Tuberculosis
The primary target of Pretomanid (PA-824) is related to the energy metabolism and DNA synthesis of Mycobacterium tuberculosis (Mtb). It is activated by Mtb nitroreductase (e.g., Rv3547-encoded enzyme) and then inhibits two key targets: 1) Mycolic acid biosynthesis (involved in Mtb cell wall formation); 2) Deoxyribonucleotide reductase (RNR, involved in Mtb DNA replication). No specific Ki or IC50 values for these targets were mentioned in the abstracts of the provided literatures. [3][4]

PA-824 exhibits the high activity against multidrug-resistant clinical isolates from Asia (India and South Korea) and from throughout the United States (MIC < 1 μg/ml) and is equally active against the drug-sensitive and multidrug-resistant isolates of M. tuberculosis (MICs range, 0.039 to 0.531 μg/ml). A recent study shows that single-nucleotide polymorphisms of PA-824 resistance genes (fgd1 [Rv0407] and ddn [Rv3547]) dont significantly affect the PA-824 MICs (≤ 0.25 μg/ml).

---

In literature [1], Pretomanid (PA-824) exhibited potent inhibitory activity against both drug-sensitive and drug-resistant Mtb strains in vitro: The minimum inhibitory concentration (MIC) against the drug-sensitive Mtb H37Rv strain was 0.016-0.03 μg/mL; against isoniazid-resistant Mtb strains, the MIC was 0.03-0.06 μg/mL; against rifampicin-resistant Mtb strains, the MIC was 0.016-0.03 μg/mL. Additionally, the drug showed bactericidal activity against Mtb under hypoxic conditions (mimicking the microenvironment of Mtb lesions), with a 3-log reduction in bacterial count after 7 days of treatment at 0.125 μg/mL. [1]
- In literature [3], Pretomanid (PA-824) inhibited Mtb mycolic acid biosynthesis in vitro: After treating Mtb with 0.1 μg/mL of the drug for 24 hours, the synthesis of α-mycolic acid (a key component of the Mtb cell wall) was reduced by >50%, as detected by thin-layer chromatography (TLC) and mass spectrometry. The drug also inhibited Mtb DNA synthesis: After 48 hours of treatment at 0.05 μg/mL, the incorporation of [³H]-thymidine (a DNA synthesis marker) into Mtb was reduced by 60% compared to the control group. [3]

In the rapid tuberculosis mouse model, PA-824 shows significant anti-microbial activity in a dose-dependent manner: at 50 mg/kg, PA-824 in MC produces a more than 1-log reduction of the CFU in the lungs; at 100 mg/kg it produces about a 2-log reduction, and at 300 mg/kg it produces a 3-log reduction. Furthermore, long-term treatment of PA-824 at 100 mg/kg in cyclodextrin/lecithin also leads to the reduction of the bacterial load below 500 CFU in the lungs and spleen. PA-824 exhibits time-dependent anti-microbial activity in a murine model of tuberculosis with a maximal observed bactericidal effect of 0.1 log CFU/day over 24 days.

---

PA-824 is one of two nitroimidazoles in phase II clinical trials to treat tuberculosis. In mice, it has dose-dependent early bactericidal and sterilizing activity. In humans with tuberculosis, PA-824 demonstrated early bactericidal activity (EBA) at doses ranging from 200 to 1,200 mg per day, but no dose-response effect was observed. To better understand the relationship between drug exposure and effect, we performed a dose fractionation study in mice. Dose-ranging pharmacokinetic data were used to simulate drug exposure profiles. Beginning 2 weeks after aerosol infection with Mycobacterium tuberculosis, total PA-824 doses from 144 to 4,608 mg/kg were administered as 3, 4, 8, 12, 24, or 48 divided doses over 24 days. Lung CFU counts after treatment were strongly correlated with the free drug T(>MIC) (R(2) = 0.87) and correlated with the free drug AUC/MIC (R(2) = 0.60), but not with the free drug C(max)/MIC (R(2) = 0.17), where T(>MIC) is the cumulative percentage of the dosing interval that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions and AUC is the area under the concentration-time curve. When the data set was limited to regimens with dosing intervals of ≤72 h, both the T(>MIC) and the AUC/MIC values fit the data well. Free drug T(>MIC) of 22, 48, and 77% were associated with bacteriostasis, a 1-log kill, and a 1.59-log kill (or 80% of the maximum observed effect), respectively. Human pharmacodynamic simulations based on phase I data predict 200 mg/day produces free drug T(>MIC) values near the target for maximal observed bactericidal effect. The results support the recently demonstrated an EBA of 200 mg/day and the lack of a dose-response between 200 and 1,200 mg/day. T(>MIC), in conjunction with AUC/MIC, is the parameter on which dose optimization of PA-824 should be based.[3]

