Pharmacokinetic-Pharmacodynamic Evaluation of Gepotidacin ... · either from mutations that alter...

Pharmacokinetic-PharmacodynamicEvaluation of Gepotidacin againstGram-Positive Organisms Using Datafrom Murine Infection Models

Catharine C. Bulik,a Ólanrewaju O. Okusanya,a* Elizabeth A. Lakota,a Alan Forrest,a

Sujata M. Bhavnani,a Jennifer L. Hoover,b David R. Andes,c Paul G. Ambrosea

Institute for Clinical Pharmacodynamics, Schenectady, New York, USAa; GlaxoSmithKline, Collegeville,Pennsylvania, USAb; University of Wisconsin, Madison, Wisconsin, USAc

ABSTRACT Gepotidacin (formerly called GSK2140944) is a novel triazaacenaphthyl-ene bacterial topoisomerase inhibitor with in vitro activity against conventional andbiothreat pathogens, including Staphylococcus aureus and Streptococcus pneumoniae.Using neutropenic murine thigh and lung infection models, the pharmacokinetics-pharmacodynamics (PK-PD) of gepotidacin against S. aureus and S. pneumoniae werecharacterized. Candidate models were fit to single-dose PK data from uninfectedmice (for doses of 16 to 128 mg/kg of body weight given subcutaneously [s.c.]).Dose fractionation studies (1 isolate/organism; 2 to 512 mg/kg/day) and dose-ranging studies (5 isolates/organism; 2 to 2,048 mg/kg/day; MIC ranges of 0.5 to 2mg/liter for S. aureus and 0.125 to 1 mg/liter for S. pneumoniae) were conducted.The presence of an in vivo postantibiotic effect (PAE) was also evaluated. Relation-ships between the change from baseline in log10 CFU at 24 h and the ratio of thefree-drug plasma area under the concentration-time curve (AUC) to the MIC (AUC/MIC ratio), the ratio of the maximum concentration of drug in plasma (Cmax) to theMIC (Cmax/MIC ratio), and the percentage of a 24-h period that the drug concentra-tion exceeded the MIC (%T�MIC) were evaluated using Hill-type models. Plasma andepithelial lining fluid (ELF) PK data were best fit by a four-compartment model withlinear distributional clearances, a capacity-limited clearance, and a first-order absorp-tion rate. The ELF penetration ratio in uninfected mice was 0.65. Since the growth ofboth organisms was poor in the murine lung infection model, lung efficacy datawere not reported. As determined using the murine thigh infection model, the free-drug plasma AUC/MIC ratio was the PK-PD index most closely associated with effi-cacy (r2 � 0.936 and 0.897 for S. aureus and S. pneumoniae, respectively). Medianfree-drug plasma AUC/MIC ratios of 13.4 and 58.9 for S. aureus, and 7.86 and 16.9for S. pneumoniae, were associated with net bacterial stasis and a 1-log10 CFU reduc-tion from baseline, respectively. Dose-independent PAE durations of 3.07 to 12.5 hand 5.25 to 8.46 h were demonstrated for S. aureus and S. pneumoniae, respectively.

KEYWORDS Staphylococcus aureus, Streptococcus pneumoniae, gepotidacin

Infections due to Gram-positive bacteria, such as Staphylococcus aureus and Strepto-coccus pneumoniae, are associated with high morbidity and mortality (1, 2). S. aureus,

including methicillin-resistant isolates, is one of the most common bacterial pathogenscausing both community- and hospital-acquired infections and is often responsible foracute bacterial skin and skin structure infections (ABSSSI) and community-acquiredbacterial pneumonia (CABP). Streptococcus spp. represent an additional group ofGram-positive pathogens associated with ABSSSI and CABP, with S. pneumoniae oftenidentified as one of the leading causes of CABP (3). Due to their demonstrated clinical

Received 14 January 2016 Returned formodification 15 February 2016 Accepted 7November 2016

Accepted manuscript posted online 21November 2016

Citation Bulik CC, Okusanya OO, Lakota EA,Forrest A, Bhavnani SM, Hoover JL, Andes DR,Ambrose PG. 2017. Pharmacokinetic-pharmacodynamic evaluation of gepotidacinagainst Gram-positive organisms using datafrom murine infection models. AntimicrobAgents Chemother 61:e00115-16. https://doi.org/10.1128/AAC.00115-16.

Copyright © 2017 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Paul G. Ambrose,[email protected].

* Present address: Ólanrewaju O. Okusanya,Food and Drug Administration, Silver Spring,Maryland, USA.

EXPERIMENTAL THERAPEUTICS

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efficacy against numerous infections, quinolones have historically been one of the mostwidely used classes of antimicrobials to treat numerous infection types, includingABSSSI and CABP (4). However, the incidence of resistance to quinolones, which resultseither from mutations that alter the drug target enzymes, most often DNA gyrase andtopoisomerase IV, or via efflux pumps or decreased uptake, has steadily increased overtime (4).

