Levonadifloxacin is a novel benzoquinolizine subclass of fluoroquinolone, active against quinolone-resistant Staphylococcus aureus. A phase 3 trial for levonadifloxacin and its oral prodrug was recently completed. The present study identified area under the concentration-time curve for the free, unbound fraction of a drug divided by the MIC (fAUC/MIC) as an efficacy determinant for levonadifloxacin in a neutropenic murine lung infection model.
KEYWORDS: ABSSSIs, MRSA, levonadifloxacin
ABSTRACT
Levonadifloxacin is a novel benzoquinolizine subclass of fluoroquinolone, active against quinolone-resistant Staphylococcus aureus. A phase 3 trial for levonadifloxacin and its oral prodrug was recently completed. The present study identified area under the concentration-time curve for the free, unbound fraction of a drug divided by the MIC (fAUC/MIC) as an efficacy determinant for levonadifloxacin in a neutropenic murine lung infection model. Mean plasma fAUC/MIC requirement for static and 1 log10 kill effects against 9 S. aureus were 8.1 ± 6.0 and 25.8 ± 12.3, respectively. These targets were employed in the selection of phase 3 doses.
TEXT
The management of acute bacterial skin and skin structure infections (ABSSSIs) in large inpatient and outpatient settings relies on the availability of well-tolerated antistaphylococcal intravenous (i.v.) and oral antibiotics, respectively. Presently, oxazolidinones and fluoroquinolones offer i.v. and oral therapy options. Fluoroquinolones offer advantages of bactericidal action and broad-spectrum coverage, which also favors its use for the treatment of serious infections such as hospital-acquired bacterial pneumonia (HABP) and bloodstream infection (BSI) caused by methicillin-resistant Staphylococcus aureus (MRSA). Levonadifloxacin is a chiral benzoquinolizine-2-carboxylic acid arginine salt with potent anti-MRSA/quinolone-resistant S. aureus (QRSA) activity (1). Unlike older fluoroquinolones such as moxifloxacin and levofloxacin, levonadifloxacin exhibits preferential affinity toward gyrase A of S. aureus. Furthermore, it is highly bactericidal to S. aureus, including levofloxacin-resistant isolates carrying double mutations in quinolone resistance determining regions (2). Levonadifloxacin exhibited an MIC50 and MIC90 of 0.03 and 1 mg/liter, respectively, against 297 U.S. community-acquired and hospital strains of S. aureus irrespective of quinolone or glycopeptide resistance (3). A phase 3 clinical trial (clinical trial registry India, CTRI/2017/06/008843) was recently completed for levonadifloxacin and its oral prodrug alalevonadifloxacin (WCK 2349) for the indication of ABSSSIs. The present study was undertaken with two objectives: first, to identify the pharmacokinetic/pharmacodynamic (PK/PD) index driving antistaphylococcal efficacy of levonadifloxacin in a neutropenic murine lung infection model, and second, to determine the magnitude of the PK/PD index required for stasis and 1 log10 kill effects against S. aureus in neutropenic murine lung infection.
Nine S. aureus isolates were employed in the neutropenic murine lung infection study. These isolates included methicillin-sensitive and -resistant and levofloxacin-resistant phenotypes. MICs of levonadifloxacin, levofloxacin, and moxifloxacin against S. aureus isolates were determined in triplicates by the reference broth microdilution method (4).
Male/female Swiss albino mice weighing 25 to 28 g (Wockhardt animal breeding facility) were utilized for the PK/PD studies. The experimental protocol was approved by Wockhardt’s animal ethics committee.
For mouse plasma pharmacokinetic analyses, Swiss albino mice were administered subcutaneous single doses of 12.5, 25, 50, 100, 200, 300, and 400 mg/kg levonadifloxacin. Blood samples were collected at 10 time points/dose (0.25, 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 h postdose). At each time point, blood samples were collected from 4 mice from the retro-orbital plexus. A terminal blood collection method was employed for collecting blood samples for each time point in all PK studies. The plasma concentrations of levonadifloxacin were estimated by a validated high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method. The lower limit of quantitation (LLOQ) was 0.156 mg/liter, and the accuracy was 96.00%, with 5.33% precision (percent relative standard deviation [%RSD]). Intra-assay accuracies in plasma at three concentrations, 0.312, 5.0, and 20 mg/liter of levonadifloxacin, were 89.76%, 96.09%, and 99.22%, with precision of 4.790, 1.722, and 1.703%, respectively. The PK parameters were estimated by noncompartmental analysis using Phoenix WinNonlin 6.2.
