Intermethod comparability analyses of gepotidacin antimicrobial susceptibility tests using a large collection of globally collected Escherichia coli and Staphylococcus saprophyticus clinical isolates.

Gepotidacin (GSK2140944) is a novel, bactericidal, first in class triazaacenaphthylene bacterial type II topoisomerase inhibitor in development for the treatment of uncomplicated urinary tract infections and gonorrhea. The performance of several antimicrobial susceptibility methods (broth microdilution, gradient diffusion, and disk diffusion) for gepotidacin were evaluated using over 5800 recent Escherichia coli and Staphylococcus saprophyticus clinical isolates. Reference broth microdilution gepotidacin MICs showed an essential agreement of 95.9 % and 98.1 % with MICs by gradient diffusion for E. coli and S. saprophyticus isolates, respectively. Gepotidacin susceptibility using disks produced by 2 manufacturers had good agreement with an R2 values of 0.95 and 99.2 % of overall zone diameters agreeing within 3 mm. A correlation with an overall R2 value of 0.72 between MICs by broth microdilution and zone diameters by disk diffusion was observed. This data should assist in the clinical development of gepotidacin and provide reliable susceptibility methods to evaluate its activity.

Effects of Bis(imino)acenaphthene (Bian)-Derived Ligands on the Cytotoxicity, DNA Interactions, and Redox Activity of Palladium(II) Bipyridine Complexes.

A series of heteroleptic bipyridine Pd(II) complexes based on 1,2-bis[(2,6-diisopropylphenyl)imino]acenaphthene (dpp-Bian) or 1,2-bis[(2,4,6-trimethylphenyl)imino]acenaphthene (tmp-Bian) were prepared. All complexes were fully characterized by spectrochemical methods, and their crystal structures were confirmed by X-ray diffraction analysis. The 72 h stability of heteroleptic bipyridine Pd(II) complexes with Bian ligands under physiological conditions was investigated using 1H NMR spectroscopy. The anticancer activity of all complexes was assessed in a panel of cancer cell lines in comparison with uncoordinated ligands and clinically used drugs cisplatin and doxorubicin. The ability of the complexes to bind DNA was investigated using several methods, including EtBr replacement assay, density functional theory calculations, circular dichroism spectroscopy, DNA gel electrophoresis, and TUNEL assay. The electrochemical activity of all complexes and the uncoordinated ligands was studied using cyclic voltammetry, and reactive oxygen species production in cancer cells was investigated using confocal microscopy. Heteroleptic bipyridine PdII-Bian complexes were cytotoxic in a low micromolar concentration range and showed some selectivity toward cancer cells in comparison with noncancerous MRC-5 lung fibroblasts.

Gepotidacin: a novel, oral, 'first-in-class' triazaacenaphthylene antibiotic for the treatment of uncomplicated urinary tract infections and urogenital gonorrhoea.

The ongoing spread of antimicrobial resistance has made the treatment of uncomplicated urinary tract infections (UTIs) and urogenital gonorrhoea increasingly difficult. New oral treatment options are urgently needed. Gepotidacin (previously GSK2140944) is a novel, bactericidal, oral, 'first-in-class' triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by blocking two essential topoisomerase enzymes. Mutations in both enzymes would likely be necessary for resistance to occur, thus raising hopes that the drug will be able to maintain long-term effectiveness. Data from Phase II clinical trials of gepotidacin in UTIs and urogenital gonorrhoea appear promising, and Phase III trials are underway. In this review we summarize the development of gepotidacin and discuss its potential role in clinical practice. If approved, gepotidacin will be the first new oral antibiotic for UTIs in more than 20 years.

Dose selection for a phase III study evaluating gepotidacin (GSK2140944) in the treatment of uncomplicated urogenital gonorrhoea.

