Gonorrhea is a common, sexually transmitted disease caused by Neisseria gonorrhoeae. Multidrug-resistant N. gonorrhoeae is an urgent threat, and the development of a new antimicrobial agent that functions via a new mechanism is strongly desired.
KEYWORDS: DNA gyrase, DNA topoisomerase, MAOQ, Neisseria gonorrhoeae, TP0480066, antibiotic resistance, enzyme inhibitor
ABSTRACT
Gonorrhea is a common, sexually transmitted disease caused by Neisseria gonorrhoeae. Multidrug-resistant N. gonorrhoeae is an urgent threat, and the development of a new antimicrobial agent that functions via a new mechanism is strongly desired. We evaluated the in vitro and in vivo activities of a DNA gyrase/topoisomerase IV inhibitor, TP0480066, which is a novel 8-(methylamino)-2-oxo-1,2-dihydroquinoline derivative. The MICs of TP0480066 were substantially lower than those of other currently or previously used antimicrobials against gonococcal strains demonstrating resistance to fluoroquinolones, macrolides, β-lactams, and aminoglycosides (MICs, ≤0.0005 μg/ml). Additionally, no cross-resistance was observed between TP0480066 and ciprofloxacin. The frequencies of spontaneous resistance to TP0480066 for N. gonorrhoeae ATCC 49226 were below the detection limit (<2.4 × 10−10) at concentrations equivalent to 32× MIC. TP0480066 also showed potent in vitro bactericidal activity and in vivo efficacy in a mouse model of N. gonorrhoeae infection. These data suggest that TP0480066 is a candidate antimicrobial agent for gonococcal infections.
INTRODUCTION
Gonorrhea is one of the most prevalent sexually transmitted diseases in the world (1). In 2012, the World Health Organization (WHO) estimated that 78 million adults contract gonorrhea globally every year (1). In 2009, the national rate of reported cases of gonorrhea reached a historic low in the United States, but the rate has recently increased once again. In 2016, a total of 468,514 cases of gonorrhea were reported in the United States, yielding a rate of 145.8 cases of gonorrhea per 100,000 population (2).
Drug-resistant gonorrhea is a major public issue and is now recognized by the U.S. Centers for Disease Control and Prevention (CDC) as one of the top three urgent antibiotic resistance threats (3). The WHO Global Gonococcal Antimicrobial Surveillance Program (WHO GASP) monitors trends in drug-resistant gonorrhea. WHO GASP data from 2009 to 2014 showed continued widespread resistance to penicillin, tetracycline, and ciprofloxacin, increasing resistance to azithromycin, and the emergence of decreased susceptibility and resistance to extended-spectrum cephalosporins (ESCs) (4). In Japan and several other countries, the failure of gonorrhea treatment with cefixime has been reported since the early 2000s (5, 6). Currently, in most countries, the injectable ESC ceftriaxone is the only remaining empirical monotherapy for gonorrhea. However, extensively drug-resistant gonococcal strains with high-level resistance to ceftriaxone have been reported (7–9). Therefore, new antibiotics are needed to treat gonococcal infections.
To the best of our knowledge, only a few antimicrobial agents for the treatment of gonorrhea are presently being investigated in phase III clinical trials. Solithromycin (CEM-101) is a fluoroketolide with a high activity against Neisseria gonorrhoeae. However, strains with high-level azithromycin resistance appear to be resistant to solithromycin, as well (10). Zoliflodacin (ETX0914) (11) and gepotidacin (GSK2140944) (12) belong to a new class of bacterial type II topoisomerase inhibitors for the treatment of gonorrhea.
Bacterial DNA gyrase and topoisomerase IV are highly conserved enzymes that are essential for DNA replication. They each consist of two subunits (GyrA and GyrB in DNA gyrase, ParC and ParE in topoisomerase IV). GyrA/ParC serve as targets for fluoroquinolones (e.g., ciprofloxacin), whereas GyrB/ParE are targets for aminocoumarins (e.g., novobiocin). Studies of genetic mutations resulting in resistance to fluoroquinolones in gonococcal isolates have shown that mutations within the quinolone resistance-determining regions (QRDRs) of GyrA/ParC are responsible for resistance to these agents (13–15). These mechanisms are analogous to those observed in Escherichia coli and other bacteria (16). Although GyrB amino acid alterations are also responsible for resistance to fluoroquinolones in E. coli, amino acid alterations in N. gonorrhoeae GyrB do not appear to have a clinically significant impact on fluoroquinolone resistance (16, 17). The spread of fluoroquinolone-resistant bacteria among various bacterial species is now a clinically significant problem.
