High In Vitro Susceptibility to the Novel Spiropyrimidinetrione ETX0914 (AZD0914) among 873 Contemporary Clinical Neisseria gonorrhoeae Isolates from 21 European Countries from 2012 to 2014

Abstract

Resistance in Neisseria gonorrhoeae against all antimicrobials available for the treatment of gonorrhea has emerged. The first gonococcal strains with high-level resistance to ceftriaxone, the last option for first-line empirical antimicrobial monotherapy, were recently described. Consequently, new treatment options are essential. In this study, the in vitro activity of the novel spiropyrimidinetrione ETX0914 (AZD0914), a DNA topoisomerase II inhibitor, was investigated among contemporary consecutive clinical N. gonorrhoeae isolates obtained in 21 European countries and compared to the activities of antimicrobials currently or previously recommended for treatment. Consecutive clinical N. gonorrhoeae isolates (n = 873) cultured in 21 European countries from 2012 to 2014 were examined for their susceptibility to ETX0914. The MICs of ETX0914 were determined using the agar dilution method. For comparison, the MICs of ceftriaxone, cefixime, azithromycin, and ciprofloxacin were determined using Etest or the agar dilution method. For ETX0914, the MIC range, modal MIC, MIC₅₀, and MIC₉₀ were ≤0.002 to 0.25 mg/liter, 0.125 mg/liter, 0.064 mg/liter, and 0.125 mg/liter, respectively. The MIC values were substantially lower than those of the fluoroquinolone ciprofloxacin and most other antimicrobials examined. No cross-resistance with any other examined antimicrobial was observed. In conclusion, the in vitro susceptibility to the novel spiropyrimidinetrione ETX0914 (AZD0914) among 873 contemporary clinical isolates from 21 European countries was high, and no cross-resistance to antimicrobials currently or previously used for gonorrhea treatment was indicated. Additional studies investigating the in vitro and in vivo induction and mechanisms of ETX0914 resistance in gonococci, pharmacokinetics/pharmacodynamics in modeling/simulations and in humans, and performance in randomized controlled gonorrhea treatment trials are essential.

INTRODUCTION

The sexually transmitted infection (STI) gonorrhea, etiological agent Neisseria gonorrhoeae, is a significant public health problem worldwide (1–4). Because no vaccine is available, appropriate prevention, sensitive and specific diagnostics, and particularly effective antimicrobial treatment are the mainstays in controlling gonorrhea. However, N. gonorrhoeae has developed resistance to all antimicrobials previously and currently used for first-line treatment. During the latest decade, in vitro resistance, including high-level resistance, to the last remaining option for first-line empirical antimicrobial monotherapy, i.e., the extended-spectrum cephalosporin ceftriaxone, has emerged (5–12). This in vitro resistance has also translated into rare clinical failures to treat pharyngeal gonorrhea with ceftriaxone in several countries (6, 7, 12–18).

In this serious situation, the World Health Organization (WHO) (2), Centers for Disease Control and Prevention (CDC) (4), and European Centre for Disease Prevention and Control (ECDC) (3) have published global and regional action plans aiming to control the spread and impact of multidrug-resistant (MDR) gonorrhea. These action/response plans strongly emphasize the necessity of novel antimicrobials or other therapeutic compounds for effective treatment of gonorrhea. The spiropyrimidinetriones comprise a newly developed class of antimicrobials that act via a new drug target and novel DNA topoisomerase II (DNA gyrase/topoisomerase IV) inhibitor mode of inhibition, which differs from the mode of action of all currently licensed antimicrobials, including other DNA topoisomerase II inhibitors such as the fluoroquinolones (19, 20). ETX0914 (also known as AZD0914) is a novel, not yet commercially available or clinically used, spiropyrimidinetrione that inhibits bacterial DNA topoisomerase II. Accordingly, ETX0914 inhibits the DNA biosynthesis and replication by an accumulation of DNA double-strand cleavages and, in contrast to fluoroquinolones, prevention of religation, resulting in a bactericidal activity (19–21). ETX0914 has been shown to have a high in vitro activity against many different bacterial species, including Gram-positive, Gram-negative, and anaerobic isolates (19). Most recently, two smaller studies also showed that susceptibility to ETX0914 among N. gonorrhoeae strains appears to be high (19, 22). The U.S. Food and Drug Administration (FDA) has now also designated ETX0914 a qualified infectious disease product (QIDP) and awarded its development program fast-track status for the treatment of uncomplicated gonorrhea (23). ETX0914 is intended for oral administration. No large N. gonorrhoeae isolate collections, including many contemporary consecutive geographically diverse clinical isolates, have been examined.

