Antitubercular agent

In vitro antimicrobial profile and frequency of resistance generation. [1]
The MIC of TBA-354 against replicating M. tuberculosis H37Rv is approximately equivalent to that observed with delamanid and about 1 order of magnitude lower than that of Pa (Table 1). All three nitroimidazoles are bactericidal against replicating cultures with MBCs equal to MICs. MIC shifts against replicating M. tuberculosis of all compounds in the presence of 10% fetal bovine serum or 4% BSA (a physiologically equivalent concentration of albumin) were minimal (Table 1), in no case exceeding 3× the MIC obtained in the standard culture medium which contains 0.5% BSA. Although all three compounds are somewhat less active against NR M. tuberculosis (determined under low-oxygen conditions) than against replicating M. tuberculosis, the relative activities of the three compounds against the NR cultures were similar to those observed with the replicating cultures, regardless of whether this was assessed by the recovery of a luminescence signal upon return to normoxia (LORA MIC) or by CFU (LORA MBC) (Table 1).

Activities for all three compounds are maintained against M. tuberculosis H37Rv isogenic strains resistant to the TB drugs isoniazid (INH), rifampin (RIF), streptomycin (STP), and kanamycin (KAN) (Table 2). For a panel of 10 drug-sensitive and drug-resistant clinical M. tuberculosis isolates sourced from China, the TBA-354 MIC was lower than that of Pa against each individual strain. The MIC range for TBA-354 across the 10 strains tested was <0.02 to 0.36 μM and that of Pa was 0.38 to 1.39 μM (Table 3). Although there was variability in the MICs for TBA-354 and Pa observed among the pansensitive clinical M. tuberculosis isolates, the MICs for the drug-resistant strains did not exceed those obtained for the pansensitive strains.

The MICs of all three compounds were >50 μM against S. aureus, E. coli, and C. albicans (Table 4), suggesting that, similar to Pa and delamanid, TBA-354 is a narrow-spectrum agent. TBA-354 and Pa, which were also assessed for activity against nontuberculous mycobacteria, maintained potent activity against M. bovis but not against most of the other mycobacterial species. TBA-354 was at least 7 times more active against M. kansasii than was Pa (Table 4). Spontaneous mutants of M. tuberculosis H37Rv that are resistant to TBA-354 arose at a frequency of approximately 3 × 10−7 (range, 2 × 10−6 to 1 × 10−7) when plated on medium containing 0.025 μM TBA-354, which is similar to the observed frequency of spontaneous mutant generation for Pa.

In vivo activity against murine TB. [1]
We previously reported that all three nitroimidazoles demonstrate efficacy against murine TB, using a low-dose aerosol infection model, when administered during the acute or chronic phase of infection, at 100 mg/kg daily for 5 of 7 days per week for 3 weeks. As illustrated in Fig. 3a, TBA-354 and delamanid exhibited significant bactericidal activity (P < 0.001) in that assay when treatment began in the acute phase of infection, reducing lung CFU by approximately 1 log10 relative to that of the pretreatment controls. Both drugs were more active than Pa (P < 0.001); Pa inhibited growth of M. tuberculosis in the lungs of the mice, relative to that in the untreated controls, but did not reduce the number of lung CFU below the pretreatment levels. When treatment was started during the chronic phase of infection (Fig. 3b), the relative activities of the three nitroimidazoles were similar to that observed when treatment was initiated during acute infection (Fig. 3a). However, superior bactericidal activity was demonstrated by TBA-354 and delamanid in the chronic infection model relative to that in the acute infection model, with a 2 to 3 log10 reduction in lung CFU evident relative to the pretreatment CFU following treatment with these compounds. Again, both TBA-354 (P = 0.007) and delamanid (P = 0.018) were more active than Pa but not significantly different from each other (P = 0.538).

To confirm and extend those findings, we evaluated the efficacy of TBA-354 against chronic murine TB, using the same low-dose aerosol infection model, with lung CFU determined after 2, 4, and 8 weeks of treatment. In this study, TBA-354 demonstrated dose- and time-dependent killing of M. tuberculosis. Bactericidal activity was significant compared to that of the untreated controls (P < 0.001) at the lowest dose tested (10 mg/kg), and at that dosage, the activity demonstrated by TBA-354 was not significantly less than that demonstrated by delamanid administered at 10 mg/kg or 30 mg/kg and was only significantly less than that shown by delamanid at 100 mg/kg when the treatment duration was 8 weeks (P = 0.033) (Fig. 4). After 8 weeks of treatment, TBA-354 at 30 mg/kg effected a larger reduction in CFU than delamanid at 30 mg/kg (P < 0.001), and this was the only significant difference observed in efficacy between the two compounds administered at equivalent dosages and for the same treatment durations in the experiment. An additional evaluation of TBA-354 in chronically infected mice was performed, in which the untreated controls achieved a bacterial load in lungs that was approximately 1 log10 lower than that for the experiments described above. TBA-354, at the lowest dose of 3 mg/kg, demonstrated a significant reduction in lung CFU after 4 weeks of treatment (P < 0.001), and the magnitudes of killing observed at this dosage and at 10 mg/kg were not significantly different from that of delamanid at 100 mg/kg (data not shown). Finally, no toxicity (as evaluated through clinical observations and monitoring of body weight) was observed during any of these efficacy studies (data not shown).