BACKGROUND: Gepotidacin is a novel, first-in-class triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by a distinct mechanism of action and is active against most strains of Neisseria gonorrhoeae (N. gonorrhoeae). Phase II data suggested higher exposures were needed for efficacy and to suppress resistance development. A translational approach using in vitro pharmacokinetic/pharmacodynamic (PK/PD) and clinical data was used to select a gepotidacin dose for a phase III study. In this narrative review of previously shown data, we summarise how a translational approach based on in vitro PK/PD and population PK modelling and simulation data was undertaken to select a dosing regimen for the ongoing phase III gepotidacin study in participants with uncomplicated urogenital gonorrhoea. METHODS: For dose selection, prior in vitro minimum inhibitory concentrations (MICs) and PK/PD data were available. PK modelling was conducted to determine a dose that would limit plasma concentrations to less than 14 µg/mL (as concentrations above this are associated with QT prolongation and effects associated with acetylcholinesterase inhibition) while maintaining ≥90% probability of target attainment (PTA) for efficacy and resistance suppression against N. gonorrhoeae isolates with gepotidacin MICs ≤1 µg/mL. RESULTS: Two 3000 mg gepotidacin doses, administered 10-12 hours apart, resulted in PTA of ≥97.5% and ≥91.7% for gepotidacin MICs ≤1 µg/mL for the ratio of the area under the free drug plasma concentration-time curve over 24 hours to the MIC (fAUC0-24/MIC) efficacy, and resistance suppression targets of 40 and 46, respectively, but limited the occurrence of maximum plasma concentrations ≥14 µg/mL. CONCLUSIONS: Two gepotidacin 3000 mg oral doses 10-12 hours apart provide ~2-fold higher systemic exposures, increase efficacy for higher gepotidacin MIC N. gonorrhoeae isolates, reduce resistance potential and limit plasma concentrations of potential safety concern, compared with higher doses.

Effects of polycyclic aromatic hydrocarbons on marine and freshwater microalgae - A review.

The first synthetic review of the PAHs effects on microalgae in experimental studies and aquatic ecosystems is provided. Phytoplankton and phytobenthos from marine and freshwaters show a wide range of sensitivities to PAHs, and can accumulate, transfer and degrade PAHs. Different toxicological endpoints including growth, chlorophyll a, in vivo fluorescence yield, membrane integrity, lipid content, anti-oxidant responses and gene expression are reported for both freshwater and marine microalgal species exposed to PAHs in culture and in natural assemblages. Photosynthesis, the key process carried out by microalgae appears to be the most impacted by PAH exposure. The effect of PAHs is both dose- and species-dependent and influenced by environmental factors such as UV radiation, temperature, and salinity. Under natural conditions, PAHs are typically present in mixtures and the toxic effects induced by single PAHs are not necessarily extrapolated to mixtures. Natural microalgal communities appear more sensitive to PAH contamination than microalgae in monospecific culture. To further refine the ecological risks linked to PAH exposure, species-sensitivity distributions (SSD) were analyzed based on published EC50s (half-maximal effective concentrations during exposure). HC5 (harmful concentration for 5% of the species assessed) was derived from SSD to provide a toxicity ranking for each of nine PAHs. The most water-soluble PAHs naphthalene (HC5 = 650 µg/L), acenaphthene (HC5 = 274 µg/L), and fluorene (HC5 = 76.8 µg/L) are the least toxic to microalgae, whereas benzo[a]pyrene (HC5 = 0.834 µg/L) appeared as the more toxic. No relationship between EC50 and cell biovolume was established, which does not support assumptions that larger microalgal cells are less sensitive to PAHs, and calls for further experimental evidence. The global PAHs HC5 for marine species was on average higher than for freshwater species (26.3 and 1.09 µg/L, respectively), suggesting a greater tolerance of marine phytoplankton towards PAHs. Nevertheless, an important number of experimental exposure concentrations and reported toxicity thresholds are above known PAHs solubility in water. The precise and accurate assessment of PAHs toxicity to microalgae will continue to benefit from more rigorously designed experimental studies, including control of exposure duration and biometric data on test microalgae.

Dose Selection for Phase III Clinical Evaluation of Gepotidacin (GSK2140944) in the Treatment of Uncomplicated Urinary Tract Infections.