Quinolone resistance in S. aureus and S. pneumoniae is thought to first occur in theParC subunit of topoisomerase IV and results in a reduced affinity of topoisomerase IVfor the quinolone (5). The prevalence of quinolone resistance in S. aureus isolates, inparticular methicillin-resistant S. aureus (MRSA) isolates, has been reported to begreater than 70% (6). In contrast to that of S. aureus, the S. pneumoniae resistance ratefor quinolones is generally considered low in North America (7). However, due to theemergence of S. pneumoniae isolates resistant to beta-lactam and macrolide antibiotics,the quinolones have been relied upon more extensively for the treatment of CABP, andas such, the potential for the emergence of quinolone resistance is greater (7). Tocombat the increase in resistance rates, additional antibiotics that are efficacious in thetreatment of patients with CABP and ABSSSI and do not demonstrate cross-resistanceto existing antibiotics are needed.

Gepotidacin (formerly called GSK2140944), a novel triazaacenaphthylene bacterialtopoisomerase inhibitor, selectively inhibits bacterial DNA replication through a novelbinding mode with the GyrA subunit of bacterial DNA gyrase and the ParC subunit ofbacterial topoisomerase IV that is distinct from the binding mode of other antibacterials(8). As a result of this novel mode of action, gepotidacin displays in vitro activity againsttarget pathogens carrying resistance determinants for established antibacterials, in-cluding the fluoroquinolones. The spectrum of in vitro activity of gepotidacin includesGram-positive pathogens and certain Gram-negative pathogens, including those thatgive rise to CABP and ABSSSI (9). Gepotidacin is currently being developed by Glaxo-SmithKline Pharmaceuticals for potential use as oral (p.o.) and intravenous (i.v.) anti-bacterial therapies (10–13).

Preclinical in vivo efficacy data for the use of gepotidacin against S. pneumoniaehave been described previously (14). However, these experiments were not designed toidentify the pharmacokinetic-pharmacodynamic (PK-PD) index associated with theefficacy of gepotidacin or to determine the magnitudes of the index associated withvarious levels of reduction in bacterial burden. Neutropenic murine thigh and lunginfection models are frequently utilized in drug development to characterize PK-PDrelationships for efficacy; PK-PD targets derived from such models can then be used tosupport dosing regimen selection. Use of such models would allow for the determi-nation of both the PK-PD index and magnitude associated with the in vivo efficacy ofgepotidacin against S. aureus and S. pneumoniae.

Using murine thigh and/or lung infection models in which gepotidacin was evalu-ated against various S. aureus and S. pneumoniae isolates, the objectives of theseanalyses were as follows: (i) to develop a pharmacokinetic (PK) model to describe thedisposition of gepotidacin in both the plasma and epithelial lining fluid (ELF) ofneutropenic uninfected mice, (ii) to identify the free-drug plasma PK-PD index mostclosely associated with the efficacy of gepotidacin, (iii) to determine the magnitudes ofthe free-drug plasma PK-PD index associated with various levels of bacterial reductionfrom baseline, and (iv) to evaluate the presence of an in vivo postantibiotic effect (PAE).

RESULTSBacterial isolates and in vitro susceptibility. The gepotidacin MIC ranges for the

isolates evaluated were 0.5 to 2 mg/liter for S. aureus and 0.125 to 1 mg/liter for S.pneumoniae. The gepotidacin MIC values for all isolates are shown in Table 1.

Pharmacokinetic analysis. The plasma and ELF PK of gepotidacin, based on thefirst study with uninfected mice, were best described by a four-compartment modelwith three compartments for plasma, one compartment for ELF, linear distributionalclearances, a parallel first-order and capacity-limited clearance, and a first-order ab-

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sorption rate. The PK data were well fit by the PK model, as evidenced by an evenscatter of the data around the line of identity and r2 values of 0.852 and 0.850 for theplasma and ELF data, respectively (see Fig. S1 and S2 in the supplemental material).Total-drug plasma and ELF concentration-time profiles for each of the evaluatedgepotidacin doses, based on the observed data, and the fitted profile based on the finalPK model are provided in Fig. S3 in the supplemental material. Table 2 shows the PKmodel parameter estimates and standard errors. As evidenced by the rate constants forthe distribution of gepotidacin into (kce) and its removal from (kec) the ELF, gepotidacinrapidly penetrates and departs the ELF compartment of mice and does not accumulate.The ELF penetration ratio for gepotidacin in noninfected neutropenic mice was 0.65across all the doses assessed. Since the kce and kec rate constants were determinedwithout variability across doses, the above-described ELF penetration ratio estimatewas independent of dose.

Evaluation of the impact of infection on the pharmacokinetic profile of gepoti-dacin. As shown in Fig. S4 in the supplemental material and evidenced by an even

TABLE 1 Gepotidacin free-drug plasma AUC/MIC ratio targets associated with various levels of bacterial reduction from baseline for S.aureus and S. pneumoniae, derived using Hill-type models based on data from a neutropenic murine thigh infection modela

Species and isolate Phenotype MIC (mg/liter)

Post hoc free-drug plasma AUC/MIC ratio estimate (%SEE)

Net bacterial stasis 1-log10 CFU reduction 2-log10 CFU reductionb

S. aureusATCC 29213 MSSA 0.5 35.5 (18.4) 103.2 (16.3) 1102 (115.3)WCUH29 MRSA 0.5 7.77 (22.2) 56.5 (40.4) —ATCC 33591 MRSA 1 4.38 (14.3) 13.9 (12.5) 117.2 (50.8)B-3 MRSA 1 21.2 (18.3) 61.2 (16.1) 397.1 (53.7)ATCC 25923 MSSA 1 3.97 (21.9) 12.6 (19.9) 103.8 (57.5)M709 MRSA 2 19.1 (19.0) 64.7 (16.5) —Median 13.4 58.9 257.2

