Toxicological Evaluation of a Probiotic-Based Delivery System for P8 Protein as an Anti-Colorectal Cancer Drug.

PURPOSE: This study aimed to toxicological evaluate a probiotics-based delivery system for p8 protein as an anti-colorectal cancer drug. INTRODUCTION: Lactic acid bacteria (LAB) have been widely ingested for many years and are regarded as very safe. Recently, a Pediococcus pentosaceus SL4 (PP) strain that secretes the probiotic-derived anti-cancer protein P8 (PP-P8) has been developed as an anti-colorectal cancer (CRC) biologic by Cell Biotech. We initially identified a Lactobacillus rhamnosus (LR)-derived anti-cancer protein, P8, that suppresses CRC growth. We also showed that P8 penetrates specifically into CRC cells (DLD-1 cells) through endocytosis. We then confirmed the efficacy of PP-P8, showing that oral administration of this agent significantly decreased tumor mass (~42%) relative to controls in a mouse CRC xenograft model. In terms of molecular mechanism, PP-P8 induces cell-cycle arrest in G2 phase through down-regulation of Cyclin B1 and Cdk1. In this study, we performed in vivo toxicology profiling to obtain evidence that PP-P8 is safe, with the goal of receiving approval for an investigational new drug application (IND). METHODS: Based on gene therapy guidelines of the Ministry of Food and Drug Safety (MFDS) of Korea, the potential undesirable effects of PP-P8 had to be investigated in intact small rodent or marmoset models prior to first-in-human (FIH) administration. The estimated doses of PP-P8 for FIH are 1.0×1010 - 1.0×1011 CFU/person (60 kg). Therefore, to perform toxicological investigations in non-clinical animal models, we orally administered PP-P8 at doses of 3.375 × 1011, 6.75 × 1011, and 13.5×1011 CFU/kg/day; thus the maximum dose was 800-8000-fold higher than the estimated dose for FIH. RESULTS: In our animal models, we observed no adverse effects of PP-P8 on clinicopathologic findings, relative organ weight, or tissue pathology. In addition, we observed no inflammation or ulceration during pathological necropsy. CONCLUSION: These non-clinical toxicology studies could be used to furnish valuable data for the safety certification of PP-P8.

A synthetic probiotic engineered for colorectal cancer therapy modulates gut microbiota.

BACKGROUND: Successful chemoprevention or chemotherapy is achieved through targeted delivery of prophylactic agents during initial phases of carcinogenesis or therapeutic agents to malignant tumors. Bacteria can be used as anticancer agents, but efforts to utilize attenuated pathogenic bacteria suffer from the risk of toxicity or infection. Lactic acid bacteria are safe to eat and often confer health benefits, making them ideal candidates for live vehicles engineered to deliver anticancer drugs. RESULTS: In this study, we developed an effective bacterial drug delivery system for colorectal cancer (CRC) therapy using the lactic acid bacterium Pediococcus pentosaceus. It is equipped with dual gene cassettes driven by a strong inducible promoter that encode the therapeutic protein P8 fused to a secretion signal peptide and a complementation system. In an inducible CRC cell-derived xenograft mouse model, our synthetic probiotic significantly reduced tumor volume and inhibited tumor growth relative to the control. Mice with colitis-associated CRC induced by azoxymethane and dextran sodium sulfate exhibited polyp regression and recovered taxonomic diversity when the engineered bacterium was orally administered. Further, the synthetic probiotic modulated gut microbiota and alleviated the chemically induced dysbiosis. Correlation analysis demonstrated that specific bacterial taxa potentially associated with eubiosis or dysbiosis, such as Akkermansia or Turicibacter, have positive or negative relationships with other microbial members. CONCLUSIONS: Taken together, our work illustrates that an effective and stable synthetic probiotic composed of P. pentosaceus and the P8 therapeutic protein can reduce CRC and contribute to rebiosis, and the validity and feasibility of cell-based designer biopharmaceuticals for both treating CRC and ameliorating impaired microbiota. Video abstract.

Probiotic-derived p8 protein induce apoptosis via regulation of RNF152 in colorectal cancer cells.

Worldwide, colorectal cancer (CRC) is one of the most common cancers and is a leading cause of cancer-related deaths. Accumulating evidence suggests that probiotics suppress the development of various cancers including CRC. Recently, we reported a Lactobacillus rhamnosus (LR)-derived 8 kDa protein (p8) that displayed anti-cancer properties in CRC cells. However, the precise anti-cancer mechanism of p8 and its target genes has not been fully examined. In the present study, we reveal that p8 leads to apoptotic cells and cleaved PARP1 expression in a mouse xenograft model of CRC. Additionally, we identified Ring finger protein 152 (RNF152) as a putative target of p8 using RNA-sequencing. Furthermore, the expression levels of RNF152 were increased following in vivo and in vitro treatment with p8. We also found that p8 leads to the accumulation of cleaved PARP1 in CRC cells. These results suggest that p8 induces apoptosis via regulation of RNF152, thus inhibiting the development of CRC.