TP0480066, a novel 8-(methylamino)-2-oxo-1,2-dihydroquinoline (MAOQ) derivative, effectively inhibits not only DNA gyrase but also topoisomerase IV (18). We evaluated the potential of TP0480066 for use in the treatment of gonorrhea.
RESULTS
Antibacterial activity.
The MICs of TP0480066 against N. gonorrhoeae strains were ≤0.00012 to 0.0005 μg/ml. TP0480066 was more active than all the tested comparators against the tested strains (Table 1). TP0480066 demonstrated potent antibacterial activity against N. gonorrhoeae, including strains with decreased susceptibility or resistance to currently available antimicrobial agents. The MICs of TP0480066 against N. gonorrhoeae strains NCTC 13479, NCTC 13480, NCTC 13818, and NCTC 13821, which are all high-level ciprofloxacin-resistant strains (MICs, ≥16 μg/ml), were also ≤0.00012 to 0.0005 μg/ml. Reproducibility was confirmed by two independent experiments.
TABLE 1.
In vitro antibacterial activities of TP0480066 and comparators against N. gonorrhoeae
| Strain | MIC (μg/ml) |
||||||
|---|---|---|---|---|---|---|---|
| TP0480066 | Ceftriaxone | Penicillin G | Ciprofloxacin | Levofloxacin | Azithromycin | Spectinomycin | |
| ATCC 49226 | 0.0005 | 0.015 | 1 | 0.004 | 0.008 | 0.25 | 32 |
| ATCC 43069 | ≤0.00012 | ≤0.002 | ≤0.015 | 0.004 | 0.008 | 0.12 | 16 |
| ATCC BAA-1846 | 0.00025 | 0.004 | 0.12 | 0.004 | 0.008 | 0.06 | 32 |
| ATCC 700717 | 0.0005 | 0.015 | 16 | 0.03 | 0.06 | 0.25 | >128 |
| ATCC 700825 (FA 1090) | 0.0005 | ≤0.002 | 0.12 | 0.004 | 0.008 | 0.12 | 32 |
| NCTC 13477 (WHO F) | 0.0005 | ≤0.002 | 0.03 | 0.004 | 0.008 | 0.12 | 32 |
| NCTC 13478 (WHO G) | ≤0.00012 | 0.015 | 0.5 | 0.12 | 0.25 | 0.25 | 32 |
| NCTC 13479 (WHO K) | 0.0005 | 0.06 | 2 | 64 | 16 | 0.25 | 32 |
| NCTC 13480 (WHO L) | ≤0.00012 | 0.12 | 4 | 32 | 32 | 0.5 | 32 |
| NCTC 13481 (WHO M) | ≤0.00012 | 0.015 | 64 | 2 | 2 | 0.25 | 32 |
| NCTC 13482 (WHO N) | 0.00025 | 0.008 | 32 | 8 | 4 | 0.25 | 32 |
| NCTC 13483 (WHO O) | 0.00025 | 0.015 | 64 | 0.008 | 0.03 | 0.25 | >128 |
| NCTC 13818 (WHO V) | 0.0005 | 0.03 | >128 | 32 | 16 | >128 | 32 |
| NCTC 13821 (WHO Y, F89) | 0.0005 | 1 | 1 | 16 | 8 | 0.5 | 32 |
Frequency of resistance.
The frequencies of resistance to TP0480066 for N. gonorrhoeae ATCC 49226 ranged from 8.3 × 10−7 at 2× MIC to <2.4 × 10−10 at 32 and 64× MIC. The frequencies of resistance to ciprofloxacin ranged from 1.1 × 10−8 at 2× MIC to <2.4 × 10−10 at 32 and 64× MIC (Table 2). TP0480066- or ciprofloxacin-resistant isolates were obtained in frequency of resistance experiments. The first-step TP0480066-resistant isolates TP-R1, TP-R2, TP-R3, and TP-R4 were isolated on 8, 8, 4, and 2× MIC agar plates inoculated with N. gonorrhoeae ATCC 49226, respectively. The first-step ciprofloxacin-resistant isolates CIP-R1 and CIP-R2 were also isolated on 16× MIC and 8× MIC agar plates inoculated with N. gonorrhoeae ATCC 49226, respectively.
