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].

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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].

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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]

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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.

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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]

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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]

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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]

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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]

Preliminary assessments of the pharmacokinetic behavior and metabolism of compound 6/surezodil in rats were encouraging. Compound 6 was well absorbed after oral administration, but significant first-pass metabolism was observed, producing sulfoxide 7 and a small amount of sulfone 8. Encouragingly, the total plasma concentrations of compounds 6 and/or 7 were fairly high and sustained; compounds 7 and 8 both showed similar in vitro anti-tuberculosis activity to compound 6. [1]

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Plasma Pharmacokinetics[3]
Figure 5 shows the plasma concentrations of surezodil and its major metabolite (PNU-101603) on day 14. Key pharmacokinetic parameters are summarized in Table 3. The AUC0-24 values of surezodil and PNU-101603 were comparable for once-daily (QD) and twice-daily (BID) dosing regimens, but once-daily dosing doubled the Cmax of surezodil, while the Cmax of the major metabolite doubled by slightly less than 2 times. When administered 600 mg twice daily, median plasma concentrations of the parent drug and major metabolite were above their respective median MICs in 71% and 89% of the dosing intervals, respectively. In contrast, a once-daily 1200 mg dosing regimen achieved median plasma concentrations above the MIC in 53% and 57% of the dosing intervals, respectively. The observed mean AUC0-24 of the parent drug and metabolite were approximately 63-71% and 86-91% of the values observed in the Phase 1 trial, respectively, while the Cmax values of the parent drug and metabolite were the same as those in healthy volunteers in the Phase 1 trial.

Treatment with sultazolide was generally safe and well-tolerated. No subjects required dose reduction or early discontinuation of treatment due to adverse events or abnormal laboratory findings. No anemia or thrombocytopenia occurred. Sultazolide had no effect on the QTc interval; the changes from baseline in the 600 mg twice-daily and 1200 mg once-daily groups were -4.2 ± 14.5 ms and -3.1 ± 12.1 ms (mean ± standard deviation), respectively. Adverse events during treatment were evenly distributed across the sultazolide treatment groups, mainly classified as mild (n = 26) or moderate (n = 13). A total of 7 subjects (14%) treated with sultazolide experienced mild or moderate elevations in alanine aminotransferase (ALT). Cases were distributed between males and females (5:2) and between the twice-daily (BID) and once-daily (QD) treatment groups (4:3). None of the cases occurred in HIV-1 seropositive individuals. No other predisposing factors were identified. The ALT levels of these subjects increased from 34±24 IU/L (mean ± standard deviation) at baseline to 173±34 IU/L (reference range, 6–48 IU/L) on day 15 (Figure 6). The increase in ALT was accompanied by a slight increase in AST, but there were no changes in alkaline phosphatase or bilirubin. None of the subjects developed symptoms of drug-induced liver injury. None of the subjects met the criteria for severe liver injury for Hy. All 7 subjects completed their assigned sultezolid treatment and successfully started standard tuberculosis treatment. Five of the 7 subjects were recalled for retesting on day 22, at which time the ALT level had decreased to 75±28 IU/L. By day 42, all patients' values had returned to normal (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 clinical trials for tuberculosis treatment. In our research on oxazolidinone antimicrobial agents, we discovered a subclass with particularly potent in vitro activity against mycobacteria. These oxazolidinone analogs 6 (U-100480), 7 (U-101603), and 8 (U-101244) are characterized by their linked thiomorpholine moiety. This paper describes the rational design, synthesis, and evaluation of the in vitro antimycobacterial activity of these analogs. Compounds 6 and 7 showed potent activity against selected strains of Mycobacterium tuberculosis (MIC ≤ 0.125 μg/mL). Oxazolidinone compounds 6 and 8 showed MIC90 values ≤ 0.50 μg/mL against a group of five drug-sensitive and five multidrug-resistant strains of Mycobacterium tuberculosis, with compound 6 exhibiting the strongest activity. Compound 6 also showed potent in vitro activity against other mycobacterial genera. For example, compound 6 showed excellent in vitro activity against multiple clinical isolates of Mycobacterium avium complex (MIC = 0.5−4 μg/mL). The in vivo efficacy of compound 6 against Mycobacterium tuberculosis and Mycobacterium avium after oral administration was similar to that of the clinical control drugs isoniazid and azithromycin, respectively. Considering these factors, as well as the favorable pharmacokinetic and chronic toxicity characteristics in rats, compound 6 (U-100480) is a promising antimycobacterial drug. [1]

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Background: Seczodamide (PNU-100480) is a linezodamide analog with excellent bactericidal activity against Mycobacterium tuberculosis in hollow fiber, whole blood and mouse models. Like linezodamide, it is not affected by mutations that confer resistance to standard anti-tuberculosis drugs. This study is the first application of seczodamide in patients with tuberculosis. Methods: Tuberculosis patients with positive sputum smears were randomly assigned to the Sutezolid group (600 mg twice daily, n = 25), the Sutezolid group (1200 mg once daily, n = 25), or the standard quadruple anti-tuberculosis drug group (n = 9) for the first 14 days of treatment. The effect of mycobacterial load in sputum was monitored by colony counts on agar plates and the time to positivity in automated liquid culture (early bactericidal activity or EBA). In addition, bactericidal activity was measured by inoculating ex vivo whole blood cultures of Mycobacterium tuberculosis H37Rv (whole blood bactericidal activity or WBA). [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.

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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.

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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.

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

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