Colorectal Cancer Therapy Using a Pediococcus pentosaceus SL4 Drug Delivery System Secreting Lactic Acid Bacteria-Derived Protein p8.

Despite decades of research into colorectal cancer (CRC), there is an ongoing need for treatments that are more effective and safer than those currently available. Lactic acid bacteria (LAB) show beneficial effects in the context of several diseases, including CRC, and are generally regarded as safe. Here, we isolated a Lactobacillus rhamnosus (LR)-derived therapeutic protein, p8, which suppressed CRC proliferation. We found that p8 translocated specifically to the cytosol of DLD-1 cells. Moreover, p8 down-regulated expression of Cyclin B1 and Cdk1, both of which are required for cell cycle progression. We confirmed that p8 exerted strong anti-proliferative activity in a mouse CRC xenograft model. Intraperitoneal injection of recombinant p8 (r-p8) led to a significant reduction (up to 59%) in tumor mass when compared with controls. In recent years, bacterial drug delivery systems (DDSs) have proven to be effective therapeutic agents for acute colitis. Therefore, we aimed to use such systems, particularly LAB, to generate the valuable therapeutic proteins to treat CRC. To this end, we developed a gene expression cassette capable of inducing secretion of large amounts of p8 protein from Pediococcus pentosaceus SL4 (PP). We then confirmed that this protein (PP-p8) exerted anti-proliferative activity in a mouse CRC xenograft model. Oral administration of PP-p8 DDS led to a marked reduction in tumor mass (up to 64%) compared with controls. The PP-p8 DDS using LAB described herein has advantages over other therapeutics; these advantages include improved safety (the protein is a probiotic), cost-free purification, and specific targeting of CRC cells.

Study on the safety of Polygala tenuifolia Willdenow root extract powder (BT-11) in young person aged from 9 to 19 years old.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygala tenuifolia Willdenow root extract (BT-11) has beneficial effects on central nervous system disorders in human. The safety of BT-11 should be elucidated in younger person further. AIM OF THE STUDY: To evaluate the safety of BT-11 in human aged from 9 to 19 years old. MATERIAL AND METHODS: The safety was evaluated in randomly assigned subjects who received the test products (61 subjects in BT-11 300 mg daily or 60 subjects in matching placebo) for 12 weeks. Adverse reactions were analyzed by the incidence rate, type, and severity. The clinical examination included hematology and blood chemistry tests, urinalysis, vital signs, body weight, and electrocardiogram (ECG). RESULTS: Eleven adverse reactions were observed in ten subjects receiving BT-11 while seven adverse reactions in six subjects receiving placebo. There were no statistical differences in the incidence of adverse reactions between the two groups. Serious adverse reactions such as acute appendicitis and acute viral gastroenteritis were observed in the BT-11 group4 and the placebo group, respectively. However, it was confirmed that they were not associated with the test product. All other adverse reactions observed during the test period were resolved completely without special treatment. No statistical difference was also observed in safety laboratory tests, vital signs, and ECG between two groups. CONCLUSIONS: This study demonstrates the safety of BT-11 in the adolescent by showing no apparent adverse reactions related to it.

A Probiotic Preparation Alleviates Atopic Dermatitis-Like Skin Lesions in Murine Models.

Atopic dermatitis (AD) is a chronic inflammatory skin disease with a complex etiology that encompasses immunologic responses. AD is frequently associated with elevated immunoglobulin (Ig) E levels, and common environmental factors contribute to its pathogenesis. Several recent studies have documented the role of specific lactic acid bacteria in the treatment and prevention of AD in humans and mice. In this study, the efficacy of Duolac ATP, a probiotic preparation, was determined in a mouse model with AD-like skin lesions. Alterations in the cytokine levels and histological staining suggested the alleviation of AD. The in vivo test showed that T helper (Th)2 cytokines, IgE, interleukin (IL)-4, and IL-5, were significantly downregulated, whereas Th1 cytokines, IL-12p40 and interferon (IFN)-γ, were upregulated in all groups of mice treated with Duolac ATP compared to that observed in the group of mice treated with 1-chloro-2,4-dinitrobenzene (DNCB) alone. Moreover, the scratch score decreased in all mice treated with Duolac ATP. Staining of the dorsal area of the mice in each group with hematoxylin and eosin and toluidine blue further confirmed the alleviation of AD in mice orally treated with Duolac ATP. These results suggest that Duolac ATP inhibits the development of AD-like skin lesions in NC/Nga mice by suppressing the Th2 cell response and increasing the Th1 cell response. Thus, Duolac ATP is beneficial and effective for the treatment of AD-like skin lesions.