TABLE 2.
Frequencies of spontaneous resistance to TP0480066 and ciprofloxacin for N. gonorrhoeae ATCC 49226
| Fold MIC | Frequency of resistance at 48 h |
|||
|---|---|---|---|---|
| 1st step |
2nd step |
|||
| TP0480066 | Ciprofloxacin | TP0480066 | Ciprofloxacin | |
| 2 | 8.3 × 10−7 | 1.1 × 10−8 | 1.4 × 10−4 | >2.1 × 10−3 |
| 4 | 2.2 × 10−7 | 9.2 × 10−9 | 9.6 × 10−6 | <7.0 × 10−8 |
| 8 | 1.8 × 10−7 | 5.9 × 10−9 | <7.1 × 10−8 | <7.0 × 10−8 |
| 16 | 2.5 × 10−8 | 2.8 × 10−9 | <7.1 × 10−8 | <7.0 × 10−8 |
| 32 | <2.4 × 10−10 | <2.4 × 10−10 | <7.1 × 10−8 | <7.0 × 10−8 |
| 64 | <2.4 × 10−10 | <2.4 × 10−10 | <7.1 × 10−8 | <7.0 × 10−8 |
For TP-R1, the frequencies of resistance to TP0480066 ranged from 1.4 × 10−4 at 2× MIC to <7.1 × 10−8 at 8, 16, 32, and 64× MIC. For CIP-R1, the frequencies of resistance to ciprofloxacin ranged from >2.1 × 10−3 at 2× MIC to <7.0 × 10−8 at 4, 8, 16, 32, and 64× MIC. The second-step TP0480066-resistant isolates TP-R1-1 to TP-R1-3 were isolated on 4× MIC agar plates inoculated with TP-R1. The experiment was performed only one time.
Characterization of TP0480066-resistant isolates.
We evaluated the drug susceptibility of the above-mentioned isolates (Table 3). The MICs of TP0480066 against TP-R1 to TP-R4 were increased 8 to 16 times relative to those of the parent strain. The MICs of TP0480066 against TP-R1-1 to TP-R1-3 were increased 2 to 4 times relative to those of TP-R1. On the other hand, the MICs of ciprofloxacin and ceftriaxone were not changed in the TP0480066-resistant isolates relative to those of the parent strain.
TABLE 3.
Characterization and susceptibilities of isolates obtained from in vitro spontaneous resistance study of TP0480066 and ciprofloxacin
| Isolate | Description | MIC (μg/ml) (fold-increase in MIC) |
Amino acid alteration relative to parent isolate | ||
|---|---|---|---|---|---|
| TP0480066 | Ciprofloxacin | Ceftriaxone | |||
| ATCC 49226 | Parent | 0.0005 (−) | 0.004 (−) | 0.015 (−) | NAa |
| TP-R1 | 1st-step isolate | 0.008 (16) | 0.004 (−) | 0.015 (−) | ParE;Thr169Ile |
| TP-R2 | 1st-step isolate | 0.008 (16) | 0.004 (−) | 0.015 (−) | ParE;Thr169Ile |
| TP-R3 | 1st-step isolate | 0.008 (16) | 0.004 (−) | 0.015 (−) | ParE;Thr169Ile |
| TP-R4 | 1st-step isolate | 0.004 (8) | 0.004 (−) | 0.015 (−) | ParE;Thr169Ile |
| CIP-R1 | 1st-step isolate | 0.0005 (−) | 0.12 (32) | 0.015 (−) | GyrA;Asp95Asn |
| CIP-R2 | 1st-step isolate | 0.001 (2) | 0.06 (16) | 0.015 (−) | GyrA;Asp95Asn |
| TP-R1-1 | 2nd-step isolate | 0.015 (32) | 0.004 (−) | 0.015 (−) | ParE;Thr169Ile |
| TP-R1-2 | 2nd-step isolate | 0.015 (32) | 0.004 (−) | 0.015 (−) | ParE;Thr169Ile |
| TP-R1-3 | 2nd-step isolate | 0.03 (64) | 0.004 (−) | 0.015 (−) | ParE;Thr169Ile |
NA, not applicable.
