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Impact of MIC Range in P. aeruingosa and S. pneumoniae on the Ceftolozane 1

In vivo PK/PD Target 2

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AJ Lepak, A Reda, K Marchillo, J Van Hecker, WA Craig, D Andes. 4

Department of Medicine, University of Wisconsin and William S. Middleton VA Hospital, 5

Madison, WI 6

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Corresponding Author: 8

David Andes, MD. 9

1685 Highland Ave, University of Wisconsin 10

email: dra@medicine.wisc.edu 11

phone: 608-263-1545 12

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Running Title: In vivo Activity of Ceftolozone Against Resistant Strains 14

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AAC Accepts, published online ahead of print on 4 August 2014Antimicrob. Agents Chemother. doi:10.1128/AAC.03572-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Abstract 19

Ceftolozane is a novel cephalosporin with activity against drug-resistant pathogens 20

including Pseudomonas aeruginosa and Streptococcus pneumoniae. The current in vivo 21

investigations test the limits of this drug against 20 PA and SP isolates across a wide 22

MIC range and defined resistance mechanisms. The T>MIC targets for stasis, 1 and 2 23

log reductions were 31%, 39%, 42% for PA and 18%, 24%, 27% for SP , respectively. 24

The 1 log endpoint was achieved for strains with MICs as high as 16 µg/ml. 25

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Text 38

Emerging drug-resistance has compromised the utility of our current antimicrobial 39

armamentarium (1-3). The development of the novel cephalosporin, ceftolozane, 40

provides a solution for a subset of drug-resistant infections. The spectrum of activity 41

includes two common multi-drug resistant respiratory pathogens, P. aeruginosa (PA) 42

and S. pneumoniae (SP) (4-9). Initial pharmacokinetic/pharmacodynamic studies with 43

this drug demonstrate that %T>MIC is the measure linked to efficacy, as with other 44

cephalosporins (10). However, studies exploring the PK/PD target associated with 45

efficacy suggest that the %T>MIC for ceftolozane is lower than for other drugs within the 46

cephalosporin class (10, 11). Mechanistic investigations suggest this may be related to 47

the rate of organism killing and perhaps affinity for the penicillin binding protein (PBP) 48

(10, 12). The goal of the current studies was to test the PK/PD limits of ceftolozane 49

efficacy in vivo against PA and SP across a wide MIC range and with a diversity of 50

resistance mechanisms. Fourteen PA and 6 SP isolates were studied (Table 1). MICs 51

were performed in triplicate according to CLSI guidelines (13). The ceftolozane MIC 52

range for the 20 isolates was 0.125-16 µg/ml (varied 128-fold). For PA, MICs ranged 53

from 2 – 16 µg/ml and for SP they ranged from 0.125 – 16 µg/ml. Strain phenotypes 54

and genotypes included ceftazidime-, carbapenem-, and ciprofloxacin-resistant PA due 55

to AmpC overproduction and/or OprD mutations and 4 penicillin-resistant SP strains. 56

The neutropenic, murine thigh infection model was used for in vivo study of ceftolozane 57

(14). Animals were maintained in accordance with the American Association for 58

Accreditation of Laboratory Animal Care (AAALAC) criteria. All animal studies were 59

approved by the Animal Research Committee of the William S. Middleton Memorial VA 60

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Hospital and the University of Wisconsin. Mice were infected with 105-6 CFU/thigh of 61

each strain. The in vivo fitness of the strains was relatively similar in untreated control 62

mice based upon a similar increase in thigh burden over the 24h period, 2.94 ± 0.49 63

log10 CFU/thigh for all isolates. Two hours after thigh infection, ceftolozane was 64

administered subcutaneously for 24 h using 6 or 7 dosing regimens that ranged from 65

0.39 mg/kg to 800 mg/kg every 6 h. A 1 or 2 log kill was achieved for most strains 66

(Figure 1A and 1C). In general, the dose-response for each strain was linked to the 67

MIC. More specifically, the drug dose required for efficacy, on a mg/kg basis, increased 68

proportionally as the MIC increased. Efficacy was observed across the resistance 69

genotypes and other drug-resistance phenotypes. Plasma pharmacokinetics from our 70

recent study in this infection model were used for %T>MIC determinations (10). Total 71

drug concentrations were used given the low degree of binding in this animal model 72

(<10%). The sigmoid Emax model was used to analyze the exposure response data. 73

The ceftolozane dose and associated %T>MIC needed to achieve net stasis, 1 log and 74

2 log kill were calculated and are shown in Table 2. Although the dose level (on a mg/kg 75

basis) associated with these endpoints varied from 62- to 138-fold, the percent T>MIC 76

varied from only 1.3- to 2.5-fold. Thus, the %T>MIC needed for efficacy was relatively 77

similar across the wide range of MICs studied. There was no difference in the PK/PD 78

target T>MIC based on MIC across for each species group (p = 0.568). As shown in 79

