**5. From PBTZ169 to Macozinone: First Results of the Clinical Studies**

Establishment of a non-profit Innovative Medicines for Tuberculosis Foundation (iM4TB Foundation) at Ecole Polytechnique Fédérale de Lausanne (EPFL) in March 2014 was a principal milestone for the further development of PBTZ169 and its transformation from a valuable TB drug candidate to the compound reaching promising results in the pilot clinical studies (Figure 3) [21]. This approach was necessary to overcome the lack of interest from pharmaceutical companies to address the disease primarily affecting low-income countries. The mission of the foundation is "to develop better and faster-acting medicines to fight tuberculosis and bridge the gap between scientific discovery and the market in order to provide affordable TB treatment to anyone in the world". In July 2014 the foundation joined in this effort with the Russian pharmaceutical company Nearmedic Plus LLC, which bought a license covering the use of PBTZ169 in most countries of the former Soviet Union, while iM4TB retained the rights for the rest of the world. Investments of Nearmedic, along with funds raised by iM4TB, including the generous support of the Bill and Melinda Gates foundation, enabled performing all necessary pre-clinical trials with PBTZ169, such as ADME (absorption, distribution, metabolism, excretion) profiling, toxicology studies, as well as production of clinical supplies of the drug, and preparation of regulatory documents necessary for entering the first clinical trials [21,22].

Clinical studies were initiated by both iM4TB and Nearmedic. In the randomized, double-blind, placebo-controlled Phase 1a study performed in Switzerland (NCT03423030) [23] the aim was to assess safety, tolerability and pharmacokinetic profile of PBTZ169 formulated as a spray-dried dispersion versus a native crystal powder. In addition, antitubercular activity of single escalating doses of PBTZ169 ex vivo was evaluated. The study included 32 healthy male volunteers, grouped to 4 panels of 8 subjects, each undergoing 2 investigation periods, during which they received either single doses of PBTZ169 at increasing dose levels up to 320 mg or a matching placebo. Promising results of this study encouraged the iM4TB team to design of the Phase 1b trial (NCT03776500) [24] aimed at evaluation of the safety, tolerability and pharmacokinetics of PBTZ169 in multiple dosing up to 1200 mg/day in healthy volunteers (32 subjects grouped in 4 consecutive panels) receiving PBTZ169 for 14 consecutive

days. An additional goal is to evaluate interactions of PBTZ16 with the human cytochrome P-450 enzyme family [24].

**Figure 3.** Pre-clinical and clinical development of macozinone.

The first Phase 1 clinical trial in Russia (Nearmedic Plus LLC) (NCT03036163, PBTZ169-Z00-C01-1) [25] with an official title "Open-label Prospective Non-comparative Study of Safety, Tolerability and Pharmacokinetics of PBTZ169 after Single and Multiple Fasting Oral Administration in Increasing Doses in Healthy Volunteers" was carried out between January and November 2016. The drug was administered in the form of capsules containing 40 mg of the drug. The study included single escalating doses (40, 80, 160, 320, and 640 mg) in fasting conditions, as well as multiple fasting doses (320 and 640 mg) for 14 days. Overall the study included 40 participants [22].

A completed open-label prospective Phase 1b study initiated by Nearmedic Plus LLC (NCT04150224, PBTZ169-Z00-C01-3) [26] was designed to assess the safety, tolerability, pharmacokinetics, and food effects on single, double, and multiple escalating doses in healthy volunteers. During the first part of the study, safety, tolerability, and pharmacokinetics of PBTZ169 in 80 mg capsules were studied in healthy volunteers who received single or double (twice daily) fasting doses, which increased sequentially (640, 960, and 1280 mg once a day and 640 mg twice a day). The food effects were studied for a single 640 mg dose. The second part of the study involved the safety, tolerability, and pharmacokinetics of PBTZ169 in 80 mg capsules in healthy volunteers who received 1280 mg daily in fed conditions, for 14 days. In total, 60 healthy volunteers (10 in each cohort) received the investigational drug and completed the study according to the protocol [22].

During these clinical trials, safety and tolerability of PBTZ169 in capsules were studied, including assessment of adverse effects, vital signs (blood pressure, heart rate, body temperature, respiratory rate), laboratory and instrumental parameters (hematology, blood chemistry, coagulation parameters, urinalysis, electrocardiography) and physical examination. In these studies (NCT03036163 and NCT04150224), good tolerability and favorable safety profile were demonstrated for PBTZ169 in the studied dose range. One event of dose-limiting toxicity (increase in the glucose level 2 hours after the drug administration) was documented after a single 80 mg dose of PBTZ169 in 40 mg capsules (NCT03036163). There were no other cases of dose-limiting toxicity in healthy volunteers in these studies. There were no serious adverse effects. There was no increase in the frequency of adverse effects associated with increased dose or with the administration regimen (fasting or in fed conditions), for single or multiple doses. Changes in some mean vital signs, laboratory, instrumental parameters, or physical examination data were not associated with a trend to increase with a growing dose [22].

