Xirong Tian ¹ Gao Yamin ¹ Wanli Ma ¹ Jingran Zhang ¹ Yanan Ju ¹ Jie Ding ¹ Sanshan Zeng ¹ H.M. Adnan Hameed ¹ Nanshan Zhong ² Greg Cook ³ Jinxing Hu ^2* Tianyu Zhang ^1*

• ¹ Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences (CAS), Guangzhou, China
• ² Guangzhou National Laboratory, Guangzhou, China
• ³ Department of Microbiology and Immunology, School of Biomedical Sciences, University of Otago, Dunedin, Otago, New Zealand

The efficacy of many compounds against Mycobacterium tuberculosis is often limited when administered via conventional oral or injection routes due to suboptimal pharmacokinetic characteristics. Inhalation-based delivery methods have been investigated to achieve high local therapeutic doses in the lungs. However, previous models, typically employing wild-type M. tuberculosis strains, were intricate, timeconsuming, labor-intensive, and with poor reproducibility. In this study, we developed an autoluminescence-based inhalation administration model to evaluate drug activity by quantifying relative light units (RLUs) emitted from live mice infected with autoluminescent M. tuberculosis. This novel approach offers several advantages: (1) it eliminates the need for anesthesia in mice during administration and simplifies the instrument manipulation; (2) it is cost-effective by utilizing mice instead of larger animals; (3) it shortens the time from several months to 16 or 17 days for obtaining result; (4) it is non-invasive by directly measuring the live RLUs of mice as a surrogate marker for CFUs for in vivo drug activity testing; up to six mice can be administrated daily and simultaneously, even 2-3 times/day; results are relatively objective and reproducible results minimizing human factors. Proof-of-concept experiments demonstrated that inhalable rifampicin, isoniazid, and ethambutol showed anti-M. tuberculosis activity at concentrations as low as 0.5, 0.5, and 0.625 mg/mL, respectively, as evidenced by comparing the live RLUs of mice. Furthermore, consistency between RLUs and colony-forming units of the autoluminescent M. tuberculosis in lungs reaffirms the reliability of RLUs as an indicator of drug efficacy, highlighting the potential of this approach for accurately assessing anti-M. tuberculosis activity in vivo.This autoluminescence-based, non-invasive inhalation model offers a substantial reduction in the time, effort, and cost required for evaluating the efficacy of screening new drugs and repurposing old drugs in vivo via inhalation administration.