The neutropenic murine lung infection study was carried out as described previously (5). Neutropenia (<50 cells/mm3) was induced by 250- and 100-mg/kg cyclophosphamide injections 4 days and 1 day before infection. Anesthetized animals were infected through the nostrils by delivering 80 μl of bacterial inoculum containing approximately 5 × 107 CFU/ml viable counts. The treatment of animals with subcutaneous doses of levonadifloxacin commenced 2 h after intranasal infection. The control group animals were vehicle dosed. The lung bacterial burden was assessed at initiation of treatment (0 h) and 24 h as previously described (5). The pharmacodynamic efficacy was assessed as the change in the lung bacterial burden at 24 h compared to baseline burden at 0 h. The control and treatment group each consisted of 6 animals.
To identify the PK/PD index that best correlates with pharmacodynamic efficacy of levonadifloxacin, exposure-response analyses with two S. aureus strains (AI 2994 and Sta 5027) were utilized. A range of total daily doses (1.56 to 200 mg/kg) were administered as single doses (every 24 h [q24h]) or in equally divided doses (q12h, q6h, and q3h). To quantify the PK/PD target requirements for static and 1 log10 kill effects, nine S. aureus strains (including AI 2994 and Sta 5027) were utilized. For this objective, total daily doses of levonadifloxacin (1.56 to 800 mg/kg) as single doses (q24h) or fractionated into two divided doses (q12h) were employed. The exposure-response analyses were performed by using sigmoidal maximum effect (Emax) model (GraphPad Prism version 7). The mean log10 CFU/lung was calculated from 6 animals for each group and employed for exposure-response analyses.
Levonadifloxacin MICs against nine S. aureus isolates ranged from 0.015 to 2 mg/liter. Levofloxacin and moxifloxacin MICs ranged 0.25 to >32 mg/liter and 0.03 to 16 mg/liter, respectively (Table 1).
TABLE 1.
In vitro activity of levonadifloxacin against S. aureus isolates employed in PK/PD studies
| Isolate | Resistotype | MIC (mg/liter)a
|
||
|---|---|---|---|---|
| Levonadifloxacin | LEVO | MOXI | ||
| AI 2994 | MSSA | 0.015 | 0.25 | 0.03 |
| ATCC 25923 | MSSA | 0.06 | 0.25 | 0.06 |
| Sta 042 | MSSA, NorA efflux | 0.06 | 1 | 0.25 |
| Sta 5027 | MRSA | 0.12 | 4 | 1 |
| Sta 097 | MRSA, QRSA | 1 | 8 | 4 |
| AI 2982 | MRSA, QRSA | 1 | 8 | 2 |
| AI 1153 | MRSA, QRSA | 1 | >32 | 8 |
| AI 2768 | MRSA, QRSA | 2 | >32 | 16 |
| AI 3231 | MRSA, QRSA | 2 | 32 | 8 |
MICs were determined in triplicates and modal MICs are provided. LEVO, levofloxacin; MOXI, moxifloxacin.
The select mouse plasma single-dose PK parameters of levonadifloxacin are shown in Table 2. Levonadifloxacin exposure increased in a dose-proportional manner across the range of doses studied. The area under the concentration-time curve (AUC) and maximum concentration of drug in serum (Cmax) were linear across the dose range (R2 = 0.9785 and 0.9669, respectively). Based on these PK parameters, the area under the concentration-time curve for the free, unbound fraction of a drug divided by the MIC (fAUC/MIC), unbound fraction of the maximum concentration of the drug in serum divided by the MIC (fCmax/MIC), and the cumulative percentage of a 24-h period that the concentration of the free, unbound fraction of the drug exceeds the MIC under steady-state pharmacokinetic conditions (%fT>MIC) for various dose regimens of levonadifloxacin were calculated.
TABLE 2.
Pharmacokinetic parameters of subcutaneously administered levonadifloxacin in Swiss albino mice
| Dose (mg/kg) | Cmax (mg/liter) | AUC0–24 (mg·h/liter) | t1/2 (h) |
|---|---|---|---|
| 12.5 | 4.37 ± 0.28 | 7.30 ± 0.26 | 1.79 ± 0.35 |
| 25 | 8.71 ± 1.45 | 15.75 ± 0.73 | 1.48 ± 0.11 |
| 50 | 19.21 ± 6.56 | 33.36 ± 9.63 | 1.48 ± 0.22 |
| 100 | 38.65 ± 14.81 | 70.86 ± 12.85 | 1.76 ± 0.10 |
| 200 | 77.29 ± 16.82 | 145.48 ± 9.78 | 1.69 ± 0.08 |
| 300 | 92.46 ± 19.23 | 286.19 ± 67.97 | 2.44 ± 0.81 |
| 400 | 115.16 ± 1.63 | 393.52 ± 23.06 | 1.74 ± 0.04 |
The lung bacterial burden at 0 h ranged from 6.50 log10 to 6.86 log10 CFU/lung in all PK/PD experiments. The bacterial load increased ≥1 log10 CFU/lung at 24 h in vehicle-dosed animals. The relationship between fAUC/MIC, fCmax/MIC, or %fT>MIC and pharmacodynamic responses for AI 2994 are illustrated in Fig. 1a to c, respectively. Similarly, the exposure-response relationship curves for Sta 5027 are shown in Fig. 2a to c. The estimated parameters of the sigmoidal Emax model are provided in Table 3. Among the three PD indices, fAUC/MIC exhibited the best correlation for both the isolates (R2 = 0.92 for AI 2994 and R2 = 0.93 for Sta 5027). The PK/PD targets in terms of magnitudes of fAUC/MIC linked to stasis and 1 log10 kill were determined by employing exposure-response analyses for each of nine S. aureus isolates. The fAUC/MIC requirement for bacteriostatic effect ranged from 2.2 to 22.1 and the mean requirement was 8.1 ± 6.0. The requirement for 1 log10 kill ranged 10.1 to 45.9 with a mean of 25.8 ± 12.3 (Table 4).