S. pneumoniaeMNO-418 FQR 0.125 8.30 (22.1) 18 (22.3) 54.3 (33.6)ATCC 10813 PSSP 0.25 35.3 (26.5) 78.8 (24.8) 286.1 (53.5)503167 PSSP, FQR 0.5 8.38 (23.2) 16.6 (22.5) 38.1 (23.9)10127 PSSP 0.5 7.42 (24) 17.2 (24.4) 85.2 (83.5)1396 PISP, macrolide resistant (mef) 0.5 1.13 (9.8) 2.57 (10.4) 16 (46)TPS3 FQR, macrolide resistant (mef) 1 4.15 (21.8) 9.76 (22.24) 91 (153.7)Median 7.86 16.9 69.8

aMRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. aureus; PSSP, penicillin-sensitive S. pneumoniae; PISP, penicillin-intermediate S. pneumoniae; FQR,fluoroquinolone resistant; SEE, standard error of the estimate.

b—, this level of bacterial reduction was not achieved for the isolate.

TABLE 2 Gepotidacin PK model parameter estimates and %SEE

Parameter Symbol (units)

Plasma and ELFdata

Estimate %SEE

First-order absorption rate Ka (h�1) 1.44 5.74Maximum rate of clearance Vmax (mg/h/kg) 3.63 13.8Apparent vol of the central compartment Vc/F (liters/kg) 0.244 32.0Apparent distributional clearance 1 CLd1/F (liters/h/kg) 1.68 30.8Apparent vol of the peripheral compartment 1 Vp1/F (liters/kg) 14.6 74.2Apparent distributional clearance 2 CLd2/F (liters/h/kg) 0.973 567Apparent vol of the peripheral compartment 2 Vp2/F (liters/kg) 2.88E�03 779Concn at which there is half-maximal clearance Km (mg/liter) 4.45E�02 59.1First-order clearance CL (liters/h/kg) 3.12 18.1Rate constant for distribution into the ELF kce (h�1) 7.91 94.8Rate constant for removal from the ELF kec (h�1) 16.0 85.3Plasma error variance model intercept SdPint 2.50E�02 FixedPlasma error variance model slope SdPslp 0.4277 FixedELF error variance model intercept SdEint 1.75E�02 468.7ELF error variance model slope SdEslp 0.121 17.56

Gepotidacin PK-PD against Gram-Positive Organisms Antimicrobial Agents and Chemotherapy





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scatter of data about the line of identity and r2 values of 0.892 and 0.940 for theuninfected and infected mice, respectively, the PK data for both uninfected andinfected mice were well fit by the respective PK models. While the observed total-drugplasma concentrations following administration of gepotidacin at 32 mg/kg weregenerally lower for uninfected mice than for infected mice, the total-drug plasmaconcentrations for these two groups overlapped (Fig. S5). The resulting geometricmean of the ratio of the total-drug plasma area under the concentration-time curvefrom 0 to 24 h (AUC0 –24) for uninfected mice to the total-drug plasma AUC0 –24 forinfected mice was 0.86 (90% confidence interval [CI], 0.72 to 1.02). Thus, gepotidacinexposures in infected and uninfected animals after administration of the same dosewere not statistically different.

Pharmacokinetic-pharmacodynamic analyses. Evaluation of the dose fraction-ation data demonstrated relationships between the change from baseline in log10 CFUat 24 h and each of the gepotidacin PK-PD indices. Examination of the log10 CFU dataat 24 h for the dosing regimen of one dose administered every 24 h (q24h) revealedsystematically higher bacterial counts than those for dosing regimens based on theother intervals studied. Due to the short half-life of gepotidacin in mice, the q24hdosing interval resulted in inadequate drug concentrations and therefore regrowthwithin the murine infection model. As such, these data were removed from theanalyses. As shown in Fig. 1A and B, which illustrate the relationships between the ratioof free-drug plasma area under the concentration-time curve from 0 to 24 h to the MIC(AUC/MIC ratio), the ratio of the maximum concentration of drug in plasma (Cmax) tothe MIC (Cmax/MIC ratio), and the percentage of a 24-h period that the drug concen-tration exceeded the MIC (%T�MIC) and the change from baseline in log10 CFU per

FIG 1 Relationships between change from baseline in log10 CFU and gepotidacin free-drug plasmaAUC/MIC ratio, Cmax/MIC ratio, and %T�MIC for S. aureus 33591 (A) and S. pneumoniae 10183 (B),excluding the q24h dosing regimens. Symbols represent changes from baseline in log10 CFU per thighfor individual mice at 24 h. The horizontal lines represent no net change from baseline.






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thigh for S. aureus and S. pneumoniae, respectively, data from the dose fractionationstudies were well described using Hill-type models. As evidenced by r2 values forfree-drug plasma AUC/MIC ratio and %T�MIC of 0.936 and 0.936, respectively, for S.aureus and 0.897 and 0.9, respectively, for S. pneumoniae, the data were well describedby both indices. Given that gepotidacin is a DNA replication inhibitor (as are otheragents, including fluoroquinolones) and that free-drug plasma %T�MIC is more diffi-cult than AUC to estimate with precision, free-drug plasma AUC/MIC ratio was consid-ered to be the more informative PK-PD index.

