Carbapenemases for use with antibiotics for the protection of the intestinal microbiome
Inventors
Kaleko, Michael • Connelly, Sheila
Assignees
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Publication Number
US-11123413-B2
Publication Date
2021-09-21
Expiration Date
Abstract
This invention relates, in part, to various compositions and methods for protecting the gastrointestinal microbiome from antibiotic disruption.
Core Innovation
The invention relates to producing a broad spectrum carbapenemase in Escherichia coli. A host E. coli cell is transformed with a vector comprising a nucleic acid sequence encoding a carbapenemase operably linked to an inducible promoter. Expression is induced during culturing, and the carbapenemase is recovered from a soluble fraction prepared from the cytoplasm or periplasmic space of the E. coli cell.
The culturing step includes a culture medium comprising zinc while inducing the expression of the carbapenemase. The encoded carbapenemase comprises at least 95% sequence identity with a specified amino acid sequence, including SEQ ID NO: 68 (P2A) and SEQ ID NO: 53 (NDM-1).
The disclosure also relates to protecting the gastrointestinal (GI) microbiome from antibiotic disruption during administration of beta-lactam antibiotics by using an antibiotic-degrading agent in conjunction with the beta-lactam antibiotics to inactivate residual or unabsorbed antibiotic within the GI tract while preserving systemic antibiotic exposure and absorption. Additional context includes GI-delivered microbiome protection, CDI-related indications, inhibitor adjuncts, co-administration or co-formulation concepts, and localized GI release or activation, including modified or delayed-release formulations and enteric or pH-dependent coatings, including colon-specific delivery systems.
Claims Coverage
The consolidated claims coverage includes two independent claims. The claims share the inventive concept of producing a broad spectrum carbapenemase in E. coli by inducing an inducible promoter with zinc-containing culture medium and recovering the carbapenemase from a soluble fraction, with sequence-identity constraints to specified amino acid sequences.
Zinc-containing inducible expression in E. coli for soluble carbapenemase recovery
A host Escherichia coli cell transformed with a vector comprising a nucleic acid sequence encoding the carbapenemase operably linked to an inducible promoter is cultured in a culture medium comprising zinc and the expression of the carbapenemase is induced, followed by recovering the carbapenemase from a soluble fraction prepared from the cytoplasm or periplasmic space of the E. coli cell.
Sequence-identity defined broad spectrum carbapenemase (P2A)
The encoded carbapenemase comprises at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 68 (P2A).
Sequence-identity defined broad spectrum carbapenemase (NDM-1)
The encoded carbapenemase comprises at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 53 (NDM-1).
The independent claims cover inducible expression of broad spectrum carbapenemase in zinc-containing culture medium in E. coli, recovery from a soluble cytoplasmic or periplasmic fraction, and sequence-identity limits to either SEQ ID NO: 68 (P2A) or SEQ ID NO: 53 (NDM-1).
Stated Advantages
Preserving systemic antibiotic exposure and absorption while inactivating residual or unabsorbed antibiotic within the GI tract.
Documented Applications
Protecting the gastrointestinal (GI) microbiome from antibiotic disruption during administration of beta-lactam antibiotics by using an antibiotic-degrading agent in conjunction with the beta-lactam antibiotics.
GI-delivered microbiome protection.
CDI-related indications.
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