Quinolone antibiotic; DNA gyrase/topoisomerase
Zoliflodacin (formerly ETX-0914; AZD-0914; ETX0914; AZD0914) possesses antibacterial activity against important fastidious Gram-negative (Haemophilus influenzae, Neisseria gonorrhoeae), atypical (Legionella pneumophila), anaerobic (Clostridium difficile), and Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae), fastidious Gram-negative (Haemophilus influenzae, Neisseria gonorrhoeae), and atypical (Legionella pneumophila) bacterial species, including isolates with documented fluoroquinolone resistance. The mechanism of action of Zoliflodacin is demonstrated to be different from that of other commercially available antibacterial compounds, such as fluoroquinolones, in that it inhibits DNA biosynthesis and accumulates double-strand cleavages, thereby exerting antibacterial activity. Under permissive conditions, zoliflodacin stabilizes and arrests the cleaved covalent complex of gyrase with double-strand broken DNA, preventing the double-strand cleaved DNA from religating to form fused circular DNA[1].

Zoliflodacin (formerly ETX-0914; AZD-0914; ETX0914; AZD0914) possesses antibacterial activity against important fastidious Gram-negative (Haemophilus influenzae, Neisseria gonorrhoeae), atypical (Legionella pneumophila), anaerobic (Clostridium difficile), and Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae), fastidious Gram-negative (Haemophilus influenzae, Neisseria gonorrhoeae), and atypical (Legionella pneumophila) bacterial species, including isolates with documented fluoroquinolone resistance. The mechanism of action of Zoliflodacin is demonstrated to be different from that of other commercially available antibacterial compounds, such as fluoroquinolones, in that it inhibits DNA biosynthesis and accumulates double-strand cleavages, thereby exerting antibacterial activity. Under permissive conditions, zoliflodacin stabilizes and arrests the cleaved covalent complex of gyrase with double-strand broken DNA, preventing the double-strand cleaved DNA from religating to form fused circular DNA[1].

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Zoliflodacin (AZD0914) exhibits potent in vitro antibacterial activity against a wide range of Gram-positive, fastidious Gram-negative, atypical, and anaerobic bacteria, including drug-resistant strains.
Against 100 Staphylococcus aureus isolates (including MRSA), the MIC range was 0.06–0.5 µg/mL, with MIC₅₀ and MIC₉₀ values of 0.25 µg/mL. Activity was maintained against levofloxacin-resistant (MIC₉₀: >16 µg/mL), vancomycin-intermediate (VISA), vancomycin-resistant (VRSA), and linezolid-resistant S. aureus isolates. [1]
Against 98 Streptococcus pneumoniae isolates, the MIC range was 0.06–0.5 µg/mL, with MIC₅₀ and MIC₉₀ values of 0.125 and 0.25 µg/mL, respectively. Activity was maintained against levofloxacin-resistant and erythromycin-resistant isolates. [1]
Against 37 Neisseria gonorrhoeae isolates, the MIC range was 0.03–0.25 µg/mL, with MIC₅₀ and MIC₉₀ values of 0.06 and 0.125 µg/mL, respectively. Activity was maintained against ciprofloxacin-resistant (MIC range: 0.03–0.125 µg/mL) and a ceftriaxone- and ciprofloxacin-resistant isolate (MIC: 0.06 µg/mL). [1]
Against 29 Haemophilus influenzae isolates, the MIC range was 0.125–1 µg/mL, with MIC₅₀ and MIC₉₀ values of 0.25 and 1 µg/mL, respectively. [1]
Against 10 Legionella pneumophila isolates, the MIC range was 0.008–0.06 µg/mL, with MIC₅₀ and MIC₉₀ values of 0.03 and 0.06 µg/mL, respectively. [1]
Against 2 Clostridium difficile isolates, the MIC was 0.125 µg/mL. [1]
Activity against Gram-negative non-fastidious pathogens like Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii was lower (MIC₉₀s ranged from 4 to >128 µg/mL). Efflux pumps (e.g., tolC in E. coli, mexABCDXY in P. aeruginosa) contributed to reduced activity in Gram-negatives. [1]
Minimum bactericidal concentration (MBC) testing against seven S. aureus strains and one S. pyogenes strain showed Zoliflodacin (AZD0914) was bactericidal, with MBCs within a 2-fold range of the MIC. [1]
In vitro time-kill studies against levofloxacin-resistant MRSA (USA100) and S. pneumoniae ATCC 49619 showed a ≥3-log₁₀ reduction in CFU/mL within 6 hours at concentrations ≥2× MIC. [1]
The post-antibiotic effect (PAE) of Zoliflodacin (AZD0914) against USA100 and S. pneumoniae ATCC 49619 ranged from 0.25 to >3.3 hours, increasing with concentration. [1]
The frequency of spontaneous resistance development in S. aureus was low (10⁻⁹ to <10⁻¹⁰ at 4–8× MIC), comparable to linezolid and lower than levofloxacin. Resistant mutants, when obtained, mapped to gyrB and showed a ≥4-fold increase in MIC. [1]
No cross-resistance was observed against isolates with known mutations in gyrA, gyrB, parC, or parE that confer resistance to fluoroquinolones, novobiocin, or coumermycin. AZD0914 MICs varied by no more than 2-fold compared to wild-type strains. [1]
In checkerboard synergy testing with 17 comparator antibacterials against five reference strains, only additive/indifferent effects (mean FICI: 0.77–1.66) were observed, with no synergy or antagonism. [1]