Results of dose-ranging studies conducted in the neutropenic murine thigh andlung infection models demonstrated activity of gepotidacin against all of the S. aureusand S. pneumoniae isolates evaluated. However, as evidenced by mean (standarddeviation [SD]) increases of 0.876 (0.472) and 0.0667 (0.468) log10 CFU relative tobaseline for the S. aureus and S. pneumoniae growth controls, respectively, in themurine lung infection model, compared to 1.64 (0.410) and 2.26 (1.07) log10 CFU,respectively, in the murine thigh infection model, the growth of S. aureus and S.pneumoniae isolates in the neutropenic murine lung infection model was lower thanthat in the murine thigh infection model. Given the �1-log10 CFU increase for either S.aureus or S. pneumoniae in the murine lung infection model, the results of the PK-PDanalyses for the murine lung infection model are not reported.

Figure 2A and B show the relationships between the free-drug plasma AUC/MICratio and the change from baseline in log10 CFU for the S. aureus and S. pneumoniaeisolates, respectively, based on data from the dose fractionation and dose-rangingstudies. The relationships between the free-drug plasma AUC/MIC ratio and the changefrom baseline in log10 CFU for S. aureus and S. pneumoniae were well described by theHill-type models, as evidenced by an r2 value of 0.925 for both pathogens. The

FIG 2 Relationships between change from baseline in log10 CFU and gepotidacin free-drug plasmaAUC/MIC ratio for six S. aureus isolates (A) and six S. pneumoniae isolates (B), with fitted functions basedon the Hill-type models overlaid on the observed data. The r2 value for both these relationships was0.925. The horizontal lines represent no net change from baseline. The observed data for each isolate arerepresented by a different symbol, with the fitted function overlaid.






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parameter estimates and associated standard errors for these models are provided inTable 3. Table 1 summarizes the magnitudes of free-drug plasma AUC/MIC ratio targetsassociated with net bacterial stasis and 1- and 2-log10 CFU reductions from baseline forS. aureus and S. pneumoniae, based on the Hill-type models presented in Table 3. Themedian free-drug plasma AUC/MIC ratio targets associated with net bacterial stasis forS. aureus and S. pneumoniae were 13.4 and 7.86, respectively. The median free-drugplasma AUC/MIC ratio targets associated with 1- and 2-log10 CFU reductions frombaseline for S. aureus were 58.9 and 257.2, respectively, while those for S. pneumoniaewere 16.9 and 69.8, respectively.

Evaluation of the presence of PAE. Figure 3A and B show the relationship betweenlog10 CFU and time overlaid with the free-drug plasma concentration-versus-timeprofiles for the gepotidacin doses evaluated and the associated PAE for S. aureus andS. pneumoniae, respectively. A dose-independent PAE was observed with gepotidacinagainst S. aureus and S. pneumoniae. The durations of PAE for S. aureus at gepotidacindoses of 16, 32, 64, and 128 mg/kg were 3.07, 10.4, 4.44, and 12.5 h, respectively. Thedurations of PAE for S. pneumoniae at the same doses were 6.68, 7.43, 5.25, and 8.46 h,respectively.

DISCUSSION

The results of the above-described studies undertaken using murine thigh and lunginfection models allowed for the following outcomes: (i) characterization of the dispo-sition of gepotidacin in the plasma and ELF of neutropenic mice and, using the PKmodel developed based on these data, reliable estimation of PK-PD indices across thedose range studied; (ii) identification of free-drug plasma AUC/MIC ratio and %T�MICas the PK-PD indices most closely associated with efficacy; (iii) calculation of themagnitudes of free-drug plasma AUC/MIC ratios associated with various levels ofreduction in bacterial burden for S. aureus and S. pneumoniae; and (iv) identification ofthe presence of a dose-independent in vivo postantibiotic effect.

The plasma and ELF PK of gepotidacin in uninfected mice were best described by afour-compartment model with three compartments for plasma, one compartment forELF, linear distributional clearances, a parallel first-order and capacity-limited clearance,and a first-order absorption rate. Results of studies conducted in infected and unin-fected mice to evaluate the impact of infection on the PK profile of gepotidacindemonstrated that the presence of infection did not have a statistically significant

TABLE 3 Parameter estimates and associated %SEE for Hill-type models for S. aureus andS. pneumoniae based on data from a neutropenic murine thigh infection modela

Species and parameterb

MeanInterisolate variability(%CV)

Estimate %SEE Estimate %SEE

S. aureusEcon 1.56 13.0 14.6 120Emax 3.54 12.7 7.58 81.6Hill coefficient 1.02 2.53 1.08 85.6EC50 13.9 40.8 95.3 92.4SDint 0.403 5.56

S. pneumoniaeEcon 2.02 16.6 39.7 61.6Emax 4.49 9.70 21.5 65.1Hill coefficient 1.20 9.78 14.6 62.8EC50

c 7.41 56.4 134 60.1SDint 0.566 5.56

aBased on data sets containing 6 S. aureus and 6 S. pneumoniae isolates.bEcon, change from baseline in log10 CFU at 24 h; Emax, maximum change in log10 CFU from Econ; EC50, free-drug plasma AUC/MIC ratio associated with half-maximal effect; SDint, intercept term for the error variancemodel; CV, coefficient of variance.

cThe EC50 was modeled as normally distributed, and as such, the arithmetic mean and standard deviationare reported.