Antibiotics are the current standard-of-care treatment for uncomplicated urinary tract infections (uUTIs). However, increasing rates of bacterial antibiotic resistance necessitate novel therapeutic options. Gepotidacin is a first-in-class triazaacenaphthylene antibiotic that selectively inhibits bacterial DNA replication by interaction with the bacterial subunits of DNA gyrase (GyrA) and topoisomerase IV (ParC). Gepotidacin is currently in clinical development for the treatment of uUTIs and other infections. In this article, we review data for gepotidacin from nonclinical studies, including in vitro activity, in vivo animal efficacy, and pharmacokinetic (PK) and pharmacokinetic/pharmacodynamic (PK/PD) models that informed dose selection for phase III clinical evaluation of gepotidacin. Based on this translational package of data, a gepotidacin 1,500-mg oral dose twice daily for 5 days was selected for two ongoing, randomized, multicenter, parallel-group, double-blind, double-dummy, active-comparator phase III clinical studies evaluating the safety and efficacy of gepotidacin in adolescent and adult female participants with uUTIs (ClinicalTrials.gov identifiers NCT04020341 and NCT04187144).

In Vitro Activity of Gepotidacin against Gram-Negative and Gram-Positive Anaerobes.

Gepotidacin (formerly GSK2140944) is a first-in-class triazaacenaphthylene antibacterial currently in phase III clinical trials. When tested against Gram-negative (n = 333) and Gram-positive (n = 225) anaerobes by agar dilution, gepotidacin inhibited 90% of isolates at concentrations of 4 and 2 µg/mL, respectively. Given gepotidacin's in vitro activity against the anaerobic isolates tested, further study is warranted to better understand the utility of gepotidacin in the treatment of infections caused by clinically relevant anaerobic organisms.

Analysis of antimicrobial susceptibility testing methods and variables and in vitro activity of gepotidacin against urogenital Neisseria gonorrhoeae in men.

Gepotidacin is a triazaacenaphthylene antibiotic with activity against Neisseria gonorrhoeae including strains resistant to current agents. We tested 145 N. gonorrhoeae isolates by agar dilution according to Gonococcal Isolate Surveillance Program and Clinical and Laboratory Standards Institute methodologies. Gepotidacin demonstrated a minimum inhibitory concentration (MIC)50 of 0.25 µg/mL and a MIC90 of 0.5 µg/mL (highest gepotidacin MIC was 1 µg/mL) against the 145 N. gonorrhoeae isolates tested. We also assessed the impact of test variables on antimicrobial susceptibility test results for gepotidacin, ciprofloxacin, and ceftriaxone against 10 N. gonorrhoeae isolates. Media type had the biggest effect but wasn't specific to gepotidacin. Gepotidacin MICs were also affected by inoculum, pH, and 10% CO2. These in vitro data indicate that further study of gepotidacin is warranted for potential use in treating gonorrhea infections and highlight the importance of controlling for media type, inoculum, CO2, and pH when performing MIC testing with gepotidacin.

Functional and Anatomical Characterization of Corticotropin-Releasing Factor Receptor Subtypes of the Rat Spinal Cord Involved in Somatic Pain Relief.