The Centers for Disease Control and the World Health Organization have issued a list of priority pathogens for which there are dwindling therapeutic options, including antibiotic-resistant Neisseria gonorrheae, for which novel oral agents are urgently needed. Zoliflodacin, the first in a new class of antibacterial agents called the spiropyrimidinetriones, is being developed for the treatment of gonorrhea. It has a unique mode of inhibition against bacterial type II topoisomerases with binding sites in bacterial gyrase that are distinct from those of the fluoroquinolones. Zoliflodacin is bactericidal, with a low frequency of resistance and potent antibacterial activity against N. gonorrheae, including multi-drug-resistant strains (MICs ranging from ≤0.002 to 0.25 μg/mL). Although being developed for the treatment of gonorrhea, zoliflodacin also has activity against Gram-positive, fastidious Gram-negative, and atypical pathogens. A hollow-fiber infection model using S. aureus showed that that pharmacokinetic/pharmacodynamic index of fAUC/MIC best correlated with efficacy in in vivo neutropenic thigh models in mice. This data and unbound exposure magnitudes derived from the thigh models were subsequently utilized in a surrogate pathogen approach to establish dose ranges for clinical development with N. gonorrheae. In preclinical studies, a wide safety margin supported progression to phase 1 studies in healthy volunteers, which showed linear pharmacokinetics, good oral bioavailability, and no significant safety findings. In a phase 2 study, zoliflodacin was effective in treating gonococcal urogenital and rectal infections. In partnership with the Global Antibiotic Research Development Program (GARDP), zoliflodacin is currently being studied in a global phase 3 clinical trial. Zoliflodacin represents a promising new oral therapy for drug-resistant infections caused by N. gonorrheae[2].

AZD0914 is a new spiropyrimidinetrione bacterial DNA gyrase/topoisomerase inhibitor with potent in vitro antibacterial activity against key Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus agalactiae), fastidious Gram-negative (Haemophilus influenzae and Neisseria gonorrhoeae), atypical (Legionella pneumophila), and anaerobic (Clostridium difficile) bacterial species, including isolates with known resistance to fluoroquinolones. AZD0914 works via inhibition of DNA biosynthesis and accumulation of double-strand cleavages; this mechanism of inhibition differs from those of other marketed antibacterial compounds. AZD0914 stabilizes and arrests the cleaved covalent complex of gyrase with double-strand broken DNA under permissive conditions and thus blocks religation of the double-strand cleaved DNA to form fused circular DNA. Whereas this mechanism is similar to that seen with fluoroquinolones, it is mechanistically distinct. AZD0914 exhibited low frequencies of spontaneous resistance in S. aureus, and if mutants were obtained, the mutations mapped to gyrB. Additionally, no cross-resistance was observed for AZD0914 against recent bacterial clinical isolates demonstrating resistance to fluoroquinolones or other drug classes, including macrolides, β-lactams, glycopeptides, and oxazolidinones. AZD0914 was bactericidal in both minimum bactericidal concentration and in vitro time-kill studies. In in vitro checkerboard/synergy testing with 17 comparator antibacterials, only additivity/indifference was observed. The potent in vitro antibacterial activity (including activity against fluoroquinolone-resistant isolates), low frequency of resistance, lack of cross-resistance, and bactericidal activity of AZD0914 support its continued development[1].