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impact on total-drug plasma concentrations. These data provide support for use ofexposures for the PK-PD analyses described herein that were determined using the PKmodel developed using data from uninfected mice.

Gepotidacin exposure in uninfected mice, as measured by the AUC0 –24 value, waslower for total-drug ELF than for free drug in plasma, as evidenced by a penetrationratio of 0.65. Comparison of this ELF penetration ratio for mice to that determined usingELF and plasma AUC values over 0 to 12 h based on PK data from healthy volunteersdemonstrated higher ELF penetration, with a ratio of 1.88 (13). This difference in ELFpenetration between mice and humans is an important factor to consider whenpredicting effective human dosing regimens for pneumonia using data from murinestudies.

For both S. aureus and S. pneumoniae, free-drug plasma AUC/MIC ratio and %T�MICwere the PK-PD indices which best described gepotidacin efficacy in dose fractionationstudies. While the mechanism of action of gepotidacin, a DNA replication inhibitor, is

FIG 3 Relationships between log10 CFU and time overlaid with the free-drug plasma concentration-versus-time profiles for gepotidacin doses evaluated and the associated PAE for S. aureus (A) and S.pneumoniae (B) using the murine thigh infection model.






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similar to that of the fluoroquinolones, for which efficacy is best predicted by AUC/MICratio, gepotidacin has a binding mode that is different from that of the fluoroquino-lones (8, 15). Given the similar mechanism of action and that free-drug plasma %T�MICis more difficult to estimate with precision than the AUC, preference was given tofree-drug plasma AUC/MIC ratio over free-drug plasma %T�MIC. When evaluating theHill-type models for free-drug plasma AUC/MIC ratios derived based on the dosefractionation and dose-ranging data, a difference in the efficacy of gepotidacin againstS. aureus and S. pneumoniae was revealed. As evidenced by the smaller maximum effect(Emax) value for S. aureus when compared to that for S. pneumoniae (3.54 versus 4.49,respectively), gepotidacin had greater in vivo efficacy against S. pneumoniae.

Attaining the PK-PD target associated with net bacterial stasis for S. aureus, basedupon data from a neutropenic murine thigh infection model, has been shown to bepredictive of high probabilities of a successful response in patients with ABSSSI treatedwith linezolid or tigecycline (16). The results of these analyses for S. aureus and S.pneumoniae (which can be used as a surrogate for Streptococcus pyogenes [William A.Craig, unpublished data]) demonstrated that median free-drug plasma AUC/MIC ratiosof 13.4 and 7.86, respectively, were associated with net bacterial stasis. When thesefree-drug plasma AUC/MIC ratio targets for the same endpoint were compared to thosefor other fluoroquinolones (free-drug plasma AUC/MIC ratios of 36 to 81 and 25 to 34for net bacterial stasis for S. aureus and S. pneumoniae, respectively), it is interesting tonote that the targets for gepotidacin were lower (16–19). Based upon the above-described free-drug plasma AUC/MIC ratio of 13.4 for S. aureus, a ratio of 14 wasselected as the PK-PD target for the evaluation of gepotidacin dosing regimens forpatients with ABSSSI. Using this free-drug plasma AUC/MIC ratio target in combinationwith phase 1 PK data and Monte Carlo simulation, PK-PD target attainment analyseswere conducted (GlaxoSmithKline Pharmaceuticals, unpublished data). The results ofthese analyses were used to select the gepotidacin dosing regimens for evaluation ina phase 2 ABSSSI study (20).

The translational value of the PK-PD target associated with a 1-log10 CFU reductionfrom baseline in a murine infection model was demonstrated in a study by Bulik et al.which evaluated the relationship between the probability of attaining the PK-PD targetassociated with this endpoint and the probability of obtaining regulatory approval forantibacterial agents for the treatment of pneumonia (21). The results of this studyshowed that the probability of regulatory drug approval increased with increasingPK-PD target attainment for the target associated with a 1-log10 CFU reduction frombaseline in the murine infection model. The murine lung infection studies describedherein were limited by the poor growth of S. pneumoniae observed at 24 h for theno-treatment control groups relative to those in the thigh infection model. The growthof the no-treatment control group at 24 h can be used as an overall indicator of thefitness of the pathogen in an infection model. If robust growth of the pathogen isobserved in the no-treatment control group at 24 h, then one can assume robustgrowth in all groups of the study over the same 24 h. Accordingly, poor growth can beassumed in all groups if poor growth is observed in the no-treatment control group at24 h. Drug efficacy in the murine infection model is measured as the change in log10

CFU from baseline at 24 h and does not consider the 24-h data from the no-treatmentcontrol group. As such, there is the potential to attribute greater efficacy to the drugthan would be warranted given the indicator of poor bacterial growth in the no-treatment control group at 24 h. A further impact of the overestimation of the drugeffect is the underestimation of the magnitudes of PK-PD targets associated withefficacy. For the above-described reasons, total-drug ELF and free-drug plasma AUC/MIC ratio targets for gepotidacin efficacy based on the murine lung infection modelwere not reported herein. The impact of poor growth, as demonstrated in the no-treatment control group, on the magnitudes of PK-PD targets for efficacy underscoresthe importance of ensuring that this study design criterion is met. Future studiesachieving sufficient growth in the no-treatment control groups with different isolatesor which use alternative methods with the current isolates (e.g., use of higher inocu-






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lums or direct introduction of the inoculum into the trachea of mice) may allow for thePK-PD of gepotidacin to be characterized using the murine lung infection model.