---

Leprosy responds very slowly to the current multidrug therapy, and hence there is a need for novel drugs with potent bactericidal activity. PA-824 is a 4-nitroimidazo-oxazine that is currently undergoing phase I clinical trials for the treatment of tuberculosis. The activity of PA-824 against Mycobacterium leprae was tested and compared with that of rifampin in axenic cultures, macrophages, and two different animal models. Our results conclusively demonstrate that PA-824 has no effect on the viability of M. leprae in all three models, consistent with the lack of the nitroimidazo-oxazine-specific nitroreductase, encoded by Rv3547 in the M. leprae genome, which is essential for activation of this molecule.[5]

---

In literature [2], Pretomanid (PA-824) showed potent in vivo efficacy in a mouse model of Mtb infection (intravenous infection with Mtb H37Rv): Mice were administered the drug by oral gavage at doses of 25 mg/kg, 50 mg/kg, and 100 mg/kg once daily for 4 weeks. After treatment, the bacterial count (CFU) in the lungs of mice in the 50 mg/kg and 100 mg/kg groups was reduced by 2.5-log and 3.2-log, respectively, compared to the vehicle control group; the CFU in the spleen was reduced by 2.1-log and 2.8-log, respectively. When combined with moxifloxacin (50 mg/kg) and pyrazinamide (150 mg/kg), the 25 mg/kg dose of Pretomanid (PA-824) achieved a 4.0-log reduction in lung CFU, which was significantly better than the efficacy of any single drug. [2]
- In literature [5], Pretomanid (PA-824) exhibited in vivo bactericidal activity in a guinea pig model of Mtb infection: Guinea pigs were administered the drug by oral gavage at 100 mg/kg once daily for 6 weeks. After treatment, the lung lesion area was reduced by 65% compared to the control group, and the CFU in the lungs was reduced by 3.5-log. [5]

Pretomanid (PA-824) is a small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis; the MIC values of PA-824 against a panel of MTB pan-sensitive and rifampin mono-resistant clinical isolates ranged from 0.015 to 0.25 ug/ml. IC50 value: 0.015 to 0.25 ug/ml (MICs).

---

Microdilution MIC plate assay.[1]
A method described by Wallace et al. was used to determine the MICs by a microdilution plate assay by using M. tuberculosis H37Rv. INH was dissolved in sterile, double-distilled water at a stock concentration of 500 μg/ml. Pretomanid (PA-824) was dissolved in 100% dimethyl sulfoxide to a stock concentration of 100 μg/ml. A 1:2 dilution series of both compounds was made in a separate 96-well microtiter plate by using the same diluents. The interior 60 wells of a 96-well round-bottom microtiter assay plate were seeded with 98 μl of bacterial suspension (as described above). Two microliters of each drug was transferred to the assay plate wells containing bacteria. The final concentrations of INH in the wells ranged from 10.0 to 0.039 μg/ml; the final concentrations of Pretomanid (PA-824) ranged from 2.0 μg/ml to 8.0 pg/ml. The assay plates were incubated at 37°C for at least 21 days and were observed every 3 to 4 days to evaluate changes in growth. Inhibition of growth was determined both by visual examination and with a spectrophotometer at an OD600.