Corticotropin-releasing factor (CRF) orchestrates our body's response to stressful stimuli. Pain is often stressful and counterbalanced by activation of CRF receptors along the nociceptive pathway, although the involvement of the CRF receptor subtypes 1 and/or 2 (CRF-R1 and CRF-R2, respectively) in CRF-induced analgesia remains controversial. Thus, the aim of the present study was to examine CRF-R1 and CRF-R2 expression within the spinal cord of rats with Freund's complete adjuvant-induced unilateral inflammation of the hind paw using reverse transcriptase polymerase chain reaction, Western blot, radioligand binding, and immunofluorescence confocal analysis. Moreover, the antinociceptive effects of intrathecal (i.t.) CRF were measured by paw pressure algesiometer and their possible antagonism by selective antagonists for CRF-R1 and/or CRF-R2 as well as for opioid receptors. Our results demonstrated a preference for the expression of CRF-R2 over CRF-R1 mRNA, protein, binding sites and immunoreactivity in the dorsal horn of the rat spinal cord. Consistently, CRF as well as CRF-R2 agonists elicited potent dose-dependent antinociceptive effects which were antagonized by the i.t. CRF-R2 selective antagonist K41498, but not by the CRF-R1 selective antagonist NBI35965. In addition, i.t. applied opioid antagonist naloxone dose-dependently abolished the i.t. CRF- as well as CRF-R2 agonist-elicited inhibition of somatic pain. Importantly, double immunofluorescence confocal microscopy of the spinal dorsal horn showed CRF-R2 on enkephalin (ENK)-containing inhibitory interneurons in close opposition of incoming mu-opioid receptor-immunoreactive nociceptive neurons. CRF-R2 was, however, not seen on pre- or on postsynaptic sensory neurons of the spinal cord. Taken together, these findings suggest that i.t. CRF or CRF-R2 agonists inhibit somatic inflammatory pain predominantly through CRF-R2 receptors located on spinal enkephalinergic inhibitory interneurons which finally results in endogenous opioid-mediated pain inhibition.

Factors of sex and age dictate the regulation of GABAergic activity by corticotropin-releasing factor receptor 1 in the medial sub-nucleus of the central amygdala.

Adolescents are phenotypically characterized with hyper-sensitivity to stress and inappropriate response to stress-inducing events. Despite behavioral distinctions from adults, investigations of developmental shifts in the function of stress peptide corticotropin-releasing factor (CRF) are generally limited. Rodent models have determined that CRF receptor 1 (CRFR1) activation within the central amygdala is associated with a stress response and induces increased GABAergic synaptic neurotransmission within adult males. To investigate age- and sex-specific function of this system, we performed whole-cell patch clamp electrophysiology in brain slices from naive adolescent (postnatal days (P) 40-49) and adult (>P70) male and female Sprague Dawley rats to assess GABAergic activity in the medial central amygdala (CeM). Our results indicate a dynamic influence of age and sex on neuronal excitability within this region, as well as basal spontaneous and miniature (m) inhibitory post-synaptic currents (IPSCs) in the CeM. In addition to replicating prior findings of CRFR1-regulated increases in mIPSC frequency in adult males, we found that the selective CRFR1 agonist, Stressin-1, attenuated mIPSC frequency in adolescent males, at a concentration that did not produce an effect in adult males. Importantly, this age-specific distinction was absent in females, as Stressin-1 attenuated mIPSC frequency in both adolescent and adult females. Finally, an increase in mIPSC frequency in response to the CRF1R antagonist, NBI 35965, was observed only in the CeM of adult males. Together, these data emphasize the robust influence of age and sex on neurophysiological function of a brain region involved in the production of the stress response.

Three new acenaphthene derivatives from rhizomes of Musa basjoo and their cytotoxic activity.

Three new acenaphthene derivatives cis-3-(4'-methoxyphenyl)-acenaphthene-1, 2-diol (1), trans-(1S, 2S)-3-phenyl-acenaphthene-1, 2-diol (2) and 8-(4-hydroxyphenyl)-2H-acenaphthylen-1-one (3) in company with six known compounds were isolated from the 70% ethanol extract of the rhizomes of Musa basjoo. Those chemical constituents were separated and purified by macroreticular resin, silica gel, Toyopearl HW-40F, SephadexLH-20 and other chromatographic methods, respectively. The chemical structures of new compounds were elucidated by HR-ESI-MS, 1H NMR, 13C NMR, HMQC, HMBC spectrum and specific optical rotations. Compound 4 was isolated for the first time from the genus Musa and compound 7 was firstly assigned the carbon spectrum. Furthermore, the cytotoxic activity of compounds 1-9 against WM9, MDA-MB231, HeLa, K562, DU145 and PC3 was screened with cisplatin as a positive control. Compound 9 showed promising cytotoxic activities with IC50 values of 2.65 ± 0.38 µM against the HeLa cell lines, while compound 8 possessed significant cytotoxicity with IC50 values of 6.51 ± 0.44, 18.54 ± 0.68 and 7.98 ± 1.44 µM against the HeLa, MDA-MB231 and WM9 cell lines, respectively.