The PAE of an antibiotic, which can be evaluated using either in vitro or in vivomethods, can be described as persistent suppression of the growth of the pathogenfollowing exposure to the antibiotic (22). While the clinical utility of the results of an invitro PAE study is questionable, the presence of an in vivo PAE has been a usefulcharacteristic for determining the length of the dosing interval required for variousantibiotics, such as aminoglycosides and quinolones (22). Gepotidacin was found todemonstrate in vivo PAE durations against both S. aureus (range, 3.07 to 12.5 h) and S.pneumoniae (range, 5.25 to 8.46 h) that are comparable to those of fluoroquinolones(23). Since the gepotidacin PAE for both organisms was not found to be dose depen-dent, it appears to be a predictable characteristic that can be used to inform decisionsregarding clinical dosing intervals. In general, the presence of a prolonged in vivo PAEallows for wider clinical dosing intervals, since bacterial regrowth would be expected tobe minimal during this period when plasma and tissue concentrations fall below theMIC value for the infecting pathogen (23).

In summary, using PK data from neutropenic mice and data from a murine thighinfection model in which mice were infected with S. aureus or S. pneumoniae, thefree-drug plasma AUC/MIC ratio was identified as the PK-PD index most associated withthe efficacy of gepotidacin. Data from these studies also allowed for characterization ofthe magnitudes of the free-drug plasma AUC/MIC ratios associated with various levelsof reduction in bacterial burden and for the characterization of PAE. Such data areuseful for supporting future drug development decisions, including dose selection, forgepotidacin.

MATERIALS AND METHODSBacterial isolates. A total of 12 Gram-positive isolates, 6 S. aureus and 6 S. pneumoniae isolates, were

used in these studies. The MIC values for gepotidacin were determined in triplicate by broth microdi-lution assay, using methods outlined by the Clinical and Laboratory Standards Institute (24). The modalMIC value was reported for each isolate.

Inoculum preparation. Frozen stocks of S. aureus were inoculated onto agar plates and grownovernight. Colonies were subcultured from these agar plates into Mueller-Hinton II broth and grownovernight to an absorbance at 580 nm of 0.3. Frozen stocks of S. pneumoniae were inoculated onto sheepblood agar plates and grown overnight. Colonies were taken directly from the sheep blood agar platesand diluted into Mueller-Hinton II broth to an absorbance at 580 nm of 0.3.

Antimicrobial test agent. Analytical-grade gepotidacin (GlaxoSmithKline, Collegeville, PA) wasutilized for all in vitro and in vivo studies. Using the supplied potency, gepotidacin powder was weighedin a sufficient quantity to achieve the required stock concentration and reconstituted using dimethylsulfoxide (DMSO), with a final DMSO concentration of 1%. Subsequent concentrations were prepared bydilution with 0.15 M NaCl before use for each of the studies.

Animal infection models. Specific-pathogen-free female CD-1 mice (Charles River, St. Constant,Canada) weighing approximately 19 to 21 g were used in all experiments. All studies were conductedafter review by the Institutional Animal Care and Use Committee of the University of Wisconsin and inaccordance with the GlaxoSmithKline policy on the care, welfare, and treatment of laboratory animals.For the PK, dose-ranging, dose fractionation, and postantibiotic effect (PAE) studies, the animals wererendered neutropenic (i.e., having no more than 100 polymorphonuclear leukocytes/mm3) by twointraperitoneal injections of cyclophosphamide, consisting of 150 mg/kg 4 days and 100 mg/kg 1 daybefore infection (25). For all but the PK studies, the animals were then infected with S. aureus or S.pneumoniae for evaluation in a murine thigh or lung infection model. The murine thigh infection model,in which mice were inoculated in each posterior thigh via a 0.1-ml intramuscular injection of bacterialsuspension containing approximately 107 CFU of bacteria 2 h prior to the initiation of antimicrobialtherapy, was used to conduct dose fractionation, dose-ranging, and PAE studies. The murine lunginfection model, in which diffuse pneumonia was induced in the mice by intranasal instillation of a 50-�lbacterial inoculum containing approximately 108 CFU of S. aureus or S. pneumoniae, was used to conductdose-ranging studies.

Pharmacokinetic studies. PK studies were undertaken in neutropenic uninfected mice administeredgepotidacin as single subcutaneous (s.c.) doses of 16, 32, 64, or 128 mg/kg. Terminal plasma sampleswere collected via cardiac puncture at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h postdose (three mice/timepoint). Bronchoalveolar lavage (BAL) fluid samples were collected at 0.5, 1, 2, 4, 6, 8, 12, and 24 hpostdose for determinations of gepotidacin ELF concentrations for all dosing cohorts. The recovered BALfluid was centrifuged to separate the alveolar macrophages (cell pellet) and the ELF (supernatant).