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New regimens based on two or more novel agents are sought in order to shorten or simplify the treatment of both drug-susceptible and drug-resistant forms of tuberculosis. PA-824 is a nitroimidazo-oxazine now in phase II trials and has shown significant early bactericidal activity alone and in combination with the newly approved agent bedaquiline or with pyrazinamide with or without moxifloxacin. While the development of PA-824 continues, a potential next-generation derivative, TBA-354, has been discovered to have in vitro potency superior to that of PA-824 and greater metabolic stability than that of the other nitroimidazole derivative in clinical development, delamanid. In the present study, we compared the activities of PA-824 and TBA-354 as monotherapies in murine models of the initial intensive and continuation phases of treatment, as well as in combination with bedaquiline plus pyrazinamide, sutezolid, and/or clofazimine. The monotherapy studies demonstrated that TBA-354 is 5 to 10 times more potent than PA-824, but selected mutants are cross-resistant to PA-824 and delamanid. The combination studies revealed that TBA-354 is 2 to 4 times more potent than PA-824 when combined with bedaquiline, and when administered at a dose equivalent to that of PA-824, TBA-354 demonstrated superior sterilizing efficacy. Perhaps most importantly, the addition of either nitroimidazole significantly improved the sterilizing activities of bedaquiline and sutezolid, with or without pyrazinamide, confirming the value of each agent in this potentially universally active short-course regimen [2].

MICs and MBCs against M. tuberculosis under aerobic and low-oxygen conditions. [1]
The M. tuberculosis H37Rv strain was used. [1]
(i) Aerobic conditions (replicating M. tuberculosis). [1]
MICs were determined using the microplate alamarBlue assay (MABA) as follows. Cultures were incubated in 200 μl 7H12 medium together with the test compound in 96-well plates for 7 days at 37°C. alamarBlue and Tween 80 were added, and incubation was continued for 24 h at 37°C. Fluorescence was determined at excitation/emission wavelengths of 530/590 nm, respectively. The MIC was defined as the lowest concentration effecting a reduction in fluorescence of 90% relative to that in the controls.

(ii) Low-oxygen conditions (NR M. tuberculosis). [1]
The low-oxygen recovery assay (LORA) was used, and the MIC was defined as the lowest concentration of the compound which reduces luminescence by 90% after 10 days of exposure to the compound under low oxygen followed by 28 h of aerobic recovery and comparison to the untreated controls. The MABA and LORA minimal bactericidal concentration (MBC) values were the lowest concentrations of test compounds which reduce CFU by 99% after 7 days (aerobic) or 10 days (low-oxygen) exposure relative to CFU at the start of the 7-day (aerobic) or 10-day (low-oxygen) exposure.

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MICs against M. tuberculosis H37Rv isogenic monoresistant strains. [1]
MICs were determined against M. tuberculosis H37Rv isogenic strains monoresistant to rifampin (ATCC 35838), isoniazid (ATCC 35822), streptomycin (ATCC 35820), and kanamycin (ATCC 35827) via the MABA as described above.

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Spectrum of activity. [1]
Mycobacterium bovis (ATCC 19210), M. scrofulaceum (ATCC 19981), M. kansasii (ATCC 12478), M. gilvum (ATCC 43909), M. fortuitum (ATCC 6841), M. triviale (ATCC 23292), and M. smegmatis (ATCC MC2155) were cultured in Middlebrook 7H9 broth with 0.2% (vol/vol) glycerol, 0.05% Tween 80, and 10% (vol/vol) albumin-dextrose-catalase (BBL Middlebrook ADC enrichment, catalog no. 212352) (7H9-ADC-TG). Staphylococcus aureus (ATCC 29213) and Escherichia coli (ATCC 25922) were cultured in cation-adjusted Mueller-Hinton (CAMH) broth and Candida albicans (ATCC 90028) in RPMI medium until an absorbance at 570 nm of 0.2 to 0.5 was achieved. Cultures were diluted 1:5,000 to 1:10,000 in fresh medium in 96-well plates and incubated at 37°C with test compounds. Incubation times were 3 days for M. smegmatis, 7 days for other mycobacteria, 36 to 48 h for C. albicans, and 16 to 20 h for S. aureus and E. coli. For C. albicans, S. aureus, and E. coli, the MIC was defined as the lowest concentration effecting a reduction of ≥90% in A570 relative to that of untreated cultures. The MABA MICs for mycobacteria were defined as described above.

