1712. In Vitro Activity of Epetraborole, a Novel Bacterial Leucyl-tRNA Synthetase Inhibitor, in Drug Combinations Against Nontuberculous Mycobacteria Including Resistance Frequency and MIC Characterization of Mycobacterium avium ATCC 700898 Epetraborole-resistant Mutants
Authors: DeStefano Michelle S , Shoen Carolyn M , Alley M R K , Cynamon Michael H 
Published: December 2022

Abstract

Background
Epetraborole (EBO) is a boron-containing, oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria (NTM). The standard of care therapy for Mycobacterium avium complex (MAC) lung disease consists of a combination of a macrolide, ethambutol (EMB) and a rifamycin. A total of 5 strains of MAC, M. abscessus ATCC 19977 and M. peregrinum ATCC 700686 were tested in checkerboard assays to evaluate the effects of combining EBO with other agents. The spontaneous resistance frequency (RF) was determined for EBO singly and in combination against M. avium ATCC 700898.

Methods
The effects of combining EBO with clarithromycin (CLR), rifabutin (RBT), EMB, amikacin (AMK) or bedaquiline (BDQ) were evaluated in 7 strains of NTM. Synergy, additive effects, indifference or antagonism was characterized in the checkerboard assay using EUCAST criteria. The RF of M. avium ATCC 700898 at 2x, 4x and 8x the MIC (8 mg/L) of EBO was determined, as was the RF of EBO combined with CLR, RBT, AMK or EMB. MICs of selected EBO mutants were determined against AMK, BDQ, CLR, RBT, EMB, and clofazimine (CFZ) and the mutants were further characterized.

Results
The RF of EBO ranged from 1.58x10-7 to 8.48x10-9 when selected on 2 - 8x agar MIC. The MIC for EBO increased 32- &gt; 256-fold for the resistant mutants; however, the MICs for the other drugs tested against EBO-resistant strains did not change significantly. The activity of EBO was not antagonized, and was mainly indifferent to the addition of CLR, RBT, AMK, or BDQ for all the NTM strains tested. Synergy with EMB was observed with 2 strains and additivity with 2 additional strains of MAC. The addition of EMB (24 mg/L), CLR (32 mg/L), RFB (2 mg/L) or AMK (128 mg/L) to agar plates containing 2x MIC of EBO lowered the RF to at least &lt; 2.13x10-10 (Table 1). Table 1.In Vitro Resistance Frequency for the Components of SOC with and without EBO in Mycobacterium avium ATCC 700898

Conclusion
In the checkerboard studies, no evidence of antagonism was observed with any strain or EBO combination; interactions were largely indifferent. EBO combined with EMB in MAC resulted in synergy or additive effects in the checkerboard assay. The addition of EMB, CLR, RFB or AMK to EBO led to a &gt; 700-fold reduction in RF. Activity of other drugs was not impacted by EBO resistance suggesting that cross-resistance did not occur.

Disclosures
Michelle S. DeStefano, n/a, AN2 Therapeutics: Grant/Research Support Carolyn M. Shoen, PhD, AN2 Therapeutics: Grant/Research Support MRK Alley, PhD, ABBOTT LABS: Stocks/Bonds|ABBVIE: Stocks/Bonds|AN2 Therapeutics: Author on epetraborole patent|AN2 Therapeutics: Salary|AN2 Therapeutics: Ownership Interest|AVANOS MED INC: Stocks/Bonds|NABRIVA THERAPEUTICS PLC: Stocks/Bonds|NOVARTIS AG: Stocks/Bonds Michael H. Cynamon, MD, AN2: Grant/Research Support|AN2: Grant/Research Support.