Leveraging Oxidative Stress to Regulate Redox Balance-Based, In Vivo Growth Selections for Oxygenase Engineering.

Directed evolution methods based on high-throughput growth selection enable efficient discovery of enzymes with improved function in vivo. High-throughput selection is particularly useful when engineering oxygenases, which are sensitive to structural perturbations and prone to uncoupled activity. In this work, we combine the principle that reactive oxygen species (ROS) produced by uncoupled oxygenase activity are detrimental to cell fitness with a redox balance-based growth selection method for oxygenase engineering that enables concurrent advancement in catalytic activity and coupling efficiency. As a proof-of-concept, we engineered P450-BM3 for degradation of acenaphthene (ACN), a recalcitrant environmental pollutant. Selection of site-saturation mutagenesis libraries in E. coli strain MX203 identified P450-BM3 variants GVQ-AL and GVQ-D222N, which have both improved coupling efficiency and catalytic activity compared to the starting variant. Computational modeling indicates that the discovered mutations cooperatively optimize binding pocket shape complementarity to ACN, and shift the protein's conformational dynamics to favor the lid-closed, catalytically competent state. We further demonstrated that the selective pressure on coupling efficiency can be tuned by modulating cellular ROS defense mechanisms.

Comparison of minimum inhibitory concentration results for gepotidacin obtained using agar dilution and broth microdilution methods.

Gepotidacin (GSK2140944) is a first in class, novel triazaacenaphthylene bacterial type II topoisomerase inhibitor in Phase 3 clinical development for the treatment of gonorrhea and uncomplicated urinary tract infections (acute cystitis). This study tested the equivalency of minimal inhibitory concentrations (MICs) obtained by two reference susceptibility testing methods, agar dilution and broth microdilution, for gepotidacin when tested against various gram-positive and gram-negative organisms. Equivalency, measured as the essential agreement >89.9%, was established between the two methods for determining gepotidacin susceptibility results against Staphylococcus spp., Streptococcus spp., and Escherichia coli. However, for Neisseria gonorrhoeae and Haemophilus influenzae, equivalency was not established. Agar dilution remains the sole dilution reference method for determining gepotidacin MICs against N. gonorrhoeae.

Stress-related endogenous neuropeptides induce neuronal excitation in the Laterodorsal Tegmentum.

Stress is a physiological response that promotes maintenance of balance against harmful stimuli. Unfortunately, chronic activation of stress systems facilitates the development of psychiatric disorders. A stress-mediated hypercholinergic state could underlie this facilitation, as cholinergic mechanisms have been suggested to play a role in anxiety, depression, and substance use disorder (SUD). Stimulation by stress hormones, urocortin (Ucn1) or corticotropin-releasing factor (CRF), of the CRF receptor type 1 (CRFR1) of acetylcholine-containing neurons of the laterodorsal tegmental nucleus (LDT) could be involved in modulation of cholinergic transmission during periods of stress hormone activation, which could play a role in psychiatric disorders as cholinergic LDT neurons project to, and control activity in, mood-, arousal- and SUD-controlling regions. The present study investigated for the first time the membrane effects and intracellular outcomes of CRFR1 activation by endogenous stress hormones on LDT neurons. Patch clamp recordings of immunohistochemically-identified cholinergic and non-cholinergic LDT neurons with concurrent calcium imaging were used to monitor cellular responses to CRFR1 stimulation with Ucn1 and CRF. Postsynaptically-mediated excitatory currents were elicited in LDT cholinergic neurons, accompanied by an enhancement in synaptic events. In addition, CRFR1 activation resulted in rises in intracellular calcium levels. CRFR1 stimulation recruited MAPK/ERK and SERCA-ATPase involved pathways. The data presented here provide the first evidence that Ucn1 and CRF exert pre and postsynaptic excitatory membrane actions on LDT cholinergic neurons that could underlie the hypercholinergic state associated with stress which could play a role in the heightened risk of psychiatric disorders associated with a chronic stress state.

