Oxazolidinone

PNU-100480, sutezolid, demonstrates remarkable in vitro efficacy against various clinical isolates of Mycobacterium avium complex (MICs ranging from 0.5 to 4 µg/mL)[1].

---

Oxazolidinones 6/Sutezolid (PNU-100480) and 7 were submitted for a preliminary evaluation of their in vitro activity against M. tuberculosis (Table 1). As shown in the table, both analogues were exceptionally active against a screening strain of M. tuberculosis, being comparable to or more active than the clinical comparator isoniazid. Compounds 6 and 8 were tested against a panel of 10 M. tuberculosis isolates (Table 2). Test isolates included five “drug-sensitive” and five “drug-resistant” strains. The resistant isolates' phenotypes covered resistance to the common antitubercular drugs isoniazid, streptomycin, rifampin, ethambutol, and ethionamide (see Table 3 for antibiograms of resistant M. tuberculosis isolates). Oxazolidinone 6 exhibited a MIC range of 0.03−0.50 μg/ mL and was the most active oxazolidinone tested. Interestingly, organisms resistant to numerous antitubercular agents were not cross-resistant with 6 and 8. Good in vitro activity was also observed for 6/Sutezolid (PNU-100480) against other mycobacterial species (Table 4). Generally, 6 was comparable to or significantly more active than the current clinical benchmark azithromycin. Analogue 6 exhibited potent in vitro activity against multiple isolates of M. avium complex, with a MIC50 of 2 μg/mL and a MIC90 of 4 μg/mL (Table 5).

With better effectiveness against M. tuberculosis in hollow-fiber, mouse, and whole-blood models, sutezolid (PNU-100480) is a thiomorpholinyl analog of linezolid[2].

---

Sutezolid (PNU-100480 [U-480]) is an oxazolidinone antimicrobial being developed for the treatment of tuberculosis. An active sulfoxide metabolite (PNU-101603 [U-603]), which reaches concentrations in plasma several times those of the parent, has been reported to drive the killing of extracellular Mycobacterium tuberculosis by Sutezolid in hollow-fiber culture. However, the relative contributions of the parent and metabolite against intracellular M. tuberculosis in vivo are not fully understood. The relationships between the plasma concentrations of U-480 and U-603 and intracellular whole-blood bactericidal activity (WBA) in ex vivo cultures were examined using a direct competitive population pharmacokinetic (PK)/pharmacodynamic 4-parameter sigmoid model. The data set included 690 PK determinations and 345 WBA determinations from 50 tuberculosis patients enrolled in a phase 2a sutezolid trial. The model parameters were solved iteratively. The median U-603/U-480 concentration ratio was 7.1 (range, 1 to 28). The apparent 50% inhibitory concentration of U-603 for intracellular M. tuberculosis was 17-fold greater than that of U-480 (90% confidence interval [CI], 9.9- to 53-fold). Model parameters were used to simulate in vivo activity after oral dosing with sutezolid at 600 mg twice a day (BID) and 1,200 mg once a day (QD). Divided dosing resulted in greater cumulative activity (-0.269 log10 per day; 90% CI, -0.237 to -0.293 log10 per day) than single daily dosing (-0.186 log10 per day; 90% CI, -0.160 to -0.208 log10 per day). U-480 accounted for 84% and 78% of the activity for BID and QD dosing, respectively, despite the higher concentrations of U-603. Killing of intracellular M. tuberculosis by orally administered sutezolid is mainly due to the activity of the parent compound. Taken together with the findings of other studies in the hollow-fiber model, these findings suggest that sutezolid and its metabolite act on different mycobacterial subpopulations.[2]

---

All patients completed assigned treatments and began subsequent standard TB treatment according to protocol. The 90% confidence intervals (CI) for bactericidal activity in sputum over the 14 day interval excluded zero for all treatments and both monitoring methods, as did those for cumulative WBA. There were no treatment-related serious adverse events, premature discontinuations, or dose reductions due to laboratory abnormalities. There was no effect on the QT interval. Seven Sutezolid-treated patients (14%) had transient, asymptomatic ALT elevations to 173±34 U/L on day 14 that subsequently normalized promptly; none met Hy's criteria for serious liver injury. Conclusions: The mycobactericidal activity of Sutezolid600 mg BID or 1200 mg QD was readily detected in sputum and blood. Both schedules were generally safe and well tolerated. Further studies of sutezolid in tuberculosis treatment are warranted.