The aim of the present study was to investigate in vitro susceptibility to the novel spiropyrimidinetrione ETX0914 (also known as AZD0914), a DNA topoisomerase II inhibitor, in consecutive clinical N. gonorrhoeae isolates (n = 873) collected in 21 European countries from 2012 to 2014. The susceptibility was compared to the susceptibility to antimicrobials previously and currently used for treatment of gonorrhea (ceftriaxone, cefixime, azithromycin, and ciprofloxacin).

MATERIALS AND METHODS

N. gonorrhoeae isolates and patients.

Consecutive clinical N. gonorrhoeae isolates cultured from 717 male, 147 female, and 9 patients of unspecified gender in 21 European countries in 2012 (n = 9), 2013 (n = 846), and 2014 (n = 18) were examined. The isolates (one isolate per patient episode of gonorrhea) were obtained in the following countries: Austria, Belgium, Cyprus, Denmark, Estonia, Germany, Greece, Hungary, Iceland, Italy, Latvia, Malta, the Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, and United Kingdom. Most of the isolates were initially obtained through the ECDC-funded European Gonococcal Antimicrobial Surveillance Programme (Euro-GASP) (3). The isolates were cultured from urogenital (n = 710), pharyngeal (n = 41), and anorectal (n = 88) specimens, and for 34 isolates the site of infection was unknown. The mean ages of the females and males were 27.1 years (median age, 24 years; range, 15 to 68 years) and 33.5 years (median age, 30 years; range, 16 to 72 years), respectively. For 27 isolates, the ages of the corresponding patients were not reported.

All isolates were collected after initial culture and species identification and preserved at −70°C. Prior to antimicrobial susceptibility testing, N. gonorrhoeae isolates were cultured on GC agar medium as previously described (24). Isolates with dubious colony morphology were verified or falsified as N. gonorrhoeae using microscopy after Gram staining, oxidase test, sugar utilization test, and/or matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonics, Bremen, Germany).

All examined gonococcal isolates were cultured and stored as part of the routine diagnostics (standard care), and no patient identification information was available in the study. Accordingly, no ethical approval was required for the present study.

Antimicrobial susceptibility testing.

The MICs (mg/liter) of ETX0914 (AstraZeneca Pharmaceuticals LP) were determined by the agar dilution technique in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines (25) and as previously described (22). Susceptibility testing for ceftriaxone, cefixime, azithromycin, and ciprofloxacin was performed using the Etest method (AB bioMérieux, Solna, Sweden) on GCVIT (Difco GC medium base agar [BD Diagnostics, Sparks, MD] supplemented with 1% IsoVitalex [BD Diagnostics] and, in one country, also with 1% hemoglobin [BD Diagnostics]) agar plates in accordance with the manufacturer′s instructions or the agar dilution technique according to CLSI guidelines (25). One of the 2008 WHO N. gonorrhoeae reference strains (26), WHO F, WHO G, or WHO P, was applied on each agar dilution plate as quality control. Susceptibility (S), intermediate susceptibility (I), and resistance (R) breakpoints from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (http://www.eucast.org/clinical_breakpoints/) were applied for ceftriaxone (S ≤ 0.12 and R > 0.12 mg/liter), cefixime (S ≤ 0.12 and R > 0.12 mg/liter), azithromycin (S ≤ 0.25 and R > 0.5 mg/liter), and ciprofloxacin (S ≤ 0.03 and R > 0.06 mg/liter). The corresponding resistance breakpoints stated by the CLSI are an I/R of >0.25 mg/liter for ceftriaxone, an I/R of >0.25 mg/liter for cefixime, and an R of >1 mg/liter for ciprofloxacin; no breakpoints are stated for azithromycin. For azithromycin, the agar dilution breakpoint technique, only distinguishing resistant and nonresistant isolates, was used for some of the examined isolates. Accordingly, in the present study, the isolates were categorized as azithromycin resistant (R) or azithromycin nonresistant (S + I) only.