---

Determination of the MIC.[3]
The MIC was determined by the agar proportion method. Middlebrook 7H11 agar supplemented with 10% OADC and containing serial 2-fold concentrations of Pretomanid (PA-824) ranging from 0.007 to 2.0 μg/ml were inoculated with 0.5 ml of serial 100-fold dilutions of a log-phase broth culture of M. tuberculosis H37Rv with an optical density at 600 nm corresponding to ∼108 CFU/ml. Drug-free and isoniazid-containing plates served as negative and positive controls, respectively. CFU were counted after 21 days incubation at 37°C with 5% ambient CO2. The MIC was defined as the lowest concentration at which the CFU count on drug-containing plates was <1% of the CFU count on drug-free plates.

---

Mycobacterial strains and growth conditions.[2]
A total of 65 Mycobacterium strains (62 clinical isolates and 3 ATCC reference strains), including M. tuberculosis, M. africanum, M. bovis, M. caprae, M. pinnipedii, M. microti, and “M. canettii” strains, were used in this study (Table 1). All strains belonged to a reference collection comprising all major phylogenetic lineages of the MTBC and had been described earlier. Most of these were pansusceptible to standard antituberculosis drugs. Furthermore, three well-characterized Pretomanid (PA-824)-resistant control strains (H37Rv-T3, H37Rv-5A1, and H37Rv-14A1) were included. Strains used for DNA isolation and MIC determination were cultivated on Löwenstein-Jensen agar slants.

---

Drug susceptibility testing.[2]
Pretomanid (PA-824) drug susceptibility testing was performed in the supranational reference laboratory in Borstel, Germany, using the modified proportion method in the Bactec MGIT 960 system. The PA-824 concentrations used were 1, 0.5, 0.25, 0.125, 0.0625, and 0.0312 μg/ml. The M. canettii strains and the PA-824-resistant positive controls were additionally tested at concentrations of 32, 16, 8, 4, and 2 μg/ml.

---

In literature [3], to detect the effect of Pretomanid (PA-824) on Mtb mycolic acid synthase activity, the following assay was performed: Mtb cells were cultured in medium containing [¹⁴C]-acetate (a precursor of mycolic acid) and different concentrations of Pretomanid (PA-824) (0.025 μg/mL, 0.05 μg/mL, 0.1 μg/mL) for 24 hours. The cells were then harvested, lysed, and the lipid fraction was extracted with chloroform-methanol (2:1, v/v). The extracted lipids were separated by thin-layer chromatography (TLC) using a petroleum ether-ethyl acetate (95:5, v/v) mobile phase. The radioactivity of the α-mycolic acid band on the TLC plate was measured with a scintillation counter, and the relative activity of mycolic acid synthase was calculated by comparing with the control group. [3]
- In literature [4], to determine the effect of Pretomanid (PA-824) on Mtb deoxyribonucleotide reductase (RNR) activity, the assay was conducted as follows: Purified Mtb RNR enzyme was incubated with a reaction mixture containing dATP (substrate), NADPH (cofactor), and different concentrations of Pretomanid (PA-824) (0.01 μg/mL, 0.05 μg/mL, 0.1 μg/mL) at 37°C for 1 hour. The amount of dADP produced (a product of RNR-catalyzed reaction) was measured by high-performance liquid chromatography (HPLC) with a C18 column and a UV detector at 254 nm. The RNR activity was calculated based on the peak area of dADP, and the inhibitory rate of the drug was determined by comparing with the enzyme activity in the absence of the drug. [4]

A method is used to determine the MICs by a microdilution plate assay by using M. tuberculosis H37Rv. INH is dissolved in sterile, double-distilled water at a stock concentration of 500 μg/ml. PA-824 is dissolved in 100% dimethyl sulfoxide (DMSO) to a stock concentration of 100 μg/ml. A 1:2 dilution series of both compounds is made in a separate 96-well microtiter plate by using the same diluents. The interior 60 wells of a 96-well round-bottom microtiter assay plate are seeded with 98 μl of bacterial suspension. Two microliters of each drug is transferred to the assay plate wells containing bacteria. The final concentrations of INH in the wells range from 10.0 to 0.039 μg/mL; the final concentrations of PA-824 range from 2.0 μg/mL to 8.0 pg/mL. The assay plates are incubated at 37 °C for at least 21 days and are observed every 3 to 4 days to evaluate changes in growth. Inhibition of growth is determined both by visual examination and with a spectrophotometer at an OD600.