---

Oxazolidinone 6/Sutezolid has been tested in a M. tuberculosis mouse infection model. The degree of efficacy seen for orally administered 6 (mean cfu reduction in spleens and lungs of 3.1 and 4.9 log units, respectively) was comparable to that observed for the clinical standard isoniazid (mean cfu reduction in spleens and lungs of 3.3 and 4.7 log units, respectively), although 6 was dosed at a higher level (100 versus 25 mg/kg, administered orally once daily, 5 days/week for 4 weeks). In a comparative dose−response study in beige mice, 6 was found to have activity against M. avium complex similar to that of azithromycin (both drugs administered orally at a level of 100 mg/kg once daily, 5 days/week for 4 weeks). [1]

A preliminary evaluation of the pharmacokinetic behavior and metabolism of 6/Sutezolid in the rat was encouraging. Compound 6 was well absorbed after oral administration, although significant first-pass metabolism to the sulfoxide 7 and, to a much lesser extent, the sulfone 8 was observed. In a gratifying result, the combined plasma concentrations of 6 and/or 7 were found to be quite high and persistent; both 7 and 8 exhibit in vitro activity against M. tuberculosis similar to that of 6.

Orally administered 6Sutezolid/ had an acceptable safety profile in rats when dosed at 50 mg/kg b.i.d. for 29 days.7 This dosing regimen was very well tolerated, and drug-related findings were considered to be of minor toxicological relevance[1].

For minimum inhibitory concentration (MIC) determinations in Tables 1−3, the compounds were incorporated into 7H10 agar medium at concentrations of 2.0, 0.50, 0.125, and 0.03 μg/mL. The M. tuberculosis test organisms were grown in 7H9 medium containing 0.05% Tween 80. After 7 days of incubation at 37 °C, the broths were adjusted to the turbidity of a 1.0 McFarland standard; the organisms were then diluted 10-fold in sterile water containing 0.10% Tween 80. The resultant bacterial suspensions were spotted onto the drug-supplemented 7H10 plates. After a 21 day cultivation at 37 °C, the growth of the organisms was scored. The MIC was defined as the lowest concentration of drug that completely inhibited growth of the organism.[1]

---

Minimal inhibitory concentration (MIC) testing [3]
MICs of Sutezolid and its major metabolite were determined using MGIT. Sterile stock solutions of Sutezolid and PNU-101603 10 mg/ml were prepared in DMSO. Testing was performed using a series of 2-fold reductions in drug concentrations from 4.0 to 0.062 µg/ml. Growth in drug-containing tubes was compared to that of a positive growth control in which the inoculum was diluted 1∶100 in saline. The MIC was defined as the lowest concentration of drug without growth (GU<100) at the time when growth was detected in the positive control.

Subjects were randomly assigned to receive Sutezolid at 600 mg twice a day (BID; n = 25) or 1,200 mg once a day (QD; n = 25) or to receive a positive control of fixed-dose combination tablets consisting of isoniazid, rifampin, pyrazinamide, and ethambutol (HRZE; Rifafour e275; n = 9). The data for HRZE-treated subjects were not included in the present analysis.[2]

---

Blood was collected for WBA at the baseline (WBA0) and for pharmacokinetic (PK) and WBA determinations on days 13 and 14 (at 0, 1, 2, 3, 6, 8, and 12 h postdosing). Plasma was separated immediately after collection and stored at −20°C for PK determinations. Total plasma concentrations of Sutezolid/PNU-100480 and PNU-101603 were determined using a validated high-pressure liquid chromatography-tandem mass spectrometry method by Advion BioServices (Ithaca, NY), as previously described. [2]