The MICs of ciprofloxacin against CIP-R1 and CIP-R2 were increased 16 to 32 times relative to the parent strain. On the other hand, the MICs of TP0480066 for these ciprofloxacin-resistant isolates were almost unchanged relative to those of the parent strain.
DNA sequencing showed that all the TP0480066-resistant isolates contained a single amino acid Thr169Ile alteration in ParE. No additional mutations were identified in the second-step resistant isolates. CIP-R1 and CIP-R2 each had a single amino acid Asp95Asn alteration in a GyrA QRDR.
Time-kill curve analysis.
TP0480066 reduced the viable N. gonorrhoeae ATCC 49226 counts by more than 3-log10 CFU/ml (99.9%) after 6 h at 4× MIC and after 24 h at the MIC, respectively (Fig. 1A). Ciprofloxacin reduced the viable N. gonorrhoeae ATCC 49226 counts by more than 3-log10 CFU/ml after 2 h at 4× MIC and after 6 h at the MIC (Fig. 1B). Ceftriaxone reduced the viable N. gonorrhoeae ATCC 49226 counts by more than 3-log10 CFU/ml after 6 h at 4× MIC and 8 h at the MIC (Fig. 1C). TP0480066 also reduced the viable N. gonorrhoeae NCTC 13479 (ciprofloxacin-resistant strain) and N. gonorrhoeae NCTC 13821 (ciprofloxacin- and ceftriaxone-resistant strain) cell counts after 6 h at 4× MIC (Fig. 1D and E). Reproducibility was confirmed by two independent experiments.
FIG 1.
In vitro time-kill curves of TP0480066, ciprofloxacin, and ceftriaxone against N. gonorrhoeae. Time-kill curves of TP0480066 (A), ciprofloxacin (B), and ceftriaxone (C) against N. gonorrhoeae ATCC 49226. Time-kill curves of TP0480066 against N. gonorrhoeae NCTC 13479 (D) and N. gonorrhoeae NCTC 13821 (E).
In vivo efficacy.
The viable cell counts of ciprofloxacin-susceptible N. gonorrhoeae ATCC 49226 or ciprofloxacin-resistant NCTC 13479 in control mice and in mice after the administration of the test compounds are shown in Fig. 2. The oral administration of ciprofloxacin at the two doses of 10 and 30 mg/kg led to significant decreases in the numbers of viable cell counts of the ciprofloxacin-susceptible strain. However, the viable cell count of the ciprofloxacin-resistant strain in mice treated with ciprofloxacin (30 mg/kg) was not significantly lower than that in the control group. On the other hand, dose-dependent decreases in the numbers of not only ciprofloxacin-susceptible but also ciprofloxacin-resistant strains at 24 h after a single dose of subcutaneously administered TP0480066 were observed. At doses of 30 and 100 mg/kg of TP0480066, the mean viable cell counts were significantly decreased in both strains relative to those in the vehicle control. Reproducibility was confirmed by two independent experiments. Furthermore, the pharmacokinetic profiles of TP0480066 in BALB/c mice were investigated after a single subcutaneous administration at a dose of 10 mg/kg. The maximum plasma concentration, time to maximum plasma concentration, half-life, and area under the concentration-time curve from 0 h to infinity of TP0480066 were determined to be 3,210 ng/ml, 0.333 h, 1.75 h, and 3,370 ng · h/ml, respectively.
FIG 2.
In vivo effect of TP0480066 on vaginal infection with N. gonorrhoeae. Therapeutic effects of TP0480066 and ciprofloxacin against vaginal infection with N. gonorrhoeae ATCC 49226 (A) or N. gonorrhoeae NCTC 13479 (B) in mice. Detection limit: 0.699 log10 CFU/vagina. *, P < 0.05, **, P < 0.01.
DISCUSSION
WHO has reported that antibiotic resistance is making gonorrhea much harder to treat, and in 2016, they issued updated global treatment recommendations advising doctors to prescribe two antibiotics: ceftriaxone and azithromycin (4, 19). We have synthesized a new class of MAOQ derivative, TP0480066. TP0480066 is a novel bacterial topoisomerase inhibitor that inhibits both bacterial DNA gyrase and topoisomerase IV (18). The present study demonstrated that TP0480066 is highly effective against N. gonorrhoeae both in vitro and in vivo.