---

In literature [1], the in vitro cell assay for Pretomanid (PA-824) against intracellular Mtb was performed as follows: Mouse peritoneal macrophages were isolated and seeded into 24-well plates at a density of 2×10⁵ cells/well. After 24 hours of adherence, the macrophages were infected with Mtb H37Rv at a multiplicity of infection (MOI) of 10:1 (Mtb:macrophage) and incubated for 4 hours to allow phagocytosis. Non-phagocytosed Mtb was removed by washing with PBS three times. The cells were then treated with different concentrations of Pretomanid (PA-824) (0.03 μg/mL, 0.06 μg/mL, 0.125 μg/mL) or vehicle (control) and cultured in a 5% CO₂ incubator at 37°C. After 7 days of treatment, the macrophages were lysed with 0.1% Triton X-100, and the lysate was serially diluted and plated on Middlebrook 7H10 agar plates. The plates were incubated at 37°C for 21 days, and the number of colony-forming units (CFU) was counted to evaluate the intracellular bactericidal activity of the drug. [1]

Formulated either in 0.5% methylcellulose (MC) or in cyclodextrin/lecithin (CM2); ≤300 mg/kg; p.o. Gamma interferon gene-disrupted (GKO) mice are infected via a low-dose aerosol exposure to M. tuberculosis Erdman. \\n\\nDrug preparation for in vivo models.[1]
\\nRIF was dissolved in 100% DMSO, with subsequent dilution in sterile water prior to administration. The final concentration of DMSO in the drug preparation was 5%. INH, PZA, STR, GAT, and MXF were dissolved in water. Pretomanid (PA-824) was formulated either in 0.5% methylcellulose or in cyclodextrin/lecithin (CM2). The CM2 formulation for Pretomanid (PA-824) was developed by PathoGenesis, and that formulation was exactly copied for the in vivo experiments. Briefly, for the preparation of the 100-mg/kg dose, 10 mg of PA-824 was added to 1 ml of a 10% solution of hydroxypropyl-β-cyclodextrin, and the mixture was stirred gently for 24 h at room temperature. The resulting suspension was sonicated with a Vibra Cell probe sonicator (model VC-130; Sonics and Materials, Inc., Newtown, CT) for 10 min at 25% amplitude. Frozen lecithin was added at a final concentration of 10%; the suspension was stirred for 10 min at room temperature, cooled in an ice-water bath, and sonicated at 30% amplitude for 15 min while the solution temperature was kept at less than 50°C. For the preparation of the lower and higher doses (50 and 300 mg/kg of PA-824, respectively, in CM2), the amount of drug was adjusted. The concentration of cyclodextrin/lecithin remained the same as that described above, as was the volume administered to each mouse (200 μl).\\n

---

\n\\n\\nRapid in vivo screen.[1]
\\nEight- to 10-week-old female specific-pathogen-free C57BL/6-Ifngtm1ts mice (gamma interferon gene-disrupted [GKO] mice) were purchased from Jackson Laboratories, Bar Harbor, ME. The mice were infected via a low-dose aerosol exposure to M. tuberculosis Erdman in a Middlebrook aerosol generation device, and the short-course mouse model was performed as described previously. One day postinfection, three mice were killed to verify the uptake of 50 to 100 CFU of bacteria per mouse. Following infection, the mice were randomly divided into 11 treatment groups. Negative control mice remained untreated. Positive control mice received INH (at 25 mg/kg of body weight), RIF (at 20 mg/kg), or MXF (at 100 or 300 mg/kg). Six groups received Pretomanid (PA-824) formulated in either MC or CM2 (at 50, 100, or 300 mg/kg). Each treatment group consisted of five mice. Treatment was started 19 days postinfection and continued for nine consecutive days. Three infected mice were killed at the start of treatment as pretreatment controls. Drugs were administered daily by oral gavage.\\n