The determination of the macrodilution MICs for zoliflodacin broth serves as the foundation for in vitro time-kill and postantibiotic-effect (PAE) assays. Using glass tubes (18 by 150 mm, without agitation), in vitro static time-kill studies are carried out with 10-mL volumes of cation-adjusted Mueller-Hinton broth containing logarithmically growing cultures (starting inoculum of 1×10⁶ CFU/mL) against both levofloxacin-susceptible and levofloxacin-resistant S. aureus. The concentrations of zoliflodacin that are tested are 0.5, 1, 2, 4, and 8 times the minimum inhibitory concentration (MIC). Samples are plated for colony counts at 0, 2, 4, 6, 8, and 24 hours using 100 μL aliquots spotted onto 25-ml sheep blood agar plates according to the previously mentioned protocol. At the lowest drug concentration that decreased viable organism counts by ≥3 log₁₀ in a 24-hour period, compounds are deemed bactericidal. Time-kill research is carried out in duplicate, with test results combined and mean values reported [1].

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In vitro time-kill studies were conducted using glass tubes containing cation-adjusted Mueller-Hinton broth. Logarithmically growing cultures of S. aureus (USA300 and USA100) or S. pneumoniae (ATCC 49619) at a starting inoculum of 1 × 10⁶ CFU/mL were exposed to Zoliflodacin (AZD0914) at concentrations equivalent to 0.5, 1, 2, 4, and 8 times the MIC. Samples were taken at 0, 2, 4, 6, 8, and 24 hours, and 100 µL aliquots were spotted onto sheep blood agar plates for colony counting. Bactericidal activity was defined as a ≥3-log₁₀ reduction in viable count within 24 hours. Studies were conducted in duplicate, and mean values were reported. [1]
Post-antibiotic effect (PAE) testing was conducted similarly. Cultures were exposed to Zoliflodacin (AZD0914) at 0.5, 1, 4, or 16 times the MIC for 1 hour. The drug was then removed by a 1:1000 dilution. The PAE was defined as the difference in time required for the drug-exposed and unexposed cultures to increase by 1 log₁₀ CFU/mL above the count immediately after dilution. [1]

Until recently, the lack of a robust animal model for N. gonorrheae has hampered the ability to understand the PK/pharmacodynamic (PD) and exposure target for this pathogen. Successful colonization is often difficult to achieve and maintain within a therapeutic window due to spontaneous eradication in the host system, which limits the utility of this model in the evaluation of new antibiotics. In vitro dynamic model systems such as the hollow-fiber used to determine the PK/PD driver and evaluate clinic dose regimens have only recently shown successful use with N. gonorrheae. Current therapies and regimens utilized clinically have often relied upon anecdotal evidence provided by clinical use of available agents, demonstrating potent susceptibility to the pathogen. Due to the similarity in the mechanism of action of zoliflodacin to other market topoisomerase inhibitors that have successfully been used to treat gonorrhea, a surrogate pathogen approach with S. aureus was utilized for zoliflodacin. The translation of experimentally derived PK/PD targets was based upon the premise that unbound plasma targets obtained from reference targets in the murine models matched those associated with clinical efficacy. Although the PK/PD driver associated with topoisomerase inhibitors has historically been the area under the concentration curve (AUC)/MIC, this correlation was confirmed with zoliflodacin itself in a series of in vitro hollow-fiber experiments in which unbound drug concentration vs time profiles were simulated and dose fractionated to distinguish among PK/PD drivers of AUC/MIC, Cmax/AUC, and T > MIC. The results of this study utilizing a methicillin-susceptible strain of S. aureus (ARC516) demonstrated AUC/MIC to be the index most closely associated with the activity of zoliflodacin, [R2] = 0.95 (Figure 4).[2]