Figures 1B and 1D, the treatment effect data regressed with the %T>MIC measure 80

resulted in a strong relationship as demonstrated by the relatively high coefficients of 81

determination (R2 = for PA 0.80 and SP 0.85, respectively). These data both confirm 82

and extend the PK/PD information for ceftolozane. First, the results affirm the 83

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importance of the %T>MIC PK/PD measure for ceftolozane (15-18). Secondly, the PD 84

targets identified in the current studies are similar to those noted in previous in vivo 85

assessment of ceftolozane. Specifically, the stasis and killing targets for the Gram 86

negative group was a T>MIC value near 30% and 40%, respectively. These values are 87

considerably lower than for other cephalosporins and may be due to more rapid killing 88

kinetics (10, 19). For example the %T>MIC stasis target for ceftazidime against P. 89

aeruginosa was found to be 40-45% in this animal infection model, which is closer to the 90

1 log kill target for ceftolozane and has also been suggested from clinical PK/PD 91

analyses (17, 20, 21). The inclusion of a wider MIC range, higher MICs, and defined 92

resistance-mechanisms in the present studies provides an opportunity to understand 93

the target pathogen “ceiling” for this new compound. The current results suggest 94

ceftolozane may be a useful treatment option for infections with few alternatives, 95

including those due to strains resistant to other cephalosporins, quinolones, and even 96

carbapenems. Efficacy was observed against organisms with MICs as high as 16 µg/ml. 97

Importantly, human kinetic studies demonstrate that a dosing regimen of 1 g every 8h 98

produces serum concentrations near this MIC value for nearly 50% of the dosing 99

interval (22, 23). Additionally, these data represent the first PK/PD exploration for 100

ceftolozane against pneumococci. Interestingly, comparison of these data with the 101

Gram negative studies show that the %T>MIC target for similar efficacy in S. 102

pneumoniae is nearly half of that for P. aeruginosa (p<0.001). This PK/PD target 103

difference has been described for other drug-bug combinations, but the mechanistic 104

explanation in this case is unknown (24). The current results verify the promising utility 105

of this new cephalosporin against multi-drug resistant pathogens and across a wide 106

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range of ceftolozane MIC. The data should be useful in guiding clinical use and the 107

development of susceptibility breakpoints. 108

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Acknowledgements 110

This study was funded by a research grant from Cubist Pharmaceuticals. We thank 111

Ron Jones and JMI for several of the strains used in these studies. 112

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Figure Legend 114

Figure 1. Panel A: Dose-response relationship for the 6 hourly dosing of ceftolozane 115

against 6 S. pneumoniae isolates. The dose is expressed as mg/kg/24 h. Each symbol 116

represents the mean log10 cfu/thigh for 2 mice (4 thighs) and the error bar represents 117

the standard deviation. Black symbols represent an MIC of 0.125 mg/L, green 0.25 118

mg/L, blue 8 mg/L, and red 16 mg/L. The dashed horizontal line represents the burden 119

of organisms in the thighs at the start of therapy. Panel B: Dose-response relationship 120

for the 6 hourly dosing of ceftolozane against 14 P. aeruginosa isolates. The dose is 121

expressed as mg/kg/24 h. Each symbol represents the mean log10 cfu/thigh for 2 mice 122

(4 thighs) and the error bar represents the standard deviation. Black Symbols represent 123

an MIC of 2 mg/L, green 4 mg/L, blue 8mg/L, and red 16 mg/L. The dashed horizontal 124

line represents the burden of organisms in the thighs at the start of therapy. Panel C: 125

Relationship between the percent time above MIC and change in cfu/thigh over 24h of 126

treatment with ceftolozane against 6 S. pneumoniae isolates. Each symbol represents 127

the mean log10 cfu/thigh for 2 mice (4 thighs). Black symbols represent an MIC of 0.125 128

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mg/L, green 0.25 mg/L, blue 8 mg/L, and red 16 mg/L to ceftolozane. R2 represents the 129

coefficient of determination. The Emax, ED50, N represent the maximal effect, 50% 130

effect, and slope of the relationship resulting from the Sigmoid Emax model, 131

respectively. The dashed horizontal line represents the burden of organisms in the 132

thighs at the start of therapy. The solid sigmoid shaped line represents the best fit line 133

using the Sigmoid Emax model, respectively. Panel D: Relationship between the 134

percent time above MIC and change in cfu/thigh over 24h of treatment with ceftolozane 135

against 14 P. aeruginosa isolates. Each symbol represents the mean log10 cfu/thigh for 136

2 mice (4 thighs). Black symbols represent an MIC of 0.125 mg/L, green 0.25 mg/L, 137

blue 8 mg/L, and red 16 mg/L to ceftolozane. R2 represents the coefficient of 138

determination. The Emax, ED50, N represent the maximal effect, 50% effect, and slope 139

of the relationship resulting from the Sigmoid Emax model, respectively. The dashed 140

horizontal line represents the burden of organisms in the thighs at the start of therapy. 141

The solid sigmoid shaped line represents the best fit line using the Sigmoid Emax 142

model. Black symbols represent an MIC of 2 mg/L, green 4 mg/L, blue 8 mg/L, and red 143