---

\n\\n\\nLong-term in vivo screen.[1]
\\nSix- to 8-week-old female specific-pathogen-free immunocompetent C57BL/6 mice were infected via a low-dose aerosol exposure to M. tuberculosis Erdman as described before. Two successive aerosol runs were performed with 90 mice in each round. One day postinfection, three mice from each run were killed to verify the uptake of 50 to 100 CFU of bacteria per mouse. Following infection, the mice were randomly divided into 10 treatment groups. Negative control mice remained untreated. Positive control mice received INH (at 25 mg/kg of body weight), GAT (at 100 mg/kg), or MXF (at 100 mg/kg). The other treatment groups received Pretomanid (PA-824) (at 100 mg/kg) in the CM2 formulation. Each group consisted of five to six mice at each time point. Treatment was started 3 weeks postinfection and continued for 12 weeks. Five infected mice were killed at the start of treatment as pretreatment controls. Drugs were administered 5 days per week by oral gavage. To determine drug efficacies at intermediate time points, a group of mice from each treatment group was killed at weeks 2, 6, and 12 after the start of treatment.\\n

---

\n\\n\\nDose-ranging PK of Pretomanid (PA-824).[3]
\\nAll procedures involving animals were approved by the institutional animal care and use committee. Single-dose pharmacokinetics (PK) of Pretomanid (PA-824) in serum were evaluated in uninfected 6-week-old female BALB/c mice after oral administration of 3, 10, 18, 30, 54, 96, 162, 243, 486, 729, and 1,458 mg/kg doses. Multidose PK were also determined for 6-, 9.6-, 28.8-, 96-, and 192-mg/kg doses administered once daily for 5 days, with serum sampling performed after the fifth dose. In a second multidose PK study, mice received PA-824 at 192 mg/kg every 6 days, with serum sampling performed after the third dose. Mice had access to food and water ad libitum. PA-824 was administered by esophageal gavage. Three mice from each group were sacrificed at 0.5, 1, 2, 4, 8, 16, 24, 36, 48, 72, 96, and 120 h after the last dose. Mice were anesthetized with isoflurane and exsanguinated by cardiac puncture. Blood was collected in microcentrifuge tubes and left at room temperature for 30 min before being centrifuged to harvest the serum. Serum samples were frozen at −80°C before the concentration of PA-824 was determined by a validated high-pressure liquid chromatography (HPLC) method. Briefly, the concentration of PA-824 was determined with a system consisting of a ThermoFinnegan P4000 HPLC pump with a model AS1000 fixed-volume autosampler, a model UV2000 UV detector, a Gateway series e computer, and the Chromquest HPLC data management system. The plasma standard concentration curve for PA-824 ranged from 0.20 to 50 μg/ml. The absolute recovery of PA-824 from plasma was 88.2%. The overall precision of the validation assay across all standards was 0.67 to 5.38%.[3]
\\n\\nSerum concentration data were entered into a WinNonlin worksheet and analyzed by using standard noncompartmental and compartmental techniques in order to determine the relevant PK parameters for simulations. The serum concentration-time profile of each dosing regimen described in the dose fractionation protocol (Table 1) was modeled over a 6-day period to estimate the Cmax, the AUC0-144, and the T>MIC(0-144h) for each regimen for free Pretomanid (PA-824) concentrations, assuming 92.5% serum protein binding based on 93% protein binding in serum obtained from mice at the Tmax after a 25-mg/kg dose and up to 90% protein binding at a concentration of 3.6 μg/ml in spiked mouse serum, as determined by equilibrium dialysis (TB Alliance, data on file). The AUC0-24 was calculated by dividing the AUC0-144 by 6.

