatly restricted by its limited drug-loading capacity and process complexity. Here, we establish a simple high yield process for preparing high drug-loaded ferritin nanomedicine for industrial production. A complex of ferritin and a target drug was obtained by incubating the mixture at an appropriate pH. An electrostatic charge potential and small ferritin cavity facilitates the passage of drug molecules through the pores, traversing the ferritin shell and enabling deposition of the drug in the ferritin cavity. Compared to the disassembly/reassembly method, the loading capacity of a doxorubicin-loaded ferritin heavy chain (DOX-FTH), constructed by our novel method, was over 3-fold higher, while doxorubicin recovery was 10-fold higher. Results of transmission electron microscopy, size exclusion chromatography, dynamic light scattering, and zeta potential indicate that DOX-FTH exhibits the same physicochemical characteristics of natural apo-ferritin. Moreover, DOX-FTH can be taken up and induce apoptosis of cancer cells overexpressing TfR1. Here, we have demonstrated the successful introduction of more than ten drug molecule types into ferritin nano-cages using a novel method. These results demonstrate that this one-step method is a powerful production process to construct a drug-loading ferritin drug delivery system carrier.

l-Serine and EPA Relieve Chronic Low-Back and Knee Pain in Adults: A Randomized, Double-Blind, Placebo-Controlled Trial.

BACKGROUND: Multisite pain, including low-back and knee pain, is a major health issue that greatly decreases quality of life. OBJECTIVES: This study analyzed the effects of l-serine, which provides necessary components for nerve function, and EPA, which exerts anti-inflammatory properties, on pain scores of adults with pain in at least the low back and knee for ≥3 mo. METHODS: This was a randomized, double-blind, placebo-controlled, parallel-group study. The Japan Low Back Pain Evaluation Questionnaire (JLEQ) and Japanese Knee Osteoarthritis Measure (JKOM) were applied as primary outcomes. The Brief Pain Inventory (BPI) and safety evaluation were secondary outcomes. We enrolled 120 participants aged ≥20 y (36 men and 84 women: mean ± SD age = 40.8 ± 10.9 y). The participants were randomly allocated to either the active group (daily ingestion of 594 mg l-serine and 149 mg EPA) or placebo group. The study period consisted of 8-wk dosing and 4-wk posttreatment observation. ANCOVA between groups for each time point was conducted using the baseline scores as covariates. RESULTS: The JLEQ scores (active compared with placebo: 14.2 ± 11.2 compared with 19.0 ± 10.2) at week 8 were lower in the active group (P < 0.001). The JKOM scores at week 4 (11.7 ± 9.0 compared with 13.9 ± 7.9), week 8 (10.4 ± 7.9 compared with 13.1 ± 7.1), and week 12 (10.3 ± 7.4 compared with 13.8 ± 7.5) were lower in the active group (P ≤ 0.04). Additionally, the active group had 11-27% better scores compared with the placebo group for BPI1 (worst pain), BPI3 (average pain), and BPI5D (pain during moving) at week 4 (P ≤ 0.028) and week 8 (P ≤ 0.019), respectively, and BPI5D was 23% better in the active group at week 12 (P = 0.007). No adverse events were observed. CONCLUSIONS: l-Serine and EPA were effective for pain relief in adults with low-back and knee pain after multiplicity adjustment.This trial was registered at the University Hospital Medical Information Network Clinical Trials Registry as UMIN000035056.

Evaluation of a Novel Boron-Containing α-D-Mannopyranoside for BNCT.

Boron neutron capture therapy (BNCT) is a unique anticancer technology that has demonstrated its efficacy in numerous phase I/II clinical trials with boronophenylalanine (BPA) and sodium borocaptate (BSH) used as 10B delivery agents. However, continuous drug administration at high concentrations is needed to maintain sufficient 10B concentration within tumors. To address the issue of 10B accumulation and retention in tumor tissue, we developed MMT1242, a novel boron-containing α-d-mannopyranoside. We evaluated the uptake, intracellular distribution, and retention of MMT1242 in cultured cells and analyzed biodistribution, tumor-to-normal tissue ratio and toxicity in vivo. Fluorescence imaging using nitrobenzoxadiazole (NBD)-labeled MMT1242 and inductively coupled mass spectrometry (ICP-MS) were performed. The effectiveness of BNCT using MMT1242 was assessed in animal irradiation studies at the Kyoto University Research Reactor. MMT1242 showed a high uptake and broad intracellular distribution in vitro, longer tumor retention compared to BSH and BPA, and adequate tumor-to-normal tissue accumulation ratio and low toxicity in vivo. A neutron irradiation study with MMT1242 in a subcutaneous murine tumor model revealed a significant tumor inhibiting effect if injected 24 h before irradiation. We therefore report that 10B-MMT1242 is a candidate for further clinical BNCT studies.