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Effect of serum/albumin on M. tuberculosis MICs. [1]
To test whether protein binding, which can reduce unbound, active compound, altered the apparent activity of TBA-354 and Pa, the MICs of the compounds against M. tuberculosis H37Rv were determined via the MABA (described above) without supplemental protein, in the presence of 4% bovine serum albumin (BSA), and in the presence of 10% fetal bovine serum (26). Note that the basal medium contains 0.5% BSA.

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MICs against drug-resistant and drug-susceptible M. tuberculosis clinical isolates. [1]
The M. tuberculosis clinical isolates were obtained from the State Laboratory of Tuberculosis Reference of China. Bacteria were cultured and tested in Middlebrook 7H9 broth with 0.2% (vol/vol) glycerol, 0.05% Tween 80, and 10% (vol/vol) albumin-dextrose-catalase. The MABA MIC was defined as described above.

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Frequency of resistance development. [1]
Four replicate cultures of M. tuberculosis H37Rv were prepared in 50-ml flasks, each containing 10 ml Middlebrook 7H9 broth plus glycerol, Casitone and oleic acid-albumin-dextrose-catalase (OADC) supplement at an initial density of M. tuberculosis of <104 CFU/ml and monitored for the presence of preexisting resistant bacteria by plating 100-μl aliquots on medium containing the test compound at 4× MIC. Cultures were then incubated for 2 to 3 weeks with shaking until an A570 of >0.9 (approximately 2 × 109 CFU/ml) was obtained. From each of four cultures, 100-μl aliquots of undiluted and 1:10 diluted suspensions (approximately 1 × 109 CFU/ml and 1 × 108 CFU/ml, respectively) were plated on 10 plates of 7H11 agar with and without 4× MIC of TBA-354. Individual mutation frequencies were calculated for each of the four cultures, and the median value was selected as representative.

Bidirectional permeability in Caco-2 cells. [1]
The Caco-2 permeability assay was developed based on the method of Hidalgo et al. In brief, a 96-well multiscreen plate with Caco-2 cells was cultured for 21 to 25 days. TBA-354 at 1 and 10 μM was incubated at 37°C for 40 or 60 min with the Caco-2 cell monolayer in Hanks' balanced salt solution plus HEPES or morpholineethanesulfonic acid (MES) (containing a final concentration of 1% dimethyl sulfoxide [DMSO] from a stock solution), in the absence and presence of ketoconazole (100 μM). The permeability through the cell barrier was measured in duplicate in both directions by taking aliquots from the apical (A) side and the basolateral (B) side. TBA-354 concentrations were analyzed by liquid chromatography-tandem mass spectroscopy (LC-MS/MS) (see “LC-MS/MS analysis methods” below).

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Plasma protein binding [1]
TBA-354 binding to proteins of human, monkey, dog, rat, and mouse plasma was conducted with a 96-well equilibrium dialysis apparatus with a 12,000- to 14,000-molecular-weight-cutoff dialysis membrane. TBA-354 was added to the plasma side at 10 μM and was shaken for 8 h at 37°C. Each sample was assessed in duplicate. TBA-354 concentrations were measured by LC-MS/MS as described below.

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In vitro metabolism. (i) Metabolism in liver microsomes. [1]
TBA-354 at 1 μM was incubated with rat, mouse, dog, monkey, or human liver microsomes (HLM) (0.3 mg/ml) at 37°C for 1 h in phosphate buffer (pH 7.4) with a NADPH regeneration system (1 mM NADP+, 5 mM glucose 6-phosphate [G6P], 1 U/ml glucose-6-phosphate dehydrogenase [G-6-PDH]). HLM were from a mixed-gender pool; all other species consisted of pooled male microsomes. Upon terminations of the incubations by addition of acetonitrile, each extract was analyzed by LC-MS/MS (see “LC-MS/MS analysis methods” below) to determine the remaining concentration of TBA-354. Positive controls (imipramine, propranolol, terfenadine, and verapamil) showed approximately 30% to 100% losses after the microsomal incubation in all species. To determine the microsomal intrinsic clearance, TBA-354 at 1 μM was incubated with human liver microsomes (0.3 mg/ml) in the presence of a NADPH regeneration system, and the percentages of the parent compound remaining were measured after 0-, 15-, 30-, 45-, and 60-min incubations. The positive controls (terfenadine and verapamil) showed high microsomal clearance (CLint of 124 and 115 ml/min/mg) in this assay.