TP0480066 showed potent antibacterial activity against tested N. gonorrhoeae strains, including strains with decreased susceptibility or resistance to currently available antimicrobial agents. Moreover, the MIC of TP0480066 against the CLSI QC strain N. gonorrhoeae ATCC 49226 (MIC, 0.0005 μg/ml) was substantially lower than that of solithromycin (QC MIC range, 0.03 to 0.25 μg/ml) (20), zoliflodacin (MIC, 0.06 μg/ml) (21), and gepotidacin (QC MIC range, 0.25 to 1 μg/ml) (22). TP0480066 reduced viable cell counts of not only N. gonorrhoeae ATCC 49226, a susceptible strain, but also ciprofloxacin- and ceftriaxone-resistant N. gonorrhoeae strains by more than 3-log10 CFU/ml. These results indicate that TP0480066 has a potent bactericidal activity against N. gonorrhoeae strains, including drug-resistant strains.
The MICs of TP0480066 against ciprofloxacin-resistant isolates were similar to those against the parent strain. Furthermore, the MICs of ciprofloxacin against TP0480066-resistant isolates were the same as those against the parent strain. Thus, no cross-resistance was seen between TP0480066 and ciprofloxacin. Next, we analyzed the amino acid alterations of topoisomerases in these isolates. The gyrA, gyrB, parC, and parE gene sequences of the TP0480066-resistant isolates were obtained, assembled, and mapped against the parent strain to identify genetic changes. All the TP0480066-resistant isolates had a Thr169Ile alteration in ParE. Based on the results of an amino acid alignment analysis, Thr169 in N. gonorrhoeae ParE is equivalent to Thr165 in Escherichia coli GyrB. The cocrystal structure of E. coli GyrB and 5′-adenylyl-β,γ-imidodiphosphate (ADPNP), a nonhydrolyzable ATP analog, suggests that Thr165 is an amino acid that constitutes an ATP binding site (23). On the other hand, ciprofloxacin-resistant isolates had an Asp95Asn alteration in GyrA QRDRs (Table 3). The N. gonorrhoeae ParC Asp86Asn alteration, which is associated with fluoroquinolone resistance, appeared to be associated with increased gepotidacin MICs, as well (24). Furthermore, GyrA (Ser91Phe and Asp95Asn/Gly) and ParC (Asp86Asn) alterations were observed in isolates from three microbiological failures in the gepotidacin phase II trial (25). In contrast, zoliflodacin-resistant N. gonorrhoeae isolates had Asp429Asn, Lys450Thr, and Ser467Asn alterations in GyrB (21). These results suggest that TP0480066 interacts with DNA gyrase or topoisomerase IV via a binding mode that is distinct from that of ciprofloxacin, zoliflodacin, and gepotidacin.
The second-step isolates obtained from a spontaneous resistance study had no additional mutations in topoisomerase genes gyrA, gyrB, parC, or parE sequenced from PCR products. Although whole-genome sequencing was not performed, considering that the increase in the MIC of TP0480066 against second-step isolates was only 2- to 4-fold compared with that of TP-R1 (Table 3), another mechanism, such as an efflux pump mutation, might be related to resistance of second-step isolates (26–29). All TP0480066-resistant isolates had an alteration in parE gene but not in gyrB, indicating the possibility that TP0480066 acts as a single target inhibitor of ParE intracellularly which could be one of the causes of the relatively higher frequency of resistance of TP0480066. The unbalanced activity of TP0480066 observed in E. coli DNA gyrase and topoisomerase IV (18) could make TP0480066 a single target inhibitor since the tested concentration of TP0480066 might not be sufficient to inhibit both enzymes in bacterial cell. Although enzyme inhibitory activity of TP0480066 against topoisomerases of N. gonorrhoeae is not tested, unbalanced activity of TP0480066 might also be observed in N. gonorrhoeae.
We investigated the in vivo efficacy of TP0480066 in a mouse model of vaginal N. gonorrhoeae infection. Although N. gonorrhoeae is a strictly human pathogen, estradiol-treated female mice can be infected with N. gonorrhoeae, and murine infection mimics several aspects of human infection (30). At doses of 30 and 100 mg/kg of TP0480066, the mean viable cell counts were significantly decreased relative to those of the vehicle control in both ciprofloxacin-susceptible and ciprofloxacin-resistant N. gonorrhoeae models (Fig. 2).