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With the PK/PD driver established for zoliflodacin against S. aureus in the hollow-fiber model, assessment of the in vivo efficacy of zoliflodacin was established using a murine thigh infection model in both immunocompetent and immunosuppressed mice using a methicillin-susceptible strain of S. aureus (ARC516). Zoliflodacin was highly efficacious in a dose-dependent fashion for both mouse models. Stasis was achieved at an AUC of 20 μg·h/mL. Accounting for the protein binding of zoliflodacin in the mouse (81% bound), a maximum 3 log10 decrease in the CFU/g bacterial burden corresponded to an unbound AUC of 23 μg·h/mL for neutropenic animals. S. aureus ARC516 used for this study had a zoliflodacin MIC of 0.0625 μg/mL with an fAUC/MIC of 368 for >3 log10 kill. Additional S. aureus strains were studied in vivo in the neutropenic thigh infection model including MRSA strains ATCC 33591, USA100 NRS382, and USA300 NR538 in order to establish mean unbound exposure requirements associated with stasis and 1 log10 kill. From the combined in vivo data set, the fAUC/MIC values associated with stasis and 1 log10 kill were 66 and 139, respectively. These values were ultimately used as PK/PD-derived exposure targets for clinical dose justification. The PK/PD index of fAUC/MIC generated in the hollow fiber with zoliflodacin against S. aureus and unbound exposure magnitudes derived from in vivo neutropenic thigh models conducted in mice were subsequently utilized in the surrogate pathogen approach to help establish dose ranges for clinical development with N. gonorrheae. To support the approach, fAUC/MIC magnitudes associated with clinical doses to treat N. gonorrheae and skin infections caused by S. aureus were obtained for reference compounds ofloxacin and ciprofloxacin as shown in Table 6. [2]

Absorption
In the fasting state, the median time to peak plasma concentration (Tmax) is 2.5 hours; in the postprandial state, it is 4.0 hours. Food intake significantly affects absorption—compatibility with moderate or high-fat foods increases Cmax by approximately 1.5 times and AUC by approximately 1.5 to 2 times compared to the fasting state. In patients with uncomplicated urogenital gonorrhea treated with a 3-gram dose, the geometric mean Cmax was 28.5 μg/mL.
Elimination Route
The primary route of elimination for zolidoxime is feces. Approximately 79.6% of drug-related substances are excreted in feces (1.5% of which is unchanged drug) and 18.2% are excreted in urine (2.5% of which is unchanged drug).
Volume of Distribution
The geometric mean apparent volume of distribution of zolidoxime is 177 L in the fasting state and 98.7 L in the postprandial state.
Clearance
The geometric mean apparent systemic clearance of zolidoxime was 19.1 L/h in the fasting state and 12.5 L/h in the fed state.
Protein Binding
Zolidoxime binds to human plasma proteins at a rate of 83%.
Metabolism/Metabolites
The main clearance mechanism of zolidoxime is through CYP-mediated and non-CYP-mediated metabolic pathways, but the non-CYP metabolic pathways are not fully elucidated. CYP phenotypic studies indicate that CYP-mediated metabolism is mainly carried out through CYP3A4/5 (68%), with smaller contributions from CYP1A2 (14%), CYP2C9 (10%), CYP2C8 (5%), and CYP2C19 (2.7%).
Biological Half-Life
The geometric mean elimination half-life of zolifludacin is 6.4 hours in the fasting state and 5.5 hours in the fed state.

[1]. In vitro antibacterial activity of AZD0914, a new spiropyrimidinetrione DNA gyrase/topoisomerase inhibitor with potent activity against Gram-positive, fastidious Gram-Negative, and atypical bacteria. Antimicrob Agents Chemother. 2015 Jan;59(1):467-74.

[2]. Zoliflodacin: An Oral Spiropyrimidinetrione Antibiotic for the Treatment of Neisseria gonorrheae, Including Multi-Drug-Resistant Isolates. ACS Infect. Dis.2020 Jun 12;6(6):1332-1345.