(ii) Metabolism in hepatocytes. [1]
TBA-354 at 1 μM was incubated with mouse, rat, rabbit, dog, monkey, or human hepatocytes (at ca. 1 × 106 cells/ml) at 37°C for 0, 0.5, 1, 2, and 4 h. The metabolic stability of TBA-354 in human hepatocytes was assessed based on the relative concentrations of the remaining TBA-354 in the 0.5-, 1-, 2-, and 4-h incubations compared to that of the 0-h incubation. The half-life of TBA-354 metabolism in hepatocytes was calculated based on the first-order decay reaction if significant loss of TBA-354 was measured. The results for the positive-control samples demonstrated that the enzymes in the prepared hepatocyte suspensions were active.

(iii) Metabolism by recombinant human CYPs. [1]
The metabolism of TBA-354 by several cytochrome P450 (CYP) isozymes as a function of time was determined by incubating 1 μM TBA-354 with human recombinant CYP2C9, CYP2C19, CYP2D6, and CYP3A4 (0.1 nmol/ml) for 0, 15, 30, 45, and 60 min in the presence of NADPH (1 mM).

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Inhibition of CYPs. [1]
TBA-354 at 10 μM (for single-concentration assessments) was incubated with individual CYP isoforms at 37°C for 20 to 30 min in pooled HLM (0.25 mg protein/ml), phosphate buffer (100 mM, pH 7.4), magnesium chloride (MgCl2) (5 mM), NADPH (1 mM), and CYP probe substrates (at the approximate Km). The CYP probe substrates and reactions were phenacetin dealkylation for CYP1A2, coumarin 7-hydroxylation for CYP2AD6, bupropion hydroxylation for CYP2B6, paclitaxel 6′-hydroxylation for CYP2C8, diclofenac 4′-hydroxylation for CYP2C9, S-mephenytoin 4′-hydroxylation for CYP2C19, bufuralol 1′-hydroxylation for CYP2D6, chlorzoxazone 6-hydroxylation for CYP2E1, and midazolam 1′-hydroxylation and testosterone 6-hydroxylation for CYP3A4. Experiments were performed in duplicate. Following the addition of acetonitrile and centrifugation at 3,000 to 4,000 rpm for 10 min at 4°C to remove protein, supernatants were analyzed for the formation of CYP probe metabolites by LC-MS/MS, and the percent inhibition was estimated. To determine the 50% inhibitory concentrations (IC50s) of TBA-354 inhibition of CYP2C8, CYP2C19, and CYP3A4 (with two substrates, midazolam and testosterone), TBA-354 (0.01, 0.1, 1, 10, and 30 μM) was incubated with HLM (0.3 mg/ml) and NADPH (1 mM). After 30 min of incubation at 37°C, formation of the metabolite of the CYP-specific substrate in the absence and presence of test compound was measured. Ketoconazole was used as a positive control for CYP3A4 inhibition. For assessment of time-dependent inhibition, HLM were preincubated with TBA-354 for 30 min in the presence of NADPH before the addition of probe substrates to measure CYP activity (preincubation mixture not diluted).

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Induction of CYPs. [1]
The potential for induction of CYP enzymes (CYP1A2, CYP2B6, CYP2C9, CYP2C19, and CYP3A) by 10 μM TBA-354 was evaluated using human hepatocytes (3 donors) cultured in medium containing 2% BSA. Freshly plated human hepatocytes (∼0.15 million cells/well in 48-well plates) were treated with the test compound for 72 h. After induction treatment, CYP enzyme activities were measured by determining the formation of CYP-specific probe metabolites by LC-MS/MS. The fold induction of the CYP enzyme activity compared with that for the vehicle control was calculated.

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LC-MS/MS analysis methods. [1]
To measure TBA-354 concentrations in the in vitro Caco-2 permeability assay, a high-performance liquid chromatography (HPLC) 20AD pump (Shimadzu) connected to an API 4000 mass spectrometer (AB Sciex) was used. A Phenomenex Luna C18(2) column (5 μm, 50 by 2 mm) was eluted with a mobile phase of water-acetonitrile (98:2 to 2:98 in 1.7 min of elution time) containing 0.1% formic acid at a flow rate of 0.5 ml/min. TBA-354 was monitored by the positive multiple reaction monitoring (MRM) mode at the transition of m/z 437 to 252. Benzyl nicotinate as an internal standard was monitored at the transition of m/z 214 to 91. In the microsomal metabolism assessments of TBA-354, the same analytical method was used to measure the remaining TBA-354.