FIG 1.
Correlations between pharmacodynamic indices and pharmacodynamic responses for S. aureus AI 2994 (a to c).
FIG 2.
Correlations between pharmacodynamic indices and pharmacodynamic responses for S. aureus Sta 5027 (a to c).
TABLE 3.
Estimated parameters of sigmoid Emax model for S. aureus AI 2994 and Sta 5027
| Model parametera | Value for strain: |
|||||
|---|---|---|---|---|---|---|
| AI 2994 |
Sta 5027 |
|||||
| fAUC/MIC | fCmax/MIC | %fT>MIC | fAUC/MIC | fCmax/MIC | %fT>MIC | |
| E0 | 2.379 | 2.566 | 2.454 | 1.287 | 1.291 | 0.9647 |
| Emax | 4.334 | 4.48 | 8.43 | 3.955 | 3.848 | 3.3817 |
| Slope | 1.125 | 0.9738 | 0.5103 | 1.741 | 1.581 | 3.671 |
| IC50 | 13.79 | 2.49 | 95.37 | 35.15 | 7.908 | 33.15 |
| R2 | 0.9209 | 0.8858 | 0.8407 | 0.928 | 0.8031 | 0.8207 |
E0, baseline effect; Emax, maximum effect; IC50, drug concentration required to produce 50% of maximal inhibition effect; R2, regression coefficient.
TABLE 4.
Pharmacodynamic/pharmacokinetic targets of levonadifloxacin
| S. aureus isolate |
fAUC/MIC for: |
|
|---|---|---|
| Static growth | 1 log10 kill | |
| ATCC 25923 | 5.7 | 10.1 |
| AI 2994 | 12.5 | 36.9 |
| Sta 042 | 5.5 | 13.2 |
| Sta 5027 | 22.1 | 45.9 |
| Sta 097 | 2.2 | 31.6 |
| AI 2982 | 8 | 29.1 |
| AI 1153 | 4.6 | 18.3 |
| AI 2768 | 7 | 32.6 |
| AI 3231 | 5.2 | 13.8 |
| Mean ± SD | 8.1 ± 6.0 | 25.8 ± 12.3 |
In the past, several studies have established fAUC/MIC as the PK/PD driver of fluoroquinolones (6, 7). For instance, in vivo efficacy of gatifloxacin was best described by fAUC/MIC in neutropenic murine thigh and lung infection models; the mean static requirement for S. aureus was 36.4 ± 9.3 (6). Delafloxacin showed a median fAUC/MIC requirement of 1.45 for S. aureus strains with low MICs (0.004 to 0.008 mg/liter) in murine neutropenic lung infection model. In the present study, levonadifloxacin’s mean requirement for stasis, an endpoint considered relevant for ABSSSIs, was 8.1 ± 6.0. By virtue of the use of high MIC strains, the identified targets of levonadifloxacin are robust and would remain clinically relevant for serious MRSA infections such as ABSSSI and HABP. Such an fAUC/MIC ratio for S. aureus with an MIC of up to 4 mg/liter is readily attainable, as the mean unbound exposures after administration of levonadifloxacin at 800 mg, q12h, is 38.94 mg·h/liter (8). Moreover, the epithelial lining fluid (ELF) area under the concentration-time curve from 0 to 24 h (AUC0–24) exposure after oral administration of prodrug ala-levonadifloxacin at 1,000 mg q12h dose is 345.2 mg·h/liter (8). Such exposures are significantly higher than the reported ELF exposures of levofloxacin and moxifloxacin (9). To date, ELF exposure data for delafloxacin have not been reported. The levonadifloxacin targets identified in this study were considered for undertaking the probability of target attainment analyses leading to clinical dose selection.
(This study was presented in part at ECCMID, Amsterdam, Netherlands, 13 to 16 April 2019 [10].)
ACKNOWLEDGMENTS
All authors are employees of the Wockhardt Research Centre. S. S. Bhagwat and M. V. Patel are employees and shareholders of Wockhardt Ltd.
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