RESULTS

The results of ETX0914 susceptibility testing for all consecutive clinical N. gonorrhoeae isolates (n = 873) obtained from 21 European countries from 2012 to 2014 are summarized in Table 1.

TABLE 1.

Susceptibility to the novel DNA topoisomerase II inhibitor ETX0914 (AZD0914) in 873 consecutive clinical Neisseria gonorrhoeae isolates obtained in 21 European countries from 2012 to 2014

Country of origin	No. of isolates	Modal MIC (mg/liter)	MIC range (mg/liter)	MIC50 (mg/liter)	MIC90 (mg/liter)
Austria	48	0.064	0.016–0.25	0.064	0.125
Belgium	50	0.064	0.016–0.25	0.064	0.125
Cyprus	9	0.125	0.008–0.125	0.125	0.125
Denmark	49	0.064	0.004–0.125	0.064	0.125
Estonia	22	0.125	0.032–0.25	0.125	0.25
Germany	49	0.064	0.032–0.125	0.064	0.125
Greece	48	0.125	0.004–0.25	0.125	0.25
Hungary	48	0.064	0.004–0.125	0.064	0.125
Iceland	4	0.125	0.032–0.125	0.125	0.125
Italy	48	0.125	0.004–0.25	0.125	0.125
Latvia	37	0.25	0.016–0.25	0.125	0.25
Malta	19	0.125	0.004–0.125	0.125	0.125
Netherlands	46	0.064	0.004–0.25	0.064	0.25
Norway	51	0.064	≤0.002–0.125	0.064	0.125
Poland	48	0.064	0.004–0.25	0.064	0.125
Portugal	49	0.125	0.008–0.125	0.125	0.125
Slovakia	50	0.125	0.004–0.125	0.064	0.125
Slovenia	51	0.125	0.004–0.25	0.125	0.25
Spain	50	0.125	0.004–0.25	0.125	0.25
Sweden	48	0.064	0.004–0.25	0.064	0.125
United Kingdom	49	0.125	0.016–0.125	0.125	0.125
All isolates	873	0.125	≤0.002–0.25	0.064	0.125

Briefly, the MICs of ETX0914 for all isolates ranged from ≤0.002 mg/liter to 0.25 mg/liter. The highest MIC value (0.25 mg/liter) was found in 60 (6.9%) of the examined isolates, which were cultured in 11 (52%) of the countries. The MIC₅₀ of ETX0914 was 0.064 mg/liter in 10 (48%) countries and 0.125 mg/liter in the remaining 11 (52%) countries. The MIC₅₀ was identical to the modal MIC in 19 (90%) of the countries. The MIC₉₀ values ranged from 0.125 mg/liter (in 15 [71%] countries) to 0.25 mg/liter (in 6 [29%] countries) (Table 1). There were no significant differences in ETX0914 MIC values in isolates obtained from females versus those obtained from males or in those according to the different ages of patients or the anatomical site of infection.

Table 2 shows the susceptibility categories for ciprofloxacin, ceftriaxone, cefixime, and azithromycin.

TABLE 2.