Lentinan diminishes apoptotic bodies in the ileal crypts associated with S-1 administration.

S-1 is an oral agent containing tegafur (a prodrug of 5-fluorouracil) that is used to treat various cancers, but adverse effects are frequent. Two pilot clinical studies have suggested that lentinan (LNT; ß-1,3-glucan) may reduce the incidence of adverse effects caused by S-1 therapy. In this study, we established a murine model for assessment of gastrointestinal toxicity associated with S-1 and studied the effect of LNT. S-1 was administered orally to BALB/c mice at the effective dose (8.3mg/kg, as tegafur equivalent) once daily (5days per week) for 3weeks. Stool consistency and intestinal specimens were examined. We investigated the effect of combined intravenous administration of LNT at 0.1mg, which is an effective dose in murine tumor models. We also investigated the effect of a single administration of S-1. During long-term administration of S-1, some mice had loose stools and an increase in apoptotic bodies was observed in the ileal crypts. An increase in apoptotic bodies was also noted after a single administration of S-1 (15mg/kg). Prior or concomitant administration of LNT inhibited the increase in apoptotic bodies in both settings. Administration of LNT also increased the accumulation of CD11b+TIM-4+ cells in the ileum, while depletion of these cells by liposomal clodronate diminished the inhibitory effect of LNT on S-1 toxicity. Combined administration of LNT with S-1 led to a decrease in apoptotic bodies in the ileal crypts, possibly because LNT promoted phagocytosis of damaged cells by CD11b+TIM-4+ cells.

Food antigen-induced immune responses in Crohn's disease patients and experimental colitis mice.

BACKGROUND: In Crohn's disease (CD), the involvement of food antigens in immune responses remains unclear. The objective of this study was to detect immune responses against food antigens in CD patients and examine the mechanism in a mouse model of colitis. METHODS: We enrolled 98 CD patients, 50 ulcerative colitis patients, and 52 healthy controls (HCs) to compare the levels of serum immunoglobulin (Ig)Gs against 88 foods. The presence of serum IgGs against foods was also examined in interleukin (IL)-10 knockout (KO) mice in which CD4(+) T cell activation by antigenic food protein was assessed. Mice transferred with IL-10 KO cells received diets with or without food antigens, and the development of colitis was evaluated. RESULTS: The prevalence of IgGs against various foods, especially vegetables, grains, and nuts, was significantly higher in CD patients than in HCs. Similarly, the prevalence of IgGs against food proteins was higher in IL-10 KO mice than in BALB/c mice. Beta-conglycinin, identified as an antigenic food proteins in IL-10 KO mice, induced CD4(+) T cell production of interferon-γ and IL-17 through dendritic cell antigen presentation. Elimination of the food antigens ameliorated the development of colitis in mice without altering the composition of their intestinal microbiota. CONCLUSIONS: In CD colitis mice, intestinal inflammation via CD4(+) T cell hyperactivation was induced by food antigens associated with high serum IgG levels and was ameliorated by the elimination of food antigens. This disrupted immunological tolerance to food antigen, which might act as an exacerbating factor, remains to be elucidated in CD patients.

Efficacy of orally administered superfine dispersed lentinan (beta-1,3-glucan) for the treatment of advanced colorectal cancer.

BACKGROUND: Lentinan (LNT) is an immune adjuvant medicine for advanced gastric cancer in Japan. Recently, an oral formulation of superfine dispersed lentinan (SDL) has become clinically available. To investigate the safety and effectiveness of SDL, a multi center clinical study in patients with advanced colorectal cancer was conducted. PATIENTS AND METHODS: Adverse events were assessed and the patients' quality of life (QOL) and the binding ability of peripheral blood monocytes (PBM) to LNT were also evaluated. RESULTS: Four grade 2 adverse events associated with SDL treatment were observed among the 80 patients. Adverse events associated with chemotherapy were observed in 9 out of the 64 chemotherapy-treated patients. Among the 48 patients assessed for QOL, the patients with low QOL scores before SDL treatment (n=23) reported a significant improvement in their QOL scores after 12 weeks of SDL administration. The rates of LNT-binding PBM in the QOL-improved group were significantly higher than those in the QOL-not-improved group (p<0.05). CONCLUSION: SDL was safe and effective for suppressing the adverse effects of chemotherapy as well as improving QOL. The binding ability of PBM to LNT appears to be a promising predictor of QOL improvement after SDL administration.