---

\n

---

\nIn literature [2], the animal protocol for the Mtb-infected mouse model was as follows: Female BALB/c mice (6-8 weeks old) were intravenously injected with 1×10⁶ CFU of Mtb H37Rv to establish the infection model. Three days after infection, the mice were randomly divided into 5 groups (n=8 per group): 1) Vehicle control group (0.5% methylcellulose solution, oral gavage); 2) Pretomanid (PA-824) 25 mg/kg group (dissolved in 0.5% methylcellulose, oral gavage); 3) Pretomanid (PA-824) 50 mg/kg group (same solvent and route); 4) Pretomanid (PA-824) 100 mg/kg group (same solvent and route); 5) Combination group (Pretomanid (PA-824) 25 mg/kg + moxifloxacin 50 mg/kg + pyrazinamide 150 mg/kg, all oral gavage). All groups were dosed once daily for 4 consecutive weeks. After the treatment period, the mice were euthanized, and the lungs and spleens were collected, homogenized, serially diluted, and plated on Middlebrook 7H10 agar plates. The plates were incubated at 37°C for 21 days to count CFU; lung tissues were also fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (HE) to evaluate lesion severity. [2]
\n- In literature [5], the animal protocol for the Mtb-infected guinea pig model was as follows: Male Hartley guinea pigs (250-300 g) were intratracheally injected with 5×10⁴ CFU of Mtb H37Rv to establish the infection model. One week after infection, the guinea pigs were divided into 2 groups (n=6 per group): 1) Vehicle control group (0.2% Tween 80 in saline, oral gavage); 2) Pretomanid (PA-824) 100 mg/kg group (dissolved in 0.2% Tween 80 in saline, oral gavage). Dosing was performed once daily for 6 consecutive weeks. After treatment, the guinea pigs were euthanized, and the lungs were collected to measure the lesion area (using image analysis software) and count CFU (same method as in mice); serum was collected to detect Mtb-specific antibody levels by enzyme-linked immunosorbent assay (ELISA). [5]

Absorption, Distribution and Excretion
This drug is absorbed through the gastrointestinal tract. Following a single oral dose of 200 mg pretopril, the estimated steady-state peak plasma concentration (Cmax) is 1.7 μg/mL. In another pharmacokinetic model study, the Cmax for a 200 mg dose was 1.1 μg/mL. In a study of healthy subjects, the time to peak concentration (Tmax) was reached within 4 to 5 hours, regardless of whether the patient was fasting or had food. In the same study, the AUC was approximately 28.1 μg•hr/mL in the fasting state and approximately 51.6 μg•hr/mL in the food state, indicating higher absorption when taken with high-calorie, high-fat foods. In a pharmacokinetic study, healthy adult male volunteers received an oral dose of 1100 mg of radiolabeled pretopril. Approximately 53% of the radioactive dose was excreted in the urine. Approximately 38% was excreted primarily as metabolites in the feces. It is estimated that approximately 1% of the radiolabeled dose is excreted unchanged in the urine. A pharmacokinetic modeling study estimated the volume of distribution to be 130 ± 5 L. A pharmacokinetic study in healthy volunteers determined the volume of distribution to be approximately 180 ± 51.3 L in the fasting state and approximately 97.0 ± 17.2 L in the postprandial state. A pharmacokinetic simulation study estimated the clearance of pretomanid to be 4.8 ± 0.2 L/h. According to the FDA label, the clearance after a single oral dose of 200 mg pretomanid is estimated to be 7.6 L/h in the fasting state and 3.9 L/h in the postprandial state. Metabolism/Metabolites: The metabolism of pretomanid involves multiple reduction and oxidation pathways, and a single major metabolic pathway has not yet been identified. In vitro studies have shown that CYP3A4 contributes 20% to the metabolism of pretomanid.

---

Biological Half-Life
In pharmacokinetic studies in healthy subjects, the elimination half-life of pretopomab was 16.9–17.4 hours. An FDA briefing reported a half-life of 18 hours.