In Vitro Activity and Microbiological Efficacy of Gepotidacin from a Phase 2, Randomized, Multicenter, Dose-Ranging Study in Patients with Acute Bacterial Skin and Skin Structure Infections.

A phase 2 study of gepotidacin demonstrated the safety and efficacy of 3 gepotidacin doses (750 mg every 12 h [q12h], 1,000 mg q12h, and 1,000 mg every 8 h [q8h]) in hospitalized patients with suspected/confirmed Gram-positive acute bacterial skin and skin structure infections (ABSSSIs). Evaluating microbiology outcomes and responses were secondary endpoints. Pretreatment isolates recovered from infected lesions underwent susceptibility testing per Clinical and Laboratory Standards Institute guidelines. Staphylococcus aureus accounted for 78/102 (76%) of Gram-positive isolates; 54/78 (69%) were methicillin-resistant S. aureus (MRSA), and 24/78 (31%) were methicillin-susceptible S. aureus (MSSA). Posttherapy microbiological success (culture-confirmed eradication of the pretreatment pathogen or presumed eradication based on a clinical outcome of success) for S. aureus was 90% for the gepotidacin 750-mg q12h group, 89% for the 1,000-mg q12h, and 73% in the 1000-mg q8h group. For 78 S. aureus isolates obtained from pretreatment lesions, gepotidacin MIC50/MIC90 values were 0.25/0.5 µg/ml against both MRSA and MSSA. Isolates recovered from the few patients with posttreatment cultures showed no significant reduction in gepotidacin susceptibility (≥4-fold MIC increase) between pretreatment and posttreatment isolates. Two of the 78 S. aureus isolates from pretreatment lesions had elevated gepotidacin MICs and had mutations known to occur in quinolone-resistant S. aureus (GyrA S84L, ParC S80Y, and ParE D422E) or to confer elevated MICs to novel bacterial topoisomerase inhibitors (GyrA D83N, both isolates; ParC V67A, one isolate). This first report of microbiological outcomes and responses of gepotidacin in patients with ABSSSIs supports further evaluation of gepotidacin as a novel first-in-class antibacterial agent. (This study has been registered at ClinicalTrials.gov under identifier NCT02045797.).

Rapid Evolution of Reduced Susceptibility against a Balanced Dual-Targeting Antibiotic through Stepping-Stone Mutations.

Multitargeting antibiotics, i.e., single compounds capable of inhibiting two or more bacterial targets, are generally considered to be a promising therapeutic strategy against resistance evolution. The rationale for this theory is that multitargeting antibiotics demand the simultaneous acquisition of multiple mutations at their respective target genes to achieve significant resistance. The theory presumes that individual mutations provide little or no benefit to the bacterial host. Here, we propose that such individual stepping-stone mutations can be prevalent in clinical bacterial isolates, as they provide significant resistance to other antimicrobial agents. To test this possibility, we focused on gepotidacin, an antibiotic candidate that selectively inhibits both bacterial DNA gyrase and topoisomerase IV. In a susceptible organism, Klebsiella pneumoniae, a combination of two specific mutations in these target proteins provide an >2,000-fold reduction in susceptibility, while individually, none of these mutations affect resistance significantly. Alarmingly, strains with decreased susceptibility against gepotidacin are found to be as virulent as the wild-type Klebsiella pneumoniae strain in a murine model. Moreover, numerous pathogenic isolates carry mutations which could promote the evolution of clinically significant reduction of susceptibility against gepotidacin in the future. As might be expected, prolonged exposure to ciprofloxacin, a clinically widely employed gyrase inhibitor, coselected for reduced susceptibility against gepotidacin. We conclude that extensive antibiotic usage could select for mutations that serve as stepping-stones toward resistance against antimicrobial compounds still under development. Our research indicates that even balanced multitargeting antibiotics are prone to resistance evolution.

