**5. Toxicology**

In vivo animal toxicity tests on delpazolid did not reveal specific toxicity profiles for six months in rats and for nine months in dogs. Furthermore, genetic toxicity tests, including the Ames test, in vitro chromosomal aberration test, and rat micronucleus test, as well as pharmacological safety tests including the hERG safety test, cardiovascular, respiratory and neurobehavioral tests, and reproductive toxicity tests were conducted, none of which revealed a specific toxicity profile (Table 3).

**Table 3.** Toxicology summary (PO: per oral /IV: intravenous). The general toxicity of delpazolid in animals lasted up to six months in rats and nine months in dogs, and no unusual findings after long-term treatment *<sup>a</sup>*.


MTD; maximum tolerated dose, NOAEL; no-observed-adverse-effect level. *<sup>a</sup>* Results were not yet published.

In a human bioavailability study, the bioavailability of the PO form was 99–100% (800 mg) of that of the IV form. Considering that the PK profiles between the IV and PO forms are similar, conversion would be relatively easy in the future. Because delpazolid is slightly polar, it exhibits low protein binding (37% in human), rapid clearance with no accumulation, and no food-related effects (Table 4).

**Table 4.** Phase 1 study: summary of delpazolid pharmacokinetic parameters. IV infusion 400 mg and PO 800 mg, cross-over study IV administration of delpazolid was generally safe and well-tolerated 800 mg (PO): Bioavailability was approximately 99% switchable between the PO and IV, with no dose adjustment.


*<sup>a</sup>* Values are the means ± standard deviation (range). Cmax, maximal drug concentration; Tmax, time to reach Cmax; T1 2 , half-life; AUC0-24, area under the concentration-24-h curve; AUCinf, AUC from time zero extrapolated to infinity; Vss, steady-state volume of distribution; Vz/F, apparent volume of distribution; CL, clearance; Cl/F, apparent oral clearance; MRTlast, mean residence time when the drug concentration is based on values up to and including the last measured concentration; Cmax, norm, Cmax divided by dose per body weight; AUCinf, norm, weight-normalised AUCinf; F, bioavailability.

#### **6. Activity Against TB and Combination Study of Delpazolid with Other Anti-TB Agents**

Studies of the early development of delpazolid focused on Gram-positive bacteria. The efficacy of delpazolid on Gram-positive bacteria was similar or slightly better than that of linezolid. For example, in animal studies of systemic infection [17], soft tissue infection, lung infection, and thigh infection models in mice, delpazolid showed greater efficacy than linezolid (data not shown).

To evaluate the efficacy of delpazolid in TB, an in vitro susceptibility test was conducted for *M. tuberculosis* H37Rv. Compared to linezolid, the minimum inhibitory concentration (MIC) for *M. tuberculosis* H37Rv was similar to that under delpazolid; however, the minimum bactericidal concentration was more than 4-fold lower under delpazolid (Table 5).


**Table 5.** Drug activities and resistance rates of linezolid and delpazolid.

*<sup>a</sup>* A total of 240 *M. tuberculosis* isolates were tested for ECOFFS and resistant rates, including 120 MDR-TB isolates and 120 XDR-TB samples in China.

The MIC90 values of delpazolid for MDR/extensively drug resistant (XDR) TB isolates were 0.25 and 1 μg/mL, respectively. However, an in vitro study of MDR/XDR TB isolates from China showed that the resistance rate varied considerably. The resistance of MDR-TB to linezolid was 6.7%, whereas that to delpazolid was 0.8%, suggesting higher potential efficacy of delpazolid in the treatment of MDR-TB, although no significant difference in resistance rates was observed between linezolid and delpazolid among XDR-TB isolates [18]. Therefore, delpazolid has been considered as a targeted application for MDR-TB treatment. Considering the significantly lower resistance rate of MDR-TB against delpazolid despite its similar structure to linezolid, further studies are needed to investigate structural variations in delpazolid to evaluate the correlations between the structures of various delpazolid derivatives and their resistance rates. In addition, intracellular MICs of delpazolid that can inhibit the growth of intracellular *M. tuberculosis* H37Rv revealed efficacy levels similar to those of linezolid under low concentrations, whereas delpazolid had greater efficacy at higher concentrations (Figure 3).

**Figure 3.** Intracellular activity of delpazolid. The activity of delpazolid on intracellular *M. tuberculosis* was compared to linezolid in bone marrow-derived macrophages (BMDMs) at three days after infection. The experiment was performed in triplicate, and the results are shown as the mean ± standard error of the mean (SEM). SC, solvent control.

The treatment of tuberculosis requires a combination of several antimicrobial agents and long-term therapy [19]. Therefore, evaluating synergy with other anti-TB agents is a crucial step in finding drugs that can be co-administered with delpazolid. As indicated in Table 6, a checkerboard assay was performed to identify pre-existing anti-TB medications with potential synergistic effects with delpazolid.


**Table 6.** MICs of selected anti-tuberculosis compounds against *M. tuberculosis* H37Rv and corresponding interaction profiles with delpazolid assessed by checkerboard.

<sup>a</sup> S: synergy, pS: partial synergy, Ad: additive, I: indifference. <sup>b</sup> Tested in acidic condition (pH 5.2)

The assay revealed that delpazolid has partial synergism with clofazimine, bedaquiline, and pyrazinamide. Based on the results, in vitro time-kill kinetics tests were conducted by combining delpazolid with clofazimine and bedaquiline (Figure 4).

**Figure 4.** In vitro combination time-kill assay with anti-TB drugs. Viability of *M. tuberculosis* H37Rv was evaluated using combinations of various concentrations of delpazolid and bedaquiline or clofazimine (μg/mL).

Using the MIC against *M. tuberculosis* H37Rv for each drug, changes in colony-forming units (CFU) with monotherapy or combination therapy were evaluated. In addition, based on the MICs, synergistic effects between delpazolid plus bedaquiline and delpazolid plus clofazimine were evaluated at varying doses. Although the CFUs decreased at the MIC of a single drug, regrowth was observed over time. However, when delpazolid was combined with bedaquiline or clofazimine, using the 0.5× MIC of each drug, no regrowth was observed (Figure 4). In addition, the combination of bedaquiline and clofazimine with delpazolid consistently suppressed the growth of *M. tuberculosis* H37Rv, exhibiting high synergistic effects with 1× MIC delpazolid (1 μg/mL) and 0.5 × MIC clofazimine (0.25 μg/mL), resulting in a 2 log CFU reduction in *M. tuberculosis* H37Rv. Synergy between two new antimycobacterial compounds, such as delpazolid and bedaquiline or clofazimine, offers an attractive foundation for a new tuberculosis regimen.