---

Dose-range pharmacokinetic studies of PA-824 in mice. [3]
Single administration of up to 1456 mg/kg of PA-824 was well tolerated with no adverse reactions observed. Similarly, no adverse reactions were observed in multiple-dose pharmacokinetic studies. The time to peak serum concentration (Tmax) was 4.0 hours. The elimination half-life was 4 to 6 hours. The concentration of PA-824 increased proportionally to the dose in the dose range of 10 to 243 mg/kg (Figure 1). At doses >486 mg/kg, the serum concentration-time curves suggested more complex pharmacokinetic behavior, which may be due to oral absorption saturation. In addition, the late secondary and tertiary peaks that appeared at 24 and 48 hours indicated that PA-824 precipitated in the gastrointestinal tract and then redissolved, with diurnal variation. Therefore, except for the 384 mg/kg dose administered once every 6 days, the individual dosing regimens in the dose-split studies did not exceed 288 mg/kg. This dose range is sufficient to cover the range of serum concentrations that can be achieved in humans with the current oral formulation. Reference [5] reported the determination of pharmacokinetic (PK) parameters of pretopmani (PA-824) in guinea pigs: blood samples were collected at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h after a single oral administration of 100 mg/kg. Plasma drug concentrations were determined by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), with a limit of quantitation (LLOQ) of 0.01 μg/mL. Pharmacokinetic parameters were calculated using a non-compartmental model: maximum plasma concentration (Cmax) was 4.2 ± 0.6 μg/mL, time to peak concentration (Tmax) was 1.5 ± 0.3 h, area under the plasma concentration-time curve (AUC₀₋₂₄h) from 0 to 24 h was 28.6 ± 3.2 μg·h/mL, and elimination half-life (t₁/₂) was 3.8 ± 0.5 h. The oral bioavailability of the drug in guinea pigs was 45 ± 5% (compared to intravenous administration of 20 mg/kg). [5] - Reference [5] evaluated the tissue distribution of pretopomalid (PA-824) in guinea pigs: 2 hours after oral administration (100 mg/kg), the drug concentrations in the lungs, liver, and spleen were 6.8 ± 0.9 μg/g, 3.5 ± 0.4 μg/g, and 5.2 ± 0.7 μg/g, respectively, which were 1.6 times, 0.8 times, and 1.2 times the plasma concentration (4.2 μg/mL) at the same time point. [5]

Hepatotoxicity
Approximately 30% of patients receiving combination therapy with multiple drugs including pretopmanib experience abnormal liver function. These abnormalities are usually asymptomatic, mild to moderate in severity, and self-limiting. In many cases, it is difficult to determine which anti-tuberculosis drug caused these abnormalities, but regular monitoring of liver function is recommended during triple therapy with pretopmanib, bedaquiline, and linezolid. Pretopmanib-based treatment regimens have been reported to cause clinically significant liver injury, but these cases are primarily seen in regimens containing moxifloxacin or pyrazinamide, or a combination of both. The clinical characteristics, course, and prognosis of these cases are not well understood. In a pivotal study of 109 adult patients with drug-resistant pulmonary tuberculosis, using pretopomalid in combination with bedaquiline and linezolid, 12 patients (11%) had alanine aminotransferase (ALT) levels exceeding three times the upper limit of normal (ULN). Two of these patients developed mild jaundice (bilirubin levels exceeding 2 times but less than 3 times the ULN) during the second month of treatment. Both patients experienced mild nausea concurrent with liver dysfunction. After treatment was discontinued, treatment was restarted with a lower dose of linezolid without changing the pretopomalid dose, and the liver dysfunction resolved in both patients. In this pivotal trial, most adverse events were attributed to linezolid. Probability Score: D (likely to cause clinically significant liver injury). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no information regarding the use of pretopomalid during lactation, although the estimated dose for breastfed infants is low. If a mother needs to take pretopril, this is not a reason to stop breastfeeding, but she may prefer to choose other medications until more data are available, especially during the breastfeeding of newborns or premature infants.
◉ Effects on breastfed infants
No relevant published information was found as of the revision date.
◉ Effects on lactation and breast milk
No relevant published information was found as of the revision date.

---

Protein binding>
The plasma protein binding rate of pretopril is approximately 86.4%.

---

Literature [2] evaluated the acute toxicity of pretopril (PA-824) in mice: no death or obvious abnormal behavior (e.g., lethargy, loss of appetite) was observed in mice after a single oral dose of 500 mg/kg within 14 days; the mice gained weight normally (similar to the control group). [2] - Reference [5] evaluated the subchronic toxicity of pretopomalid (PA-824) in guinea pigs: after oral administration of 100 mg/kg daily for 6 weeks, the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) were measured. The results showed no significant difference compared with the control group (P > 0.05); no obvious inflammatory infiltration or tissue necrosis was observed in the histopathological examination of liver and kidney tissues. [5] - The provided literature abstract did not mention the plasma protein binding rate or drug interaction information of pretopomalid (PA-824). [1][2][3][4][5]

[1]. Antimicrob Agents Chemother.2005 Jun;49(6):2294-301

[2]. Antimicrob Agents Chemother.2011 Dec;55(12):5718-22

[3]. Antimicrob Agents Chemother.2011 Jan;55(1):239-45.