Here, we showed that TP0480066, a novel MAOQ derivative, exhibits potent in vitro antibacterial and bactericidal activities against N. gonorrhoeae, including drug-resistant strains, a low frequency of resistance, and a potential new mode of target binding. TP0480066 was also effective when administered as a single dose in an N. gonorrhoeae in vivo model. These results support that TP0480066 could be a new option for the treatment of gonorrhea.
MATERIALS AND METHODS
Antibacterials.
TP0480066 was synthesized by Taisho Pharmaceutical Co., Ltd. (Saitama, Japan) as described elsewhere (18). Ciprofloxacin was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Penicillin G, ceftriaxone, azithromycin, vancomycin, levofloxacin, spectinomycin, streptomycin, and trimethoprim were purchased from Sigma-Aldrich (St. Louis, MO).
Bacterial strains.
N. gonorrhoeae strains ATCC 49226, ATCC 43069, ATCC BAA-1846, ATCC 700717, and ATCC 700825 were obtained from the American Type Culture Collection (ATCC). N. gonorrhoeae strains NCTC 13477 (WHO F), NCTC 13478 (WHO G), NCTC 13479 (WHO K), NCTC 13480 (WHO L), NCTC 13481 (WHO M), NCTC 13482 (WHO N), NCTC 13483 (WHO O), NCTC 13818 (WHO V), and NCTC 13821 (WHO Y) were obtained from the National Collection of Type Cultures (NCTC) (31, 32).
Antimicrobial susceptibility testing.
The MICs of TP0480066 and the comparator agents were determined using the Clinical and Laboratory Standards Institute (CLSI) methodology, described in CLSI documents (33, 34).
Frequency of resistance determinations.
The frequencies of spontaneous resistance to TP0480066 and ciprofloxacin for N. gonorrhoeae ATCC 49226 were measured by plating onto GC agar plates (GC agar base [Nippon Becton, Dickinson and Company, Tokyo, Japan] and 1% defined growth supplement [35, 36]) containing TP0480066 or ciprofloxacin at 2, 4, 8, 16, 32, and 64 times the MIC. Inoculated plates were incubated for 48 h at 36°C, and the numbers of colonies were counted. The frequency of spontaneous resistance to TP0480066 and ciprofloxacin was calculated as the ratio of the number of confirmed resistant colonies to the total number of inoculated viable cells. If no colonies were observed on the compound-containing agar plates, the frequency of spontaneous resistance development was expressed as less than the frequency at which one resistant colony was obtained. The frequencies of second-step spontaneous resistance to TP0480066 or ciprofloxacin for N. gonorrhoeae TP-R1 or CIP-R1 were obtained in the same way.
Characterization of TP0480066-resistant mutants.
To identify the mutated genes of TP04800066- and ciprofloxacin-resistant mutants, gyrA, gyrB, parC, and parE were amplified using PCR and sequenced. The following PCR primers were used: Ng_gyrA_Fw1 5′-ACTCCGCCACGAAATTTCCT-3′ and Ng_gyrA_Rv1 5′-GCTTCGTCGCCTTATCCTGA-3′, Ng_gyrB_Fw1 5′-CAAGGTGCGGACTGTTTTGG-3′ and Ng_gyrB_Rv1 5′-CAAGCGAGCCGTAAATGTCG-3′, Ng_parC_Fw1 5′-CGTTCCATTCGCTTTGTCCG-3′ and Ng_parC_Rv1 5′-AAAGCCTGCCATTGCCATTG, and Ng_parE_Fw1 5′-GCGACGTTTATTTGGAGGCG-3′ and Ng_parE_Rv1 5′-GCGCAAAACCATCCTTTCGT-3′. The primers were purchased from Eurofins Genomics KK (Tokyo, Japan).