Efficacy of PBTZ169 based on the early bactericidal activity (EBA), was studied in a Phase 2a clinical trial (PBTZ169-A15-C2A-1, NCT03334734) [27] by Nearmedic Plus LLC in patients with newly diagnosed smear-positive tuberculosis of the respiratory tract, with preserved sensitivity to isoniazid and rifampicin. Although the trial was terminated early because of slow enrollment, relevant information was obtained from 16 patients who participated in the study. The drug was administered in 80 mg capsules, orally as a monotherapy for 14 days, as follows: 160 mg/day dose—4 patients; 320 mg/day dose—4 patients; 640 mg/day dose—7 patients. Isoniazid tablets were used as a control treatment at 600 mg/day dose in one patient.

The primary efficacy of the treatment was evaluated by two methods:

(i) According to quantification of colony forming units in sputum by its inoculation on agar plates, in the group of patients receiving PBTZ169 in the 640 mg dose, EBA 0–14 (mean of the two measurements) and EBA 0–14 (the higher of the two measurements) were 0.071 log10 cells/ml/day [95% confidence interval (CI) 0; 0.143] and 0.080 log10 cells/ml/day [95% CI 0.002; 0.158], respectively.

(ii) According to quantitative polymerase chain reaction, in the same group of patients, EBA 0–14 (mean of the two measurements) and EBA 0–14 (the higher of the two measurements) were 0.098 log10 cells/ml/day [95% CI 0.021; 0.175] and 0.100 log10 cells/ml/day[95% CI 0.021; 0.180], respectively.

Thus, the analysis of efficacy based on the data obtained in a pilot Phase 2a clinical study revealed statistically significant early bactericidal activity of PBTZ169 14 days after the start of monotherapy (EBA0-14) in the group of PBTZ169 with the 640 mg dose. During this clinical study, there were no cases of death or severe adverse effects related to the PBTZ169. No adverse effects were considered as definitely related to the drug and, at the study completion, all adverse effects were resolved. There was no increase in adverse effects frequency with growing dose [22].

During the clinical studies it was confirmed that PBTZ169 after single, double, or multiple doses was detectable in the blood plasma of all volunteers and patients who had received the drug. However, it should be noted that pharmacokinetic studies in animals and in healthy volunteers (in Phase 1 clinical studies) demonstrated that PBTZ169, being more soluble in an acidic environment, is absorbed primarily in the stomach. These data agree with the results of in vitro studies, which were conducted by Nearmedic Plus LLC, assessing cellular permeability and solubility in biorelevant media, which mimicked native gastric and intestinal juices. They confirmed that the highest solubility of PBTZ169 is observed at the pH range of 1 to 2, which coincides with the pH in the lower part of the stomach, and its solubility gradually decreases with the growing pH. In the media with pH higher than 5, PBTZ169 was virtually insoluble. According to the test of solubility in biorelevant media, the drug level in the gastric juice was much higher than in the intestinal juice. So, one of the possible methods to increase bioavailability and exposure of PBTZ169 is to prolong its presence in the stomach.

After a single fasting dose, PBTZ169 absorbed rapidly, and the speed of absorption was independent of the administered dose (median Tmax was 1.5–2.5 hours for the dose range 40–640 mg (NCT03036163), and 1.5–1.75 hours for the dose range 640–1280 mg (NCT04150224). Meantime of retention of the drug in the body was 13.02–19.30 hours and was independent of the administered dose (NCT03036163) [22].

According to the obtained pharmacokinetic data, dose proportionality of Cmax and AUC0-t was demonstrated for single, as well as for multiple administrations of the investigational drug in the dose range 40–640 mg (NCT03036163, NCT03334734). After single fasting escalating doses (640, 960, and 1280 mg), Cmax, AUC0-24, AUC0-<sup>∞</sup> were less than dose proportional. Thus, it can be concluded that there is a trend for linear pharmacokinetics for single and multiple doses up to 640 mg.

When pharmacokinetics of different administration regimens for 1280 mg daily dose was studied (640 mg twice daily or 1280 mg once in fasting conditions), the pharmacokinetic parameters AUC0-<sup>∞</sup> and AUC0-t increased 1.5–1.6-fold (NCT04150224). The relation of mean AUC0-<sup>∞</sup> and AUC0-t in cohorts receiving the investigational drug as 640 mg twice a day and 1280 mg once a day, was 153.68% and 160.32%. For Cmax, a slight decrease (1.1-fold) was observed for the 640 mg twice a day (the relation of means was 90.84%). All observed differences were statistically significant.

According to the results of analysis of relative bioavailability and relative degree of absorption, the main pharmacokinetic parameters of PBTZ169 substantially and statistically significantly increased when 640 mg dose was administered in fed conditions: AUC0-∞increased 3.45-fold, AUC0-t (f') increased 3.50-fold, Cmax increased 2.29-fold. The drug absorbed and cleared from the body significantly more slowly, when it was administered in fed conditions compared to the fasting conditions (median Tmax

increased 2-fold, mean T1/<sup>2</sup> increased 1.44-fold). These data allow to conclude that it is preferable to administer PBTZ169 in fed conditions [22].

In October 2018, the international non-proprietary name "macozinone" was given to PBTZ169 by WHO [28]. In addition to the clinical studies, macozinone is undergoing further in vitro and in vivo testing focused on improvement of its pharmacological properties by structure-based design [29], or on the interactions with anti-TB drugs [30]. Importantly, an efficient method for monitoring metabolism of macozinone in human plasma was recently developed. It enables simultaneous measurement of concentrations of macozinone and its five active metabolites in the clinical samples, which is important for comprehensive pharmacokinetic/pharmacodynamic analyses [31].