---

After providing written informed consent, subjects were randomly assigned in blocks of 7–9 to Sutezolid 600 mg BID (N = 25) or 1200 mg QD (N = 25), or to a positive control of weight adjusted fixed dose combination tablets consisting of isoniazid, rifampin, pyrazinamide, and ethambutol (HRZE, N = 9). The purpose of this group was to ascertain comparability of laboratory methods to other EBA trials. Sutezolid was administered as 200 mg tablets. All treatments were administered on an inpatient basis. Neither subjects nor investigators were blinded to assigned treatment. Laboratory staff performing the sputum and whole blood assays was unaware of treatment allocation. After discharge all subjects were referred for a full course of standard antituberculosis treatment.[3]

A preliminary evaluation of the pharmacokinetic behavior and metabolism of 6/Sutezolid in the rat was encouraging. Compound 6 was well absorbed after oral administration, although significant first-pass metabolism to the sulfoxide 7 and, to a much lesser extent, the sulfone 8 was observed. In a gratifying result, the combined plasma concentrations of 6 and/or 7 were found to be quite high and persistent; both 7 and 8 exhibit in vitro activity against M. tuberculosis similar to that of 6. [1]

---

Plasma pharmacokinetics [3]
Plasma concentrations of Sutezolid and its major metabolite (PNU-101603) on day 14 are shown in figure 5. Key PK parameters are summarized in table 3. While the AUC0 −24 values were comparable between the QD and BID dosing regimens for both sutezolid and PNU-101603, administration of sutezolid as a single daily dose resulted in a doubling of the Cmax but slightly less than a doubling of that of the major metabolite. When dosed at 600 mg BID, median plasma concentrations of parent and major metabolite remained above their respective median MIC values for 71% and 89% of the dosing interval. In contrast, dosing at 1200 mg QD resulted in median supra-MIC plasma concentrations for 53% and 57% of the dosing interval, respectively. The observed mean AUC0–24 of parent and metabolite appeared to range from 63–71% and 86–91%, respectively, of values observed in phase 1, whereas Cmax values of parent and metabolite equaled those observed in healthy volunteers in phase 1.

Treatment with Sutezolid was generally safe and well tolerated. No subject required dose reduction or premature discontinuation due to adverse events or abnormal laboratory parameters. There were no instances of anemia or thrombocytopenia. There was no effect of sutezolid on the QTc interval, with changes from baseline to day 14 of −4.2±14.5 msec and −3.1±12.1 msec (mean±SD) in the 600 mg BID and 1200 mg QD arms, respectively. Treatment-emergent adverse events were distributed evenly across the Sutezolid arms and were mainly classified as mild (n = 26) or moderate (n = 13) in severity. A total of 7 Sutezolid-treated subjects (14%) experienced mild or moderate increases in alanine transaminase (ALT). Cases were distributed in both men and women (5∶2) and in both the BID and QD treatment arms (4∶3). None occurred in HIV-1 seropositive individuals. No other predisposing factors were identified. ALT values for these subjects increased from 34±24 IU/L at baseline (mean±SD) to 173±34 IU/L on day 15 (reference range, 6–48 IU/L) (figure 6). ALT increases were accompanied by smaller increases in AST, but not by changes in alkaline phosphatase or bilirubin. None of the subjects experienced symptoms of drug-induced liver injury. None met Hy's criteria for serious liver injury. All 7 subjects completed their assigned Sutezolid treatment and began standard TB therapy without interruption. Five of the 7 subjects were recalled for repeat testing on day 22, by which time values had declined to 75±28 IU/L. Values for all patients had returned to normal on day 42 (24±9 IU/L).[3]

[1]. Identification of a novel oxazolidinone (U-100480) with potent antimycobacterial activity. J Med Chem. 1996;39(3):680-685.

[2]. Population pharmacokinetic/pharmacodynamic analysis of the bactericidal activities of sutezolid (PNU-100480) and its major metabolite against intracellular Mycobacterium tuberculosis in ex vivo whole-blood cultures of patients with pulmonary tuberculosis. Antimicrob Agents Chemother. 2014;58(6):3306-3311.

