Translational Development of Microbiome-Based Therapeutics: Kinetics of E. coli Nissle and Engineered Strains in Humans and Nonhuman Primates

Abstract

Understanding the pharmacology of microbiome-based therapeutics is required to support the development of new medicines. Strains of E. coli Nissle (EcN) were genetically modified and administered to cynomolgus monkeys at doses of 1 × 10⁹ and 1 × 10¹² colony-forming units (CFU)/day for 28 days. A clinical study to evaluate the exposure and clearance of EcN in healthy volunteers was also performed. Healthy subjects received oral doses of EcN, 2.5 to 25 × 10⁹ CFU 3 times daily for 28 days or a single day. In cynomolgus monkeys, replicating strains yielded higher fecal concentrations than nonreplicating strains and persisted for longer following cessation of dosing. In the clinical study, all subjects cleared EcN following cessation of dosing with median clearance of 1 week. Quantitative methodology can be applied to microbiome-based therapeutics, and similar kinetics and clearance were observed for EcN in cynomolgus monkeys and humans.

Figure 1

Quantitation of E. coli Nissle‐derived strains in cynomolgus monkeys using quantitative PCR. The red color indicates a dose of 1 × 10⁹ CFU and the green color indicates a dose of 1 × 10¹² CFU; the colored solid lines indicate the geometric means for animals administered SYN975, while the colored dashed lines indicate animals administered SYNB1010; the gray background indicates the dosing period; the gray dashed lines indicate the LOQ, BLOQ, and Qual Neg. BLOQ is plotted as LOQ/2, and Qual Neg is plotted as LOQ/4. BLOQ, below the limit of quantitation with quantitative PCR but positive with qualitative PCR; EcN, E. coli Nissle; LOQ, limit of quantitation estimated as the lowest measured copies of EcN/mL; Qual Neg, one or both qualitative PCR primers were negative; SD, standard deviation.

Figure 2

Consort diagram for the clinical study.

Figure 3

E. coli Nissle clearance via qualitative assay with two negative results required to define clearance. The horizontal axis represents days since the last dose, and the vertical axis is the fraction of subjects continuing follow‐up (i.e., had not yet cleared E. coli Nissle). The solid and dashed lines indicate the Kaplan–Meier estimates for time to clearance and its 95% confidence interval; the arrows at the top indicate times when monitoring occurred with color indicating dosing regimen.

Figure 4

Time course of median E. coli Nissle quantitative PCR for single and multiple dose regimens. EcN, E. coli Nissle; PCR, polymerase chain reaction.