In the plasma protein binding assays, the TBA-354 concentration was measured under similar LC-MS/MS conditions. An Agilent HP1100 LC system connected to a Waters Quattro Micro mass spectrometer was used. An Atlantis C18 column (3 μm, 10 × 2.1 mm) was eluted with a mobile phase consisting of water and methanol containing 0.1% formic acid and 10 mM ammonium formate (100% water to 100% methanol in 2 min of elution time). TBA-354 was monitored under the positive MRM mode at the transition m/z of 437.3 to 252.5.

TBA-354 concentrations in hepatocyte metabolism incubations were measured using a Shimadzu liquid chromatograph connected to a Q-Trap 4000 MS/MS system (AB/MDS Sciex). A Phenomenex Luna C18 column (5.0 μm, 50 × 2.0 mm) was used for separation and eluted with a mobile phase consisting of water-acetonitrile (100% water to 100% acetonitrile in 7 min) containing 0.1% formic acid at a flow rate of 1 ml/min. TBA-354 was monitored under the positive ion mode with the transition m/z of 437 to 252. Pa was used as an internal standard monitored at the transition m/z of 360 to 175. The same LC-MS/MS system was used in the CYP inhibition and induction studies.

In the mouse PK study, a Thermo Finnigan LC-TSQ mass spectrometer system was used. TBA-354 was monitored with the single reaction monitoring (SRM) positive mode at the transition of m/z 437.3 to 252.5. The detection linear range was from 10 ng/ml to 1,000 ng/ml.

Pharmacokinetics of TBA-354 in mice. [1]
\nThe PK of TBA-354 was studied in female BALB/c mice following oral gavage of TBA-354, formulated in 0.5% CMC, at 3, 30, and 100 mg/kg. Plasma samples were collected from three mice per time point over the course of 48 h using sodium heparin as an anticoagulant. In a separate study, mice were administered 2 mg/kg TBA-354 (formulated in 40% hydroxypropyl-β-cyclodextrin and 50 mM citrate buffer at pH 3) by intravenous (i.v.) bolus, and blood samples (0.25 ml) were collected (three per time point) over the course of 24 h into potassium EDTA-containing tubes. Plasma concentrations were determined by LC-MS/MS (see “LC-MS/MS analysis methods” below). Noncompartmental PK analysis was performed with WinNonlin Phoenix v6.2 using composite data.\n

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\n\nIn vivo efficacy in mice. [1]
\nFemale ∼20-g BALB/c mice were infected by aerosol with a low dose of M. tuberculosis Erdman as previously described. The protocol results in the deposition of approximately 50 to 100 bacilli into the lungs, and the course of infection was then followed by plating homogenates of the lungs on 7H11 agar and determining CFU. Controls consisted of mice treated with the vehicle only. The compounds such as TBA-354 were prepared weekly by suspension in 0.5% (wt/vol) carboxymethylcellulose (CMC) such that the target dosages were obtained by once-daily dosing by oral gavage of a 200-μl suspension. Groups of 6 or 7 mice were dosed for 5 consecutive days each week. The suspensions were stored at 4°C between daily doses. Mice were sacrificed 3 days after the final dose to minimize carryover from the lung homogenates to the plating medium. Both lungs were homogenized and diluted in Hanks' balanced salt solution (HBSS)-Tween, and aliquots were plated on Middlebrook 7H11 medium. CFU were determined after 3 weeks of incubation at 37°C. For statistical analysis of efficacy data, multiple comparisons among pairs were performed by the Bonferroni method.\n

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\n\nAntimicrobials. [2]
\nINH, RIF, PZA, BDQ, PA-824, PNU, and CFZ were obtained and formulated for oral administration, as previously described, except that in experiment 4, CFZ was formulated in the same acidified 20% hydroxypropyl-β-cyclodextrin solution as BDQ. TBA-354 and delamanid were formulated in the same cyclodextrin micelle (CM-2) formulation as PA-824.\n

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\n\nChemotherapy. [2]
\nThe mice were block randomized by run to experimental arms prior to treatment. Treatment was initiated 13 to 14 days after infection in experiments 1, 3, and 4, and treatment began 42 days after infection in experiment 2. The treatment was administered once daily, 5 days per week, by gavage. The drug doses were 10 mg/kg of INH, 10 mg/kg of RIF, 150 mg/kg of PZA, 25 mg/kg of BDQ, 50 mg/kg of PNU, and 20 mg/kg of CFZ.\n