Mechanistic and Structural Basis for the Actions of the Antibacterial Gepotidacin against Staphylococcus aureus Gyrase.

Gepotidacin is a first-in-class triazaacenaphthylene novel bacterial topoisomerase inhibitor (NBTI). The compound has successfully completed phase II trials for the treatment of acute bacterial skin/skin structure infections and for the treatment of uncomplicated urogenital gonorrhea. It also displays robust in vitro activity against a range of wild-type and fluoroquinolone-resistant bacteria. Due to the clinical promise of gepotidacin, a detailed understanding of its interactions with its antibacterial targets is essential. Thus, we characterized the mechanism of action of gepotidacin against Staphylococcus aureus gyrase. Gepotidacin was a potent inhibitor of gyrase-catalyzed DNA supercoiling (IC50 ≈ 0.047 µM) and relaxation of positively supercoiled substrates (IC50 ≈ 0.6 µM). Unlike fluoroquinolones, which induce primarily double-stranded DNA breaks, gepotidacin induced high levels of gyrase-mediated single-stranded breaks. No double-stranded breaks were observed even at high gepotidacin concentration, long cleavage times, or in the presence of ATP. Moreover, gepotidacin suppressed the formation of double-stranded breaks. Gepotidacin formed gyrase-DNA cleavage complexes that were stable for >4 h. In vitro competition suggests that gyrase binding by gepotidacin and fluoroquinolones are mutually exclusive. Finally, we determined crystal structures of gepotidacin with the S. aureus gyrase core fusion truncate with nicked (2.31 Å resolution) or intact (uncleaved) DNA (2.37 Å resolution). In both cases, a single gepotidacin molecule was bound midway between the two scissile DNA bonds and in a pocket between the two GyrA subunits. A comparison of the two structures demonstrates conformational flexibility within the central linker of gepotidacin, which may contribute to the activity of the compound.

Microbiological Analysis from a Phase 2 Randomized Study in Adults Evaluating Single Oral Doses of Gepotidacin in the Treatment of Uncomplicated Urogenital Gonorrhea Caused by Neisseria gonorrhoeae.

We evaluated microbiological correlates for the successful treatment of Neisseria gonorrhoeae isolates from a phase 2 study of gepotidacin, a novel triazaacenaphthylene antibacterial, for therapy of uncomplicated urogenital gonorrhea. Culture, susceptibility testing, genotypic characterization, and frequency of resistance (FoR) were performed for selected isolates. Microbiological success was defined as culture-confirmed eradication of N. gonorrhoeae Against 69 baseline urogenital isolates, gepotidacin MICs ranged from ≤0.06 to 1 µg/ml (MIC90 = 0.5 µg/ml). For gepotidacin, the ratio of the area under the free-drug concentration-time curve to the MIC (fAUC/MIC) was associated with therapeutic success. Success was 100% (61/61) at fAUC/MICs of ≥48 and decreased to 63% (5/8) for fAUC/MICs of ≤25. All 3 isolates from microbiological failures were ciprofloxacin resistant, had a baseline gepotidacin MIC of 1 µg/ml, and carried a preexisting ParC D86N mutation, a critical residue for gepotidacin binding. In a test-of-cure analysis, the resistance to gepotidacin emerged in 2 isolates (MICs increased ≥32-fold) with additional GyrA A92T mutations, also implicated in gepotidacin binding. Test-of-cure isolates had the same sequence type as the corresponding baseline isolates. For 5 selected baseline isolates, all carrying a ParC D86N mutation, the in vitro FoR to gepotidacin was low (10-9 to 10-10); the resistant mutants had the same A92T mutation as the 2 isolates in which resistance emerged. Five participants with isolates harboring the ParC D86N mutation were treatment successes. In summary, fAUC/MICs of ≥48 predicted 100% microbiological success, including 3 isolates with the ParC D86N mutation (fAUC/MICs ≥ 97). Pharmacokinetic/pharmacodynamic determinations may help to evaluate new therapies for gonorrhea; further study of gepotidacin is warranted. (This study has been registered at ClinicalTrials.gov under identifier NCT02294682.).