Susceptibility to ciprofloxacin, ceftriaxone, cefixime, and azithromycin among the examined 873 consecutive clinical Neisseria gonorrhoeae isolates obtained in 21 European countries from 2012 to 2014

Country of origin	Ciprofloxacin S/I/R (%)a	Ceftriaxone S/R (%)	Cefixime S/R (%)	Azithromycin S + I/R (%)
Austria	22.9/4.2/72.9	100/0	100/0	97.9/2.1
Belgium	48.0/0.0/52.0	100/0	96.0/4.0	100/0
Cyprus	11.1/0/88.9	100/0	100/0	66.7/33.3
Denmark	49.0/0/51.0	100/0	81.6/18.4	91.8/8.2
Estonia	77.3/0/22.7	95.5/4.5	100/0	72.7/27.3
Germany	49.0/0/51.0	100/0	98.0/2.0	98.0/2.0
Greece	29.2/0/70.8	100/0	77.1/22.9	68.7/31.3
Hungary	25.0/0/75.0	100/0	95.8/4.2	100/0
Iceland	75.0/0/25.0	100/0	100/0	100/0
Italy	33.3/0/66.7	100/0	100/0	100/0
Latvia	75.7/0/24.3	100/0	97.3/2.7	83.8/16.2
Malta	68.4/0/31.6	100/0	100/0	100/0
Netherlands	60.9/0/39.1	100/0	100/0	97.8/2.2
Norway	21.6/0/78.4	100/0	98.0/2.0	90.2/9.8
Poland	28.3/0/71.7	100/0	100/0	83.3/16.7
Portugal	51.0/0/49.0	100/0	100/0	77.6/22.4
Slovakia	52.0/0/48.0	100/0	96.0/4.0	98.0/2.0
Slovenia	41.2/0/58.8	100/0	98.0/2.0	100/0
Spain	44.0/0/56.0	98.0/2.0	88.0/12.0	88.0/12.0
Sweden	45.8/0/54.2	100/0	100/0	89.6/10.4
United Kingdom	73.5/0/26.5	100/0	100/0	100/0
All isolates	44.8/0.3/54.9	99.8/0.2	95.9/4.1	91.6/8.4

^aS indicates susceptibility, I intermediate susceptibility, and R resistance, according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (http://www.eucast.org/clinical_breakpoints/). Ciprofloxacin, S ≤ 0.03 and R > 0.06 mg/liter; ceftriaxone, S ≤ 0.12 and R > 0.12 mg/liter; cefixime, S ≤ 0.12 and R > 0.12 mg/liter; and azithromycin, S ≤ 0.25 and R > 0.5 mg/liter. For azithromycin, the agar dilution breakpoint technique, only distinguishing resistant (MIC > 0.5 mg/liter) and nonresistant isolates (MIC ≤ 0.5 mg/liter), was used for some of the examined isolates. Accordingly, the isolates were categorized as azithromycin resistant or azithromycin nonresistant (S + I) only.

In total, the resistance levels to ciprofloxacin, azithromycin, cefixime, and ceftriaxone were 55%, 8.4%, 4.1%, and 0.2%, respectively. Fifty-five percent of the isolates were resistant to the previously recommended fluoroquinolone ciprofloxacin, and the resistance levels ranged from 23% (in Estonia) to 89% (in Cyprus). Azithromycin resistance was identified in 14 (67%) of the countries, ranging from 2.0% (in Germany and Slovakia) to 33% (in Cyprus). Cefixime resistance was found in 10 (48%) of the countries and ranged from 2.0% (in Germany, Norway, and Slovenia) to 23% (in Greece). Finally, only 2 (0.2%) ceftriaxone-resistant isolates were identified (one each in Estonia and Spain) (Table 2). The MICs of ETX0914 were similar in isolates susceptible and resistant to all these other antimicrobials tested.

The MIC distributions for ETX0914, the fluoroquinolone ciprofloxacin, cefixime, ceftriaxone, and azithromycin are shown in Fig. 1.

FIG 1.