\nIn experiment 1, the control mice received RIF+INH+PZA. The test mice received PA-824 alone (10, 30, 100, 300, or 600 mg/kg) or TBA-354 or delamanid alone (3, 10, 30, or 100 mg/kg). Treatment was administered for up to 8 weeks. In experiment 2, all mice received RIF+INH+PZA for the first 4 weeks of treatment. After that, the negative controls received no treatment, the positive controls received RIF+INH, and the test groups received 50 mg/kg of PA-824 alone or 10 mg/kg of TBA-354 alone. The total treatment duration was 12 weeks.\n

\nIn experiment 3, the positive-control mice received 8 weeks of RIF+INH+PZA, followed by up to 8 weeks of RIF+INH. The remaining mice received either BDQ+PZA or BDQ+PNU, alone or in combination with either 50 mg/kg of PA-824 or 10 or 50 mg/kg of TBA-354. In experiment 4, the mice received BDQ+PZA+CFZ or BDQ+PZA+PNU, alone or in combination with 50 mg/kg of PA-824 or 25 or 50 mg/kg of TBA-354. An additional group received BDQ+PZA+CFZ+PNU. PA-824 and TBA-354 or a sham treatment with the CM-2 vehicle was administered immediately after the dose of BDQ and/or PZA, which were formulated together, in order to be consistent with prior combination experiments. PNU or CFZ was administered ≥4 h later. For the BDQ+PZA+CFZ+PNU regimen, BDQ, PZA, and CFZ were formulated and administered together, and PNU was given 4 h later. The drugs were administered for up to 16 weeks in experiment 3 and up to 8 weeks in experiment 4.\n

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\n\nSelection of drug-resistant isolates. [2]
\nAfter 8 weeks of treatment in experiment 1, the lung homogenates were also plated directly on 7H11 agar containing PA-824, delamanid, or TBA-354 at 1, 0.25, or 0.25 μg/ml, respectively, to assay for drug-resistant mutants. To evaluate for cross-resistance between the nitroimidazoles, resistant colonies isolated from 2 mice each from the 2 highest dose groups were scraped together and homogenized with glass beads. The resulting suspension was left to settle for 30 min before being plated in serial 10-fold dilutions on plates containing 1 μg/ml PA-824 or 0.25 μg/ml delamanid or TBA-354.

ADME and PK characteristics. (i) In vitro evaluation of ADME properties. [1]
The uptake potential of TBA-354 and its interaction with P-glycoprotein (P-gp) were assessed using an in vitro Caco-2 cell bidirectional permeability assay. In this assay, TBA-354 showed high permeability at both 1 μM and 10 μM concentrations, with apparent permeability (Papp) values (from apex to basal end, i.e., A to B) > 10 × 10−6 cm/s (Table 5). The permeability values were similar in the presence or absence of the P-gp inhibitor ketoconazole, and the efflux ratios of 1 μM and 10 μM TBA-354 were <2 in both the presence and absence of ketoconazole. The results indicate that TBA-354 is not a substrate of the P-gp transporter. Digoxin, used as a positive control for P-gp substrates, showed a Papp B/A/A/B ratio of 68-fold, which was inhibited by ketoconazole to 1.4-fold. TBA-354 exhibited moderate to high protein binding at 10 μM concentrations, with mean plasma protein binding rates of 96.5%, 94.6%, 96.2%, 95.9%, and 92.6% in human, monkey, dog, rat, and mouse plasma, respectively. The recoveries in this study ranged from 86% to 120%. TBA-354 was metabolically stable or underwent only moderate metabolism during in vitro incubation with liver microsomes, recombinant cytochrome P450 (CYP), and hepatocytes. The metabolic effects of the tested CYP enzymes (CYP2C9, CYP2C19, CYP2D6, and CYP3A4) on TBA-354 were undetectable. TBA-354 was stable in in vitro microsomal incubation, and no measurable metabolism was detected after 1 hour of incubation with human, monkey, dog, rat, or mouse liver microsomes in the presence of a NADPH regeneration system. TBA-354 was moderately metabolized in hepatocytes of six different species (Figure 1). After 4 hours of incubation of 1 μM TBA-354 with mouse, rat, rabbit, dog, monkey, and human hepatocytes, unmetabolized TBA-354 remained the major component, accounting for 77.1%, 87.7%, 98.6%, 82.9%, 70.7%, and 54.5%, respectively. The estimated half-life of TBA-354 in human hepatocytes was 4.6 hours. Identification of metabolites after incubation with hepatocytes is important and is planned.