In order to evaluate the impact of infection on the PK profile of gepotidacin, a second plasma PKstudy was conducted in both neutropenic infected and neutropenic uninfected mice administered






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gepotidacin as a single s.c. dose of 32 mg/kg and in neutropenic infected mice administered gepotidacinat 16, 64, or 128 mg/kg.

Plasma and BAL fluid samples for the first PK study were assayed for gepotidacin concentrations ontwo separate occasions by GlaxoSmithKline, using a validated high-performance liquid chromatography–tandem mass spectrometry method, with lower limits of quantification of 0.05 mg/liter and 0.1 mg/literfor the plasma samples and 0.0025 mg/liter and 0.005 mg/liter for the ELF samples. Plasma samples forthe second PK study were assayed for gepotidacin concentrations by GlaxoSmithKline, using the samemethodology as that described above, with a lower limit of quantification of 0.010 mg/liter (GlaxoSmith-Kline Pharmaceuticals, unpublished). The assay was reproducible, with precision between 0.16 and 2.31%for the plasma samples and between 2.13 and 7.30% for the BAL fluid samples. The assay also showedgood accuracy, with gepotidacin concentrations within 97 to 107% of the expected plasma concentra-tions and within 92 to 105% of the expected BAL fluid concentrations (GlaxoSmithKline Pharmaceuticals,unpublished).

Plasma and ELF urea concentrations were used to correct the ELF drug concentrations by use of themethod described by Rennard et al. (26). Urea concentrations were determined in triplicate by use of acommercial colorimetric assay kit (Pointe Scientific, Inc.).

Efficacy as assessed by changes in bacterial density. Two hours after infection for the murinethigh infection model studies and 4 h after infection for the murine lung infection model studies,gepotidacin treatment regimens were administered as 0.2-ml s.c. injections and continued for 24 h in allstudies. Control animals received sterile normal saline, using the same volume, route, and schedule asthose for the active-treatment regimens. Non-drug-treated control animals were sacrificed just prior toantibiotic initiation (0 h) and after 24 h. All animals were euthanized by carbon dioxide asphyxiationfollowed by cervical dislocation. After sacrifice, the thighs of mice in the murine thigh infection modelstudies were removed and individually homogenized in normal saline. Serial dilutions of the thighhomogenates were plated for CFU determination and incubated overnight at 37°C. Counts for each thighwere considered independent observations. Similarly, for the murine lung infection model, the lungswere aseptically excised following euthanasia, homogenized, diluted, plated, and incubated. Viable platecounts were determined as log10 CFU per thigh or lung. Efficacy was calculated as the change in the log10

CFU obtained for antibiotic-treated mice at 24 h compared to the preantibiotic baseline CFU measuredfor the 0-h control animals.

Dose fractionation studies. Dose fractionation studies were conducted in neutropenic mice in-fected in each thigh with S. aureus 33591 or S. pneumoniae 10813. Groups of two mice each wereadministered gepotidacin at a total daily dose of 2, 8, 32, 128, or 512 mg/kg s.c., divided evenly every 3,6, 12, or 24 h. Untreated mice used for the growth control assessment were euthanized 2 and 24 h afterinoculation.

Dose-ranging studies. (i) Murine thigh infection model dose-ranging studies. Dose-rangingstudies were conducted in neutropenic mice infected in each thigh with one of five S. aureus isolates orone of five S. pneumoniae isolates. Groups of two mice each were administered gepotidacin at a totaldaily dose of 2, 8, 32, 128, 512, or 2,048 mg/kg s.c., divided into four equal doses administered every 6h. Untreated infected mice used for the growth control assessment were euthanized 2 and 24 h afterinoculation for each of the 10 isolates studied.

(ii) Murine lung infection model dose-ranging studies. Dose-ranging studies were conducted inneutropenic mice infected in the lung via intranasal instillation with one of three S. aureus isolates or oneof three S. pneumoniae isolates. Groups of three mice each were administered gepotidacin at a total dailydose of 2, 8, 32, 128, 512, or 2,048 mg/kg s.c., divided into four equal 0.2-ml doses administered every6 h. Untreated infected mice used for the growth control assessment were euthanized 4 and 24 h afterinoculation for each of the six isolates studied.

Pharmacokinetic analyses. (i) Pharmacokinetic model development. Candidate models were fitto plasma and ELF concentration data from uninfected mice from the first PK study using the maximumlikelihood objective function, as implemented in S-ADAPT 1.57 (27). Weighting of the plasma and ELFconcentrations was based on the reciprocal of the estimated observation variance (the error modelstandard deviation [SD] squared), which was predicted as a function of the fitted concentrations. Giventhat the study design for PK sampling was destructive in nature (i.e., animals were sacrificed at theevaluated time points), the data were combined and fit using a pooled PK analysis approach.