MIC (mg/liter) distributions for the novel DNA topoisomerase II inhibitor ETX0914, the fluoroquinolone ciprofloxacin, cefixime, ceftriaxone, and azithromycin for 873 consecutive clinical Neisseria gonorrhoeae isolates obtained in 21 European countries from 2012 to 2014. Note that for azithromycin, only 565 isolates were included, i.e., all isolates with exact MICs available.

Briefly, the contemporary consecutive clinical European N. gonorrhoeae isolates (n = 873) appeared to mainly represent a wild-type MIC distribution for ETX0914, indicating a general lack of ETX0914 resistance mutations (Fig. 1). The correlation coefficient between the MICs of ETX0914 and ciprofloxacin in all individual isolates was −0.0006, that is, nearly zero, which further confirms the lack of associations between the MICs of ETX0914 and the previously used DNA gyrase/topoisomerase IV inhibitor fluoroquinolone ciprofloxacin. No obvious correlations between the MICs of ETX0914 and any of the other tested antimicrobials were identified either.

DISCUSSION

We report the first comprehensive evaluation of the in vitro activity of the novel spiropyrimidinetrione ETX0914 (also known as AZD0914), a DNA topoisomerase II inhibitor, against a large and geographically diverse collection of contemporary clinical N. gonorrhoeae isolates, that is, isolates obtained in 21 European countries from 2012 to 2014. The resistance levels for ciprofloxacin, azithromycin, cefixime, and ceftriaxone were 55%, 8.4%, 4.1%, and 0.2%, respectively. However, the susceptibility to ETX0914 was high among all examined N. gonorrhoeae isolates, that is, with a narrow MIC range and low modal MIC, MIC₅₀, and MIC₉₀ values (≤0.002 to 0.25 mg/liter, 0.125 mg/liter, 0.064 mg/liter, and 0.125 mg/liter, respectively). Furthermore, the MIC distribution for ETX0914 appeared to represent mainly a wild-type MIC distribution, which indicated a general lack of in vitro resistance and resistance mechanisms for ETX0914. In general, ETX0914 also had similar activity against N. gonorrhoeae isolates susceptible and resistant to all four additional antimicrobials examined, indicating a general lack of cross-resistance with these antimicrobials.