[3]. Mycobactericidal activity of sutezolid (PNU-100480) in sputum (EBA) and blood (WBA) of patients with pulmonary tuberculosis. PLoS One. 2014 Apr 14;9(4):e94462.

Sutezolid has been used in trials studying the treatment of Tuberculosis.

---

During the course of our investigations in the oxazolidinone antibacterial agent area, we have identified a subclass with especially potent in vitro activity against mycobacteria. The salient structural feature of these oxazolidinone analogues, 6 (U-100480), 7 (U-101603), and 8 (U-101244), is their appended thiomorpholine moiety. The rational design, synthesis, and evaluation of the in vitro antimycobacterial activity of these analogues is described. Potent activity against a screening strain of Mycobacterium tuberculosis was demonstrated by 6 and 7 (minimum inhibitory concentrations or MIC's ≤0.125 μg/mL). Oxazolidinones 6 and 8 exhibit MIC90 values of 0.50 μg/mL or less against a panel of organisms consisting of five drug-sensitive and five multidrug-resistant strains of M. tuberculosis, with 6 being the most active congener. Potent in vitro activity against other mycobacterial species was also demonstrated by 6. For example, 6 exhibited excellent in vitro activity against multiple clinical isolates of Mycobacterium avium complex (MIC's = 0.5−4 μg/mL). Orally administered 6 displays in vivo efficacy against M. tuberculosis and M. avium similar to that of clinical comparators isoniazid and azithromycin, respectively. Consideration of these factors, along with a favorable pharmacokinetic and chronic toxicity profile in rats, suggests that 6 (U-100480) is a promising antimycobacterial agent.[1]

---

Rationale: Sutezolid (PNU-100480) is a linezolid analog with superior bactericidal activity against Mycobacterium tuberculosis in the hollow fiber, whole blood and mouse models. Like linezolid, it is unaffected by mutations conferring resistance to standard TB drugs. This study of sutezolid is its first in tuberculosis patients. Methods: Sputum smear positive tuberculosis patients were randomly assigned to sutezolid 600 mg BID (N = 25) or 1200 mg QD (N = 25), or standard 4-drug therapy (N = 9) for the first 14 days of treatment. Effects on mycobacterial burden in sputum (early bactericidal activity or EBA) were monitored as colony counts on agar and time to positivity in automated liquid culture. Bactericidal activity was also measured in ex vivo whole blood cultures (whole blood bactericidal activity or WBA) inoculated with M. tuberculosis H37Rv.[3]

C16H20N3O3FS

353.4117

353.121

C, 54.38; H, 5.70; F, 5.38; N, 11.89; O, 13.58; S, 9.07

168828-58-8

465951

A crystalline solid

1.321g/cm3

609ºC at 760 mmHg

322.1ºC

8.97E-15mmHg at 25°C

1.584

2.361

1

6

4

24

476

1

O=C(O[C@H]1CNC(C)=O)N(C1)C2=CC(F)=C(N3CCSCC3)C=C2

FNDDDNOJWPQCBZ-ZDUSSCGKSA-N

InChI=1S/C16H20FN3O3S/c1-11(21)18-9-13-10-20(16(22)23-13)12-2-3-15(14(17)8-12)19-4-6-24-7-5-19/h2-3,8,13H,4-7,9-10H2,1H3,(H,18,21)/t13-/m0/s1

(S)-N-((3-(3-fluoro-4-thiomorpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide

PNU 100480; PNU100480; Sutezolid; 168828-58-8; PNU-100,480; Oxazolidininone; U-100,480; (S)-N-((3-(3-fluoro-4-thiomorpholinophenyl)-2-oxooxazolidin-5-yl)methyl)acetamide; PNU 100,480; Sutezolid [INN]; U-100480; U 100480; U100480; PF-02341272; PF02341272; PF 02341272; PNU-100480;

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 : ~50 mg/mL (~141.48 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.07 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (7.07 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (7.07 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
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.

---

Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.5 mg/mL (7.07 mM)

---

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