Improvement of atopic dermatitis-like skin lesions by IL-4 inhibition of P14 protein isolated from Lactobacillus casei in NC/Nga mice.

Atopic dermatitis (AD) is a chronic inflammatory skin disease, with a complex etiology encompassing immunologic responses. AD is frequently associated with elevated serum immunoglobulin (Ig) E levels and is exacerbated by a variety of environmental factors, which contribute to its pathogenesis. However, the etiology of AD remains unknown. Recently, reports have documented the role of lactic acid bacteria (LAB) in the treatment and prevention of AD in humans and mice. The LAB, Lactobacillus casei (LC), is frequently used in the treatment of AD. To identify the active component of LC, we screened fractions obtained from the ion exchange chromatography of LC extracts. Using this approach, we identified the candidate protein, P14. We examined whether the P14 protein has anti-atopic properties, using both in vitro and in vivo models. Our results showed that the P14 protein selectively downregulated serum IgE and interleukin-4 cytokine levels, as well as the AD index and scratching score in AD-like NC/Nga mice. In addition, histological examination was also effective in mice. These results suggest that the P14 protein has potential therapeutic effects and that it may also serve as an effective immunomodulatory agent for treating patients with AD.

Use of selected lactic acid bacteria in the eradication of Helicobacter pylori infection.

Helicobacter pylori is among the major pathogenic bacteria that cause chronic gastritis and peptic ulcer disease and is related to the development of gastric cancer. Several chemicals, including antibiotics, have been used to eradicate H. pylori; however, they do not always curb the infection. Ten representative type strains of lactic acid bacteria (LAB) were screened for antagonism toward H. pylori via inhibition of urease activity. Strains inhibiting the binding of H. pylori to human gastric cell line cells and suppressing H. pylori-induced interleukin-8 (IL-8) production were also screened. Of these, Pediococcus pentosaseus (SL4), which inhibited the adhesion of H. pylori to MKN-45 gastric cancer cells, Bifidobacterium longum (BG7), with urease inhibiting activity, and Lactococcus lactis (SL3), and Enterococcus faecalis (SL5), which suppressed H. pylori-induced IL-8 production within MKN-45 and AGS cells, were selected. In mouse model, these LAB stains in combination significantly suppressed IL-8 levels in serum. Gastric pH also recovered to normal values after the administration of these LAB. These stains effectively suppressed H. pylori viability, although not to the extent of antibiotic treatment. When used as probiotics, LAB may help decrease the occurrence of gastritis and reduce the risk of H. pylori infection without, inducing side effects.

A Probiotic Preparation Duolac-Gold Ameliorates Dextran Sulphate Sodium-induced Mouse Colitis by Downregulating the Expression of IL-6.

Probiotics are live microorganisms that confer a health benefit on the host. Duolac-Gold is a mixture of six probiotic bacteria containing three species of Bifidobacteria, two species of Lactobacillus, and Streptococcus thermophilus [corrected]. The aim of this study was to assess the anti-inflammatory effects of Duolac-Gold in an inflammatory bowel disease (IBD) mouse model. IBD was induced by administering 1.5% dextran sulfate sodium (DSS) for 10 days. After induction of DSS-induced colitis, Duolac-Gold was orally administered at three different concentrations. Interestingly, Duolac-Gold treatment accelerated IBD healing, and anti-inflammatory activity was assessed by weight loss, length of the colon, and a microscopic damage score by histology. The expression of inflammatory related cytokines was measured in colon tissues and serum. Of these cytokines, the expression of interleukin-6 decreased remarkably after Duolac-Gold treatment. Taken together, these results suggest that Duolac-Gold treatment is effective in IBD healing by regulating IL-6.

A study on the prevention of salmonella infection by using the aggregation characteristics of lactic Acid bacteria.

Salmonella is one of the major pathogenic bacteria that cause food poisoning. This study investigated whether heat-killed as well as live Lactobacillus protects host animal against Salmonella infection. Live and heat-killed Lactobacillusacidophilus was administered orally to Sprague-Dawley rats for 2 weeks before the rats were inoculated with Salmonella. Rise in body temperature was moderate in the group that was treated with heat-killed bacteria as compared to the Salmonella control group. The mean amount of feed intake and water consumption of each rat in the heat-killed bacteria group were nearly normal. The number of fecal Salmonellae was comparable between the live and the heat-killed L. acidophilus groups. This finding shows that L. acidophilus facilitates the excretion of Salmonella. Moreover, the levels of pro inflammatory cytokines, including tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta, in the heat-killed L. acidophilus group were significantly lower when compared to the levels in the Salmonella control group. These results indicate that nonviable lactic acid bacteria also could play an important role in preventing infections by enteric pathogens such as Salmonella.