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

Abstract

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.

Keywords:

• biologics
• therapeutic protein
• probiotics derived anti-cancer protein P8
• Pediococcus pentosaceus SL4
• Lactobacillus rhamnosus
• colorectal cancer
• drug delivery system
• protein secretion system
• oral administration
• non-clinical trial
• toxicology finding

Acknowledgments

This study was supported by the World Class 300 Project, funded by the Small and Medium Business Administration [SMBA, S2367890 (S2416714)], Korea.

Abbreviations

LAB, Lactic acid bacteria; PP, Pediococcus pentosaceus SL4; LR, Lactobacillus rhamnosus; PP-P8, Pediococcus pentosaceus SL4 (PP) secretes the probiotic-derived anti-cancer protein P8; CRC, Colorectal cancer; IND, Investigational new drug; MFDS, The Ministry of Food and Drug Safety; FIH, First-in-human; IBD, Inflammatory bowel disorder; UC, Ulcerative colitis; CD, Crohn’s disease; DDS, Drug delivery system; r-P8, Recombinant P8 protein; 6×His, Hexa-histidine; OD, Optical density; PK, Pyruvate kinase promoter; MF, Microfiltration; TMP, Trans-membrane pressure; UF, Ultrafiltration; IAC, Immuno-affinity chromatography; Q-TOF, Quadrupole time-of-flight; DRF, Dose range-finding; Chemon IACUC, Chemon Institutional animal care and use committee; AAALAC International, Assessment and Accreditation of Laboratory Animal Care International; OMIF, Osong Medical Innovation Foundation; SOP, Standard Operating Procedure; CFU, Colony-forming units; ECGs, Electrocardiograms; GLU, Glucose; BIL, Bilirubin; KET, Ketone body; SG, Specific gravity; PRO, Protein; URO, Urobilinogen; NIT, Nitrite; BLO, Occult blood; LEU, Leukocyte; EOS, Eosinophil; MCHC, Mean corpuscular hemoglobin concentration; HCT, Hematocrit; NEU, Neutrophil; HGB, Hemoglobin; MCV, Mean corpuscular volume; LUC, Large unstained cell; LYM, Lymphocyte; RBC, Red blood cell; WBC, White blood cell; PLT, Platelet; MCH, Mean corpuscular hemoglobin; MONO, Monocyte; MPV, Mean platelet volume; BASO, Basophil; RDW, Red cell distribution width; HDW, Hemoglobin distribution width; ALB, Albumin; ALP, Alkaline phosphatase; AST, Aspartate aminotransferase; A/G ratio, Albumin/globulin; BUN, Blood urea nitrogen; TP, Total protein; CRE, Creatinine; TBIL, Total bilirubin; Ca₂^+, Calcium ion; TG, Triglyceride; K^+, Potassium ion; Cl^−, Chloride ion; TCHO, Total cholesterol; CPK, Creatine phosphokinase; ALT, Alanine aminotransferase; GLU, Glucose; Na^+, Sodium ion; IP, Inorganic phosphorus; PT, Prothrombin time; APTT, Thromboplastin time.

Consent for Publication

All authors agree with publication of this study.

Disclosure

All authors have no conflicts of interest to report.