Clinical efficacy of superfine dispersed lentinan (beta-1,3-glucan) in patients with hepatocellular carcinoma.

BACKGROUND/AIMS: Recently, complementary alternative medicine is actively performed for cancer therapy. We investigated the effectiveness of supplementary food containing superfine dispersed lentinan (beta-1,3-glucan) in patients with unresectable or recurrent hepatocellular carcinoma in a multi-center study. METHODOLOGY: Peripheral blood was collected prior to the test food ingestion and was incubated with fluorescein-labeled lentinan. The rates of lentinan-binding CD14+ monocytes were determined by flow cytometry. Patient survival times were followed up for 3 years. RESULTS: Thirty-six patients were eligible among 40 enrolled patients. Median survival time of eligible patients was 13.6 months (95% confidence interval, 8.7-18.9 months). Survival times of patients who ingested test food for a mean period of 47 weeks (range, 26 to 145 weeks) were significantly longer than that of patients who ingested for 7 to 12 weeks (p < 0.05). The rates of lentinan-binding cells in CD14+ monocytes showed individual variations (0.1-19.7%; Median, 1.6%). Survival times (median survival time, 16.3 months) of lentinan-high-binding group were significantly longer than those (median survival time, 12.5 months) of lentinan-low-binding group (p < 0.05). CONCLUSIONS: A superfine dispersed lentinan-containing supplementary food is effective for hepatocellular carcinoma patients' survival. Long-time ingestion is preferable. Assessment of lentinan-binding CD14+ monocytes is a promising prognostic predictor.

Potentiation of intestinal immunity by micellary mushroom extracts.

Mushroom (shiitake) extracts were dispersed with lecithin micelles to prepare superfine particles (0.05 to 0.2 microm in diameter) of beta-1,3-glucan (micellary mushroom extracts). When mice were fed with these micelles of beta-glucan (0.75 mg/day/mouse, smaller amounts of beta-glucan), the number of lymphocytes yielded by the small intestine increased by up to 40%. More interestingly, the ratio of CD8alphabeta(+)TCRalphabeta(+) cells/CD8alphaalpha(+)TCRalphabeta(+) cells increased prominently. In parallel with this deviation in the distribution of lymphocyte subsets, tumor cytotoxicity against P815 cells and cytokine productions were also augmented. In other words, phylogenetically developed lymphocytes (CD8alphabeta(+), TCRalphabeta(+)) were much more effectively activated by the oral administration of micellary beta-glucan. These results suggest that smaller amounts of micellary beta-glucan might be useful for the potentiation of intestinal immunity.

Combination effect of AC-7700, a novel combretastatin A-4 derivative, and cisplatin against murine and human tumors in vivo.

The in vivo combination effect of AC-7700, a novel combretastatin A-4 derivative, and cisplatin (CDDP) was examined. The combination of AC-7700 and CDDP increased antitumor activity against murine colon 26 tumor in mice and cured the mice. This combination effect was found over wide dosage ranges of AC-7700 (20-80 mg/kg) and CDDP (2.5-5 mg/kg). Moreover, this combination augmented antitumor activity against murine S180 and M109 tumors, and human LX-1 and LS180 tumor xenografts in mice. The effect was the strongest when AC-7700 and CDDP were administered simultaneously. To study this combination effect, we measured the concentrations of CDDP in tumors, plasma and kidneys of the mice with colon 26 tumor. In the combination with AC-7700, the concentration of CDDP in the tumors increased from 0.5 to 96 h after administration, but did not change or decrease in plasma or kidneys. Against human LS180 xenografts in mice, the combination similarly increased the concentration of CDDP in the tumors. These results suggest that AC-7700 may specifically augment the accumulation of CDDP in tumors, and thus has the potential to be useful in combination chemotherapy with CDDP.