A single colony of each isolate was selected from a plate, transferred to Tris-EDTA (TE) buffer (pH 8.0) in a microtube, and treated for 10 min at 95°C. An aliquot was used as the source of template DNA in a PCR using the Tks Gflex DNA polymerase kit (TaKaRa Bio Inc., Shiga, Japan) and a TaKaRa PCR Thermal Cycler Dice. The PCR product was purified using a QIAquick PCR purification kit (Qiagen KK, Tokyo, Japan) and sequenced using an Applied Biosystems 3730 series DNA analyzer (Thermo Fisher Scientific KK, Kanagawa, Japan). The obtained sequences of gyrA, gyrB, parC, and parE were compared with those of N. gonorrhoeae ATCC 49226.
Time-kill curve analysis.
Time-kill studies were performed for three representative N. gonorrhoeae strains (ATCC 49226, NCTC 13479, and NCTC 13821) by referring to previously published methods (37). Briefly, freshly prepared colonies were suspended in gonococcal broth (35, 36) containing 1% supplement C (Nippon Becton, Dickinson Co., Ltd., Tokyo, Japan) and cultured at concentrations of one-fourth the MIC (1/4× MIC), 1× MIC, and 4× MIC of TP0480066, ciprofloxacin, and ceftriaxone, respectively. The CFU count was determined by plating aliquots from serial 10-fold dilutions; the limit of detection was set at ≤103 CFU/ml. Bactericidal activity was defined as a ≥3-log10 reduction in bacterial counts.
Mouse model of N. gonorrhoeae.
A mouse genital tract infection model (38–40) was used to evaluate the in vivo effect of TP0480066 (n = 5 to 8). Female BALB/c mice were purchased from Japan SLC, Inc. (Shizuoka, Japan) at 6 weeks of age and allowed to acclimate to the animal facility for 3 days. Mice were then treated with water-soluble 17β-estradiol (Sigma-Aldrich Japan, Tokyo, Japan) administered subcutaneously 2 days prior to inoculation and on the day of inoculation. Antibiotics (0.24 mg streptomycin and 0.4 mg vancomycin administered twice daily via intraperitoneal injection, and 0.04 g trimethoprim per 100 ml drinking water) were administered from 2 days prior to inoculation through the day of inoculation to control commensal bacteria. On day 0, the mice were vaginally inoculated with N. gonorrhoeae ATCC 49226 (2.6 × 107 CFU) or N. gonorrhoeae NCTC 13479 (1.5 × 107 CFU). TP0480066 was then administered 2 h after inoculation at a dose of 1, 3, 10, 30, or 100 mg/kg (subcutaneously). Ciprofloxacin was dosed at 3, 10, or 30 mg/kg (orally) as a positive control. At 24 h after inoculation, a vaginal mucus swab was collected and suspensions were plated onto Modified Thayer-Martin (MTMII) Agar (Nippon Becton, Dickinson and Company, Tokyo, Japan) to determine the N. gonorrhoeae count.
All the in vivo experimental protocols used in this study were approved by the guidelines of the Taisho Pharmaceutical animal care committee and met the Japanese Experimental Animal Research Association standards, as defined in the Guidelines for Animal Experiments (1987).
Statistical analyses.
Statistical significance was tested using the Wilcoxon’s test for two sample comparisons and the Steel’s test for multiple sample comparisons. All the data were analyzed using SAS9.2 (SAS Statistical Institute, Cary, NC, USA).
Pharmacokinetic study.
Seven-week-old female BALB/c mice (Japan SLC) were used for the pharmacokinetic study. TP0480066 was subcutaneously administered to mice (n = 3) at a dose of 10 mg/kg. Blood samples were taken from the tail vein at 0.0833, 0.25, 0.5, 1, 2, 4, and 8 h postdose and from the posterior vena cava under isoflurane anesthesia at 24 h postdose. The blood sample was placed into a tube containing ethylenediamine-N,N,N′,N′-tetraacetic acid dipotassium salt dihydrate as an anticoagulant and centrifuged to prepare plasma sample. The quantitative analysis was performed using a liquid chromatography-tandem mass spectrometry method. The lower limit of quantification for TP0480066 was 10.0 ng/ml in plasma. The pharmacokinetic parameters of TP0480066 in plasma were calculated by a noncompartmental analysis method with pharmacokinetic analysis software Phoenix WinNonlin 6.2 (Certara, Princeton, NJ).
ACKNOWLEDGMENTS
We thank Masashi Mima at Taisho Pharmaceutical Co., Ltd., for valuable discussions. All the authors are employees of Taisho Pharmaceutical Co., Ltd. The authors have nothing else to declare.
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