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Mouse Pharmacokinetics. [1]
Pharmacokinetic studies were conducted in mice. The formulation, mouse strain, and route of administration were the same as those used in the mouse efficacy studies. Following a single oral dose of 3, 30, or 100 mg/kg of TBA-354, the time to peak serum concentration (Tmax) ranged from 2 to 6 hours, with a relatively long terminal half-life (t1/2) of 8 to 12 hours (Figure 2 and Table 6). Both the maximum serum concentration (Cmax) and the area under the concentration-time curve (AUC0–inf) increased with increasing dose, but the linear relationship with dose increase differed slightly; AUC0–inf increased from 22.7 μg·h/ml to 242 μg·h/ml, and Cmax increased from 1.6 μg/ml to 12.8 μg/ml. An intravenous bolus of 2 mg/kg TBA-354 allows for assessment of the volume of distribution and estimation of absolute bioavailability. Comparison of AUC0–inf values at similar dose levels and concentration ranges (3 mg/kg orally and 2 mg/kg intravenously) indicated that the absolute bioavailability in mice was approximately 40%. The apparent volume of distribution (Vz/F) in mice was 1.61 L/kg, suggesting that TBA-354 may be distributed in tissues outside of body fluids.

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Potential drug interactions. [1]
The direct and time-dependent inhibitory effects of TBA-354 on CYP2C8, CYP2C19 and CYP3A4 were evaluated in human liver microsomes (HLM). TBA-354 showed a weak inhibitory effect on CYP3A4-catalyzed 6-hydroxylation of testosterone (40% inhibition rate at 30 μM), while no inhibitory effect on 1′-hydroxylation of midazolam was observed. TBA-354 did not directly inhibit other CYPs; the IC50 values for inhibition of these CYPs by TBA-354 were all >30 μM. In another study, the inhibition rates of CYP enzyme activities in human liver microsomes (HLM) by 10 μM TBA-354 were: CYP2A6 <10%, CYP1A2 14%, CYP2B6 20%, CYP2C9 33%, CYP2D6 26%, and CYP2E1 24%. In the presence of NADPH, after pre-incubating TBA-354 with HLM for 30 minutes, the CYP3A4-catalyzed 6β′-hydroxylation of testosterone was inhibited with an IC50 value of 24 μM, which may suggest a weak time-dependent inactivation of CYP3A4. However, no time-dependent inhibition of the CYP3A4-catalyzed 1′-hydroxylation of midazolam was observed. In the three human hepatocyte donors tested, 10 μM TBA-354 did not significantly induce the expression of CYP1A2, CYP2B6, CYP2C9, CYP2C19, or CYP3A (the induction fold was less than or close to 2-fold compared to the solvent control group). Under these experimental conditions, TBA-354 had no significant effect on cell viability.

Finally, no toxicity was observed in any of the efficacy studies (assessed by clinical observation and weight monitoring) (data not shown) [1]. As with all potential tuberculosis drugs, the safety and toxicological information of TBA-354, as well as its clinical pharmacokinetic characteristics, are needed to determine whether the plasma concentrations required to achieve the expected clinical efficacy can be achieved, and whether they can be achieved safely. Delamani has been reported to prolong the QT interval, which has become a side effect of many tuberculosis drugs, complicating the implementation of combination therapy regimens. The safety profile of TBA-354 and all aspects thereof need to be carefully considered in conjunction with possible combination therapies in order to properly assess the potential use of this compound. However, the existing data suggest that TBA-354 has the potential to be a next-generation nitroimidazole drug for the treatment of tuberculosis and should be further investigated for a more comprehensive evaluation. Further studies are currently underway to evaluate its efficacy in preclinical models of tuberculosis, as well as pharmacokinetic, safety and toxicological studies. [1]

[1]. In Vitro and In Vivo Activities of the Nitroimidazole TBA-354 against Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2015 Jan;59(1):136-44.

[2]. Contribution of the Nitroimidazoles PA-824 and TBA-354 to the Activity of Novel Regimens in Murine Models of Tuberculosis. Antimicrob Agents Chemother. 2015 Jan;59(1):129-35.