Zolifludacin has been used in clinical trials for basic science and treatment research on gonorrhea. To address the unmet medical needs of multidrug-resistant Neisseria gonorrhoeae, zolifludacin (formerly known as AZD0914, ETX0914) was developed as a novel antimicrobial drug—a spiropyrimidine trione—for the treatment of uncomplicated gonorrhea. Zolifludacin is an orally bioavailable antimicrobial drug whose bactericidal mechanism is through inhibition of bacterial type II topoisomerase. This review presents preclinical and early clinical data of this novel oral antibiotic. [2] Zolifludacin is a novel spiropyrimidine trione antibiotic developed by Entasis Therapeutics for the treatment of uncomplicated gonorrhea and is the first novel topoisomerase inhibitor. Due to its unique mechanism of action, zolifludacin does not exhibit cross-resistance with fluoroquinolones and may therefore be effective in treating infections caused by fluoroquinolone-resistant Neisseria gonorrhoeae strains. A phase II study confirmed its safety and efficacy, supporting this view. Entasis has partnered with the Global Antibiotics Development Programme (GARDP) to continue the development of zoliflodacin and recently announced the initiation of a global Phase III trial (https://gardp.org/news-resources/gardp-and-entasis-therapeutics-initiate-global-phase-3-trial-of-zoliflodacin-a-first-in-class-oral-antibiotic-for-the-treatment-of-gonorrhoea/). This is an open-label trial comparing the efficacy of a single oral dose of 3 grams of zoliflodacin versus ceftriaxone combined with azithromycin (2:1 randomization). Similar to the Phase II study, the Phase III trial also uses genitourinary infections caused by Neisseria gonorrhoeae as the inclusion criterion. However, this trial will also investigate extragenitourinary infections as a secondary endpoint, hoping that these data will also help us understand the efficacy of zoliflodacin in treating these infections. This clinical study is expected to enroll approximately 1,000 adult patients with urogenital gonorrhea from the United States, the Netherlands, Thailand and South Africa (ClinicalTrials.gov registration number: NCT03959527), with results expected in 2021. If the trial yields positive results in the 2020s, it will suggest that zolidoxime can be used as an oral monotherapy for the treatment of gonorrhea. [2]

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Zolidoxime (AZD0914) is a novel oral active spiropyrimidine trione antibiotic designed to treat infections caused by drug-resistant Gram-positive and difficult-to-culture Gram-negative bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), drug-resistant Streptococcus pneumoniae and multidrug-resistant Neisseria gonorrhoeae. [1]
Its mechanism of action involves inhibiting DNA biosynthesis by stabilizing the cleaved DNA gyrase-DNA complex, which is quite different from the mechanism of action of fluoroquinolones. [1]
This compound exhibits potent activity against fluoroquinolone-resistant and ceftriaxone-resistant strains, with low resistance rates, no cross-resistance, and bactericidal properties, all of which support its continued development. [1]
This compound has obtained Qualified Infectious Disease Product (QIDP) designation and Fast Track status, and its efficacy in treating sexually transmitted infections caused by Neisseria gonorrhoeae is currently being evaluated. [1]

C22H22N5O7F

487.438

487.15

C, 54.21; H, 4.55; F, 3.90; N, 14.37; O, 22.98

1620458-09-4

76685216

White to off-white solid powder

1.6±0.1 g/cm3

1.685

1.77

2

10

1

35

962

4

O=C(NC(C12[C@]([C@H](C)O[C@H](C)C3)([H])N3C4=C(C=C5C(ON=C5N6C(OC[C@@H]6C)=O)=C4F)C2)=O)NC1=O

ZSWMIFNWDQEXDT-ZESJGQACSA-N

InChI=1S/C22H22FN5O7/c1-8-7-33-21(32)28(8)17-12-4-11-5-22(18(29)24-20(31)25-19(22)30)16-10(3)34-9(2)6-27(16)14(11)13(23)15(12)35-26-17/h4,8-10,16H,5-7H2,1-3H3,(H2,24,25,29,30,31)/t8-,9+,10-,16+/m0/s1

(4'R,6'S,7'S)-17'-fluoro-4',6'-dimethyl-13'-[(4S)-4-methyl-2-oxo-1,3-oxazolidin-3-yl]spiro[1,3-diazinane-5,8'-5,15-dioxa-2,14-diazatetracyclo[8.7.0.02,7.012,16]heptadeca-1(17),10,12(16),13-tetraene]-2,4,6-trione

Zoliflodacin; ETX0914; AZD-0914; ETX-0914; AZD0914; ETX 0914; AZD 0914; AZD0914; AZD-0914; Zoliflodacin [USAN]; FWL2263R77;

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: ~140 mg/mL (~287.2 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.27 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 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.27 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 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.27 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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