The results of the present study were in accordance with the results from two previous smaller studies examining the susceptibility to ETX0914 among N. gonorrhoeae isolates, and no N. gonorrhoeae isolates with higher MICs than 0.25 mg/liter have yet been identified (19, 22). The frequency of resistance mutations induced or selected in N. gonorrhoeae isolates by exposure to increasing concentrations of ETX0914 has also been shown to be low. The selected resistant gonococcal 1st step mutants had ETX0914 MICs of 0.25 to 2 mg/liter, and 2nd-step resistant mutants had MICs of 2 to 8 mg/liter. Particularly, one of two mutations (D429N or K450T) in the C terminus of GyrB caused the increased ETX0914 MICs. However, the 2nd-step mutants with an MIC of 8 mg/liter had GyrB D429N and S467N mutations (27). Nevertheless, understanding of the effects on the MIC of ETX0914 by other mutations in the gyrB gene and possible mutations in gyrA, parC, parE, and porB (encoding the PorB porin) genes as well as overexpression of efflux pumps is mainly lacking. It has been shown that overexpression of several efflux pumps, particularly MtrCDE, significantly contributes to higher MICs of many antimicrobials (28). Accordingly, it would be exceedingly valuable to perform additional genetic studies examining induced or selected resistance mutations and the frequency of these in the antimicrobial targets and porB and mutations resulting in an overexpression of the MtrCDE efflux pump, particularly, but also additional efflux pumps. Furthermore, it is important to perform time-kill curve analysis of N. gonorrhoeae wild-type strains and selected resistance mutants combined with pharmacodynamic simulations and modeling. In the developing situation of resistance to ceftriaxone, the last option for empirical first-line gonorrhea treatment, recommendations of dual antimicrobial therapy (ceftriaxone, 250 to 500 mg, and azithromycin, 1 to 2 g) have been introduced in Europe, the United States, and some additional countries (7, 29–31). However, the susceptibility to ceftriaxone in N. gonorrhoeae isolates has decreased globally, and resistance to azithromycin is relatively frequent in many countries. Accordingly, these dual antimicrobial regimens may not be long-term solutions. Dual antimicrobial regimens, including high-quality ceftriaxone and azithromycin, might not be affordable in many less-resourced settings, where it can be difficult to have access to affordable high-quality ceftriaxone alone, and which also suffer from the highest burdens of gonorrhea. Due to this reason, currently used dual antimicrobial regimens may not be used universally and, consequently, will not significantly mitigate the antimicrobial resistance emergence and spread on a global perspective (7, 31). Accordingly, an effective and, most importantly, cost-effective single-antimicrobial regimen, ideally for oral administration, would be exceedingly valuable (2–4, 6, 7, 31). ETX0914 appears to have a high in vitro activity against N. gonorrhoeae. ETX0914 administered orally also seems to have good target tissue penetrance, good bioavailability, and sufficiently high safety and tolerability (an oral dose ranging from 200 mg to 4 g was well tolerated in healthy adult subjects in fed and fasted states) as indicated from initial animal toxicology studies and a phase 1, randomized, placebo-controlled trial conducted in 48 healthy subjects (19, 32, 33). Accordingly, in the phase 1 trial, ETX0914 was well tolerated at doses predicted to be clinically effective for the treatment of gonorrhea. Furthermore, in the dose escalation study, all adverse events were mild and nonserious, with none leading to ETX0914 discontinuation. The most frequent adverse events in subjects receiving ETX0914 were transient dysgeusia of the drug suspension and headache. There were no clinically significant changes in electrocardiogram values or in clinical laboratory values or observed hematology. An open-label, randomized, multicenter phase 2 clinical trial, to assess efficacy and safety, is also currently recruiting participants, females and males aged 18 to 55 years, with uncomplicated gonorrhea. The study aims to enroll 180 participants, and treatment with 2 g of ETX0914 orally (n = 70) and 3 g of ETX0914 orally (n = 70) will be evaluated with 500 mg of ceftriaxone (n = 40) as the active comparator (www.clinicaltrials.gov). Finally, the in vitro activity of ETX0914 has also been indicated to be high against a low number of Chlamydia trachomatis (34) and Mycoplasma genitalium (35) strains, which suggests that the antimicrobial may be active against several of the main bacterial STIs. However, additional in vitro studies, including a higher number of C. trachomatis and M. genitalium strains and azithromycin- and moxifloxacin-resistant isolates, are imperative.

In conclusion, the in vitro susceptibility to the novel spiropyrimidinetrione ETX0914 (also known as AZD0914), a DNA topoisomerase II inhibitor, was high among consecutive clinical N. gonorrhoeae isolates (n = 873) from 21 European countries obtained from 2012 to 2014. ETX0914 appeared to have mainly a wild-type distribution and similar in vitro activity against gonococcal isolates susceptible and resistant to ciprofloxacin, ceftriaxone, cefixime, and azithromycin, indicating a general lack of resistance and resistance determinants to ETX0914 as well as cross-resistance to other antimicrobials currently or previously used for the treatment of gonorrhea. These in vitro results suggest that ETX0914 might be an effective antimicrobial for future treatment of gonorrhea. Accordingly, additional N. gonorrhoeae studies further evaluating in vitro induction/selection and in vivo emergence and mechanisms of ETX0914 resistance, pharmacokinetics/pharmacodynamic properties in vitro (time-kill curve analysis with subsequent pharmacodynamic modeling) and in humans, and appropriate phase 3 randomized and strictly controlled clinical trials including patients with anogenital and pharyngeal gonorrhea, would be exceedingly valuable.