TBA-354 is a small molecule drug currently in Phase I clinical trials with one investigational indication. Nitroimidazole drugs are a promising class of novel anti-tuberculosis drugs. The nitroimidazole-oxazine drug delmani (OPC-67683, trade name Deltaba) is undergoing a Phase III clinical trial for the treatment of multidrug-resistant tuberculosis, while the nitroimidazole-oxazine drug PA-824 is about to enter a Phase III clinical trial for the treatment of drug-sensitive and drug-resistant tuberculosis. TBA-354 (SN31354[(S)-2-nitro-6-((6-(4-trifluoromethoxy)phenyl)pyridin-3-yl)methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine]) is a pyridine-containing biaryl compound that has shown excellent efficacy and good bioavailability against chronic mouse tuberculosis in preliminary rodent studies. Following extensive medicinal chemistry studies, TBA-354 has been selected as a potential next-generation anti-tuberculosis nitroimidazole drug. This article further evaluates the pharmacokinetic properties of TBA-354 and its activity against Mycobacterium tuberculosis. TBA-354 exhibits narrow-spectrum bactericidal activity against both replicating and non-replicating Mycobacterium tuberculosis, and in vitro experiments show that its potency is similar to that of deramani and higher than that of PA-824. The addition of serum protein or albumin does not significantly alter its activity. TBA-354 maintains activity against Mycobacterium tuberculosis H37Rv homologous monodrug-resistant strains as well as clinically drug-sensitive and drug-resistant isolates. The frequency of spontaneously resistant mutants is 3 × 10⁻⁷. In vitro and in vivo (mouse) studies confirm that TBA-354 has high bioavailability and a long elimination half-life. In vitro studies indicate a low risk of drug interactions with TBA-354. Low-dose aerosol infection of mice in acute and chronic tuberculosis models showed that TBA-354 had time- and dose-dependent in vivo bactericidal activity, with efficacy at least comparable to deramani and superior to PA-824. Its superior efficacy and predictive pharmacokinetic profile for once-daily oral administration suggest that TBA-354 warrants further investigation as a potential next-generation nitroimidazole drug. [1] In summary, these experiments confirm and extend previous studies, demonstrating the potential of novel drug combinations containing BDQ, PNU, and nitroimidazoles to lay a solid foundation for universally effective short-course treatment regimens for tuberculosis that are not affected by existing drug resistance. The addition of this bactericidal agent when the infecting strain remains sensitive to pyrazinamide is expected to further shorten the course of treatment. Although the potential of these regimens to significantly shorten the course of treatment in mice cannot be directly extrapolated to human tuberculosis, the results presented in this paper undoubtedly support future clinical trials to investigate the efficacy of these and other similar regimens. This study also confirms previous findings that TBA-354 is more effective than PA-824. In both monotherapy and combination therapy, this new generation of nitroimidazole is superior to PA-824 at equivalent doses, including its contribution to bactericidal efficacy. Further studies will determine whether TBA-354 is more effective than PA-824 and delamani. Future clinical and preclinical studies are needed to assess whether adequate drug exposure can be achieved at safe and well-tolerated doses. [2]

C19H15F3N4O5

436.341414690018

436.099

C, 52.30; H, 3.47; F, 13.06; N, 12.84; O, 18.33

1257426-19-9

1257426-19-9;

49836057

White to off-white Solid powder

1.5±0.1 g/cm3

570.0±60.0 °C at 760 mmHg

298.5±32.9 °C

0.0±1.6 mmHg at 25°C

1.623

3.02

0

10

5

31

614

1

FC(F)(F)OC1=CC=C(C2=CC=C(CO[C@H]3CN4C(OC3)=NC([N+]([O-])=O)=C4)C=N2)C=C1

ZXSGSFMORAILEY-HNNXBMFYSA-N

InChI=1S/C19H15F3N4O5/c20-19(21,22)31-14-4-2-13(3-5-14)16-6-1-12(7-23-16)10-29-15-8-25-9-17(26(27)28)24-18(25)30-11-15/h1-7,9,15H,8,10-11H2/t15-/m0/s1

(S)-2-nitro-6-((6-(4-(trifluoromethoxy)phenyl)pyridin-3-yl)methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine

TBA-354; TBA 354; TBA-354; 1257426-19-9; 911T37M2WY; UNII-911T37M2WY; ((6S)-6-((6-(4-(Trifluoromethoxy)phenyl)-3-pyridyl)methoxy)-6,7-dihydro-5H-imidazo(2,1-b)(1,3)oxazin-2-yl)azinic acid; (sn31354((S)-2-Nitro-6-((6-(4-trifluoromethoxy)phenyl)pyridine-3-yl)methoxy)-6,7-dihydro-5H-imidazo(2,1-b)(1,3)oxazine)); SN31354; TBA354; SN31354; SN-31354; SN 31354

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~229.18 mM)

Solubility in Formulation 1: 2.5 mg/mL (5.73 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (5.73 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: 2.5 mg/mL (5.73 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: 2.5 mg/mL (5.73 mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)