(ii) Calculation of ELF penetration ratio. Using the PK model described above, which wasdeveloped using data from uninfected mice from the first PK study, fitted total-drug plasma and ELFconcentration-time profiles were examined for each dose evaluated (16, 32, 64, and 128 mg/kg/day).Using total-drug plasma and ELF concentration-time profiles from 0 to 24 h on day 1, AUC0 –24 valueswere calculated for each dose. Gepotidacin murine plasma protein binding was determined by Glaxo-SmithKline Pharmaceuticals using plasma from normal, uninfected CD-1 mice and equilibrium dialysisover a range of gepotidacin concentrations (5, 9, 18, and 36 �M) (GlaxoSmithKline Pharmaceuticals,unpublished). Concentration-dependent protein binding was negligible. Based on these data, the meanestimate for plasma protein binding was 0.237 (GlaxoSmithKline Pharmaceuticals, unpublished). Usingthe corresponding mean unbound gepotidacin estimate of 0.763, free-drug plasma AUC0 –24 values weredetermined. The ELF penetration ratio was calculated by dividing the total-drug ELF AUC0 –24 value by thefree-drug plasma AUC0 –24 value.

(iii) Evaluation of the impact of infection on the pharmacokinetic profile of gepotidacin. UsingNONMEM, version 7.1.2, PK models were developed based on the data for uninfected and infected miceevaluated in the second PK study, using a first-order conditional estimation with interaction. Total-drugplasma AUC0 –24 values based on the PK data for the dose of 32 mg/kg were calculated by integrating






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the respective PK profiles. The geometric mean total-drug plasma AUC0 –24 value was computed for bothinfected and uninfected mice. The ratio of the uninfected to infected geometric mean AUC0 –24 valuesand the associated 90% confidence interval around this ratio were computed.

Pharmacokinetic-pharmacodynamic data analyses. Using the final PK model, plasma and ELFconcentration-time profiles for total gepotidacin doses ranging from 2 to 2,048 mg/kg were generated.These concentration-time profiles and the free-fraction estimate of 0.763 for gepotidacin were used togenerate the PK-PD indices. Free-drug plasma and total-drug ELF AUC0 –24 values were calculated foreach dose by numerically integrating the individual fitted functions for the free-drug plasma andtotal-drug ELF concentration-time profiles, respectively. These were then divided by the gepotidacin MICvalue for each isolate to obtain free-drug plasma and total-drug ELF AUC/MIC ratios. Maximum free-drugplasma and total-drug ELF concentration values (Cmax) for each dose were divided by the MIC value foreach isolate to obtain free-drug plasma and total-drug ELF Cmax/MIC ratios. Free-drug plasma andtotal-drug ELF %T�MIC values were also calculated for each isolate by numerical integration of the fittedfunctions.

(i) Dose fractionation studies. Relationships between efficacy, as measured by changes frombaseline in log10 CFU, and the gepotidacin free-drug plasma PK-PD indices AUC/MIC ratio, Cmax/MIC ratio,and %T�MIC were evaluated to determine the PK-PD index most associated with efficacy by using datafrom the murine thigh infection model. These relationships were evaluated by fitting Hill-type models tothe data by using maximum likelihood estimation in S-ADAPT 1.57 (27).

(ii) Dose-ranging studies. (a) Murine thigh infection model dose-ranging studies. Data from thedose fractionation and dose-ranging studies carried out using the neutropenic murine thigh infectionmodel in which mice were infected with S. aureus were combined for analysis. The corresponding datafor S. pneumoniae were also combined for analysis. Relationships between efficacy, as measured bychange from baseline in log10 CFU, and the PK-PD index most associated with the efficacy of gepotidacinwere evaluated. These relationships were evaluated by fitting Hill-type models to the S. aureus and S.pneumoniae data independently by using a population PK-PD analysis approach and, as such, accountingfor interisolate variability in parameter estimates. Using the post hoc parameter estimates from theHill-type models, free-drug plasma PK-PD targets associated with net bacterial stasis and 1- and 2-log10

CFU reductions from baseline were identified.(b) Murine lung infection model dose-ranging studies. The relationships between the change from

baseline in log10 CFU and the total-drug ELF and free-drug plasma PK-PD indices most associated withthe efficacy of gepotidacin were evaluated using data obtained from the neutropenic murine lunginfection model. These relationships were evaluated by fitting Hill-type models to the S. aureus and S.pneumoniae data independently by using a population PK-PD analysis approach in S-ADAPT 1.57 (27).Using the relationships described by these models, PK-PD targets that were associated with net bacterialstasis and 1- and 2-log10 CFU reductions from baseline for S. aureus and S. pneumoniae were identified.

(iii) Postantibiotic effect study. Postantibiotic effect studies were conducted using S. aureus 33591and S. pneumoniae 10813 in a neutropenic murine thigh infection model. In this model, the mice wereadministered gepotidacin at single doses of 16, 32, 64, or 128 mg/kg s.c. Groups of three mice per dosewere euthanized 2, 4, 6, 12, and 24 h after the start of dosing. The PAE for each dose was calculated usingthe following formula (28): PAE � T � C � M, where T is the time required for the bacterial counts oftreated mice to increase by 1 log and was calculated using linear interpolation; C is the time required forbacterial counts to increase by 1 log in the control animals; and M is the time for which free-drug plasmaconcentrations exceed the MIC value, determined using the murine PK model.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/AAC.00115-16.

TEXT S1, PDF file, 0.3 MB.

ACKNOWLEDGMENTSThis study was undertaken with funds supplied by GlaxoSmithKline, Collegeville, PA.

This project has been funded in whole or in part with federal funds awarded by theDefense Threat Reduction Agency under agreement HDTRA1-07-9-0002.

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