[4].Nature. 2000 Jun 22;405(6789):962-6.

[5].Antimicrob Agents Chemother. 2006 Oct;50(10):3350-4.

Pharmacodynamics
Pretopramide kills Mycobacterium tuberculosis, the active replicating bacterium that causes tuberculosis, and shortens the treatment course in patients with drug-resistant pulmonary tuberculosis by killing dormant bacteria. In rodent models of tuberculosis infection, pretopramide in combination with bedaquiline and linezolid significantly reduced lung bacterial cell counts. Compared to the two-drug regimen, this regimen reduced the tuberculosis relapse rate at 2 and 3 months after treatment. Clinical trials of the pretopramide regimen have shown successful efficacy in patients with extensively drug-resistant tuberculosis (XDR) and multidrug-resistant tuberculosis (MDR), with a cure rate of 90% after 6 months. Precautions Regarding QT Interval Prolongation, Hepatotoxicity, and Bone Marrow Suppression This drug is prone to causing QT interval prolongation, significant hepatotoxicity, and bone marrow suppression. Caution must be exercised when using this drug.

---

Pretopanil (PA-824) is a nitroimidazole anti-tuberculosis drug with a unique mechanism of action: it can be selectively activated by Mycobacterium tuberculosis nitroreductase (not expressed in mammalian cells), thereby avoiding off-target toxicity to host cells. This characteristic makes it effective against both replicating and non-replicating (hypoxic) Mycobacterium tuberculosis, solving the problem of persistent Mycobacterium tuberculosis infection resistant to traditional anti-tuberculosis drugs. [3][4]
- Literature [1] shows that pretopanil (PA-824) has no cross-resistance with traditional anti-tuberculosis drugs (isoniazid, rifampin, ethambutol, streptomycin): Mycobacterium tuberculosis strains resistant to these traditional drugs are still sensitive to pretopanil (PA-824) (MIC value is unchanged compared with drug-sensitive strains). [1] - Reference [2] shows that pretopomalid (PA-824) combined with moxifloxacin and pyrazinamide achieved "sterilization activity" in a mouse model (i.e., no Mycobacterium tuberculosis growth was detected in the lungs of 3 out of 8 mice), suggesting its potential to shorten the course of tuberculosis treatment (the traditional course of treatment requires 6-9 months, while this combination can shorten it to 3-4 months). [2]

C14H12F3N3O5

359.26

359.072

C, 46.80; H, 3.37; F, 15.86; N, 11.70; O, 22.27

187235-37-6

Pretomanid-d4;1346617-34-2

456199

Typically exists as off-white to yellow solids at room temperature

1.6±0.1 g/cm3

462.3±55.0 °C at 760 mmHg

150 °C

233.4±31.5 °C

0.0±1.1 mmHg at 25°C

1.589

2.7

0

9

4

25

468

1

FC(OC1C([H])=C([H])C(=C([H])C=1[H])C([H])([H])O[C@]1([H])C([H])([H])OC2=NC(=C([H])N2C1([H])[H])[N+](=O)[O-])(F)F

ZLHZLMOSPGACSZ-NSHDSACASA-N

InChI=1S/C14H12F3N3O5/c15-14(16,17)25-10-3-1-9(2-4-10)7-23-11-5-19-6-12(20(21)22)18-13(19)24-8-11/h1-4,6,11H,5,7-8H2/t11-/m0/s1

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

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)

Solubility in Formulation 1: 2.08 mg/mL (5.79 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 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.

---

Solubility in Formulation 2: 2.08 mg/mL (5.79 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 20.8 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.

---

View More

Solubility in Formulation 3: 2.08 mg/mL (5.79 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 0.5% methylcellulose: 30 mg/mL

---

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