In vitro activity of the novel triazaacenaphthylene gepotidacin (GSK2140944) against MDR Neisseria gonorrhoeae.

Objectives: Increased antimicrobial resistance surveillance and new effective antimicrobials are crucial to maintain treatable gonorrhoea. We examined the in vitro activity of gepotidacin, a novel triazaacenaphthylene, and the effect of efflux pump inactivation on clinical Neisseria gonorrhoeae isolates and international reference strains (n = 252) and compared gepotidacin with antimicrobials currently or previously recommended for gonorrhoea treatment. Methods: MICs (mg/L) were determined by agar dilution (gepotidacin) or by Etest (seven other antimicrobials). The gyrA and parC genes were sequenced and the impact of inactivation of the MtrCDE, MacAB and NorM efflux pumps on gepotidacin MICs was examined. Results: Gepotidacin showed potent in vitro activity against all gonococcal isolates (n = 252; MIC ≤4 mg/L). The modal MIC, MIC50, MIC90 and MIC range of gepotidacin were 0.5, 0.5, 1 and 0.032-4 mg/L, respectively. Inactivation of the MtrCDE efflux pump, but not MacAB or NorM, decreased the gepotidacin MICs for most strains. No significant cross-resistance between gepotidacin and any other antimicrobials, including the fluoroquinolone ciprofloxacin, was identified. However, the ParC D86N mutation (possibly together with additional antimicrobial resistance mutation), which is associated with fluoroquinolone resistance, was associated with increased gepotidacin MICs. Conclusions: Gepotidacin demonstrated high in vitro activity against gonococcal strains, indicating that gepotidacin could potentially be an effective option for gonorrhoea treatment, particularly in a dual antimicrobial therapy regimen and for patients with resistance or allergy to extended-spectrum cephalosporins. Nevertheless, elucidating in vitro and in vivo resistance emergence and mechanisms in detail, together with further gonorrhoea clinical studies, ideally also including chlamydia and Mycoplasma genitalium are essential.

Gepotidacin for the Treatment of Uncomplicated Urogenital Gonorrhea: A Phase 2, Randomized, Dose-Ranging, Single-Oral Dose Evaluation.

Background: In this phase 2 study, we evaluated the efficacy and safety of oral gepotidacin, a novel triazaacenaphthylene bacterial type II topoisomerase inhibitor, for the treatment of uncomplicated urogenital gonorrhea. Methods: Adult participants with suspected urogenital gonorrhea were enrolled and completed baseline (day 1) and test-of-cure (days 4-8) visits. Pretreatment and posttreatment urogenital swabs were collected for Neisseria gonorrhoeae (NG) culture and susceptibility testing. Pharyngeal and rectal swab specimens were collected if there were known exposures. Participants were stratified by gender and randomized 1:1 to receive a 1500-mg or 3000-mg single oral dose of gepotidacin. Results: The microbiologically evaluable population consisted of 69 participants, with NG isolated from 69 (100%) urogenital, 2 (3%) pharyngeal, and 3 (4%) rectal specimens. Microbiological eradication of NG was achieved by 97%, 95%, and 96% of participants (lower 1-sided exact 95% confidence interval bound, 85.1%, 84.7%, and 89.1%, respectively) for the 1500-mg, 3000-mg, and combined dose groups, respectively. Microbiological cure was achieved in 66/69 (96%) urogenital infections. All 3 failures were NG isolates that demonstrated the highest observed gepotidacin minimum inhibitory concentration of 1 µg/mL and a common gene mutation. At the pharyngeal and rectal sites, 1/2 and 3/3 NG isolates, respectively, demonstrated microbiological cure. There were no treatment-limiting adverse events for either dose. Conclusions: This study demonstrated that single, oral doses of gepotidacin were ≥95% effective for bacterial eradication of NG in adult participants with uncomplicated urogenital gonorrhea. Clinical Trials Registration: NCT02294682.