16 mg/L to ceftolozane. 144

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References 150

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13. Institute CLS. 2007. Methods for dilution antimicrobial susceptibility tests for bacteria that grow 194 aerobically; approved standard. Clinical Laboratory Standards Institute, Wayne PA. 7th ed CLSO 195 document M7-A7. 196

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24. Craig WA. 1998. Pharmacokinetic/pharmacodynamic parameters: rationale for antibacterial 228 dosing of mice and men. Clin Infect Dis 26:1-10; quiz 11-12. 229

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Table 1. Study organisms, phenotype, genotype, and ceftolozane MICs. 232

Organism MIC (µg/ml) Phenotype Genotype SP ATCC 10813 0.125 PSSP NA SP 145 0.125 PRSP NA SP 146 0.25 PRSP NA SP 1329 8 PRSP NA SP 1020 8 PRSP NA SP 49619 16 PISP NA PA 3B 2 NA NA PA 2638 2 DOR R; IPM R, MEM R,

FEP R, CAZ R, TZP R, NA

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CIP R, TOB R PA 4304A 2 NA NA PA 3068 4 DOR R; IPM R, MEM R,

FEP R, CAZ S, TZP R, CIP R

NA

PA 3070 4 DOR R; IPM R, MEM R, FEP I, CAZ R, TZP S, CIP R

OprD mutant, AmpC hyperproducer

PA 2757 4 NA AmpC hyperproducer PA 2627 4 NA NA PA 9139 4 DOR S; IPM S, MEM S,

FEP R, CAZ S, TZP R, CIP S, TOB S

NA

PA 3072 4 DOR R; IPM R, MEM R, FEP S, CAZ S, TZP S, CIP R, TOB R

OprD mutant, AmpC hyperproducer

PA 3071 8 DOR R; IPM R, MEM R, FEP R, CAZ R, TZP R, CIP R

OprD mutant, AmpC hyperproducer

PA 823 8 DOR S; IPM S, MEM S, FEP R, CAZ R, TZP R, CIP S, TOB S

NA

PA 26975 8 DOR S; IPM S, MEM S, FEP R, CAZ R, TZP R, CIP S, TOB R

NA

PA 3076 16 DOR R; IPM R, MEM R, FEP I, CAZ R, TZP R, CIP R, TOB S

OprD mutant, AmpC hyperproducer

PA 24530 16 DOR R, IPM R, MEM R, FEP R, CAZ R, TZP R, CIP S, TOB S

NA

SP, Streptococcus pneumoniae; PA, Pseudomonas aeruginosa; PSSP, penicillin-susceptible 233 Streptococcus pneumoniae; PRSP, penicillin-resistant Streptococcus pneumoniae; PISP, 234 penicillin-intermediate Streptococcus pneumoniae; DOR, doripenem; IPM, imipenem; MEM, 235 meropenem; FEP, cefepime; CAZ, ceftazidime; TZP, piperacillin-tazobactam; CIP, ciprofloxacin; 236 TOB, tobramycin; S, susceptible; I, intermediate; R, resistant; NA, not available 237

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Table 2. Dose and %T>MIC values for stasis, 1 log kill, and 2 log kill for 241 ceftolozane against six S. pneumoniae and 14 P. aeruginosa strains. 242

Organism Static Dose (mg/kg/24h)

Stasis %T>MIC

1 Log Kill (mg/kg/24h)

1 Log Kill %T>MIC

2 Log Kill (mg/kg/24h)

2 Log Kill %T>MIC

SP 10813 10.3 20.9 18.9 23.8 44.4 27.7 SP 145 4.44 17.0 8.38 20.0 30.2 25.9 SP 146 6.02 15.2 20.0 20.8 66.0 26.4 SP 1329 642 25.4 1101 30.0 >1600 NA SP 1020 271 17.1 696 26.2 >1600 NA SP 49619 231 12.6 677 22.0 >1600 NA

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Mean 194 18.1 420 23.8 46.8 26.7 Median 121 17.1 349 22.9 44.4 26.4 SD 250 4.52 468 3.75 18.0 0.94 PA 3B 55.7 15.9 140 20.1 389 27.6 PA 2638 1473 40.1 2538 43.2 4501 46.4 PA 4304A 907 36.2 5029 47.0 >12800 NA PA 3068 717 30.3 1958 37.8 6942 44.9 PA 3070 1814 37.3 10228 47.1 >12800 NA PA 2757 1848 37.5 4672 42.7 >12800 NA PA 2627 2040 38.0 7558 45.4 >12800 NA PA 9139 456 25.6 1597 36.6 9242 46.5 PA 3072 608 28.7 >12800 - >12800 NA PA 3071 780 27.2 11226 43.7 >12800 NA PA 823 444 21.5 1847 33.5 >12800 NA PA 26975 974 29.9 3021 36.3 12800 44.5 PA 3076 5602 35.9 >12800 - >12800 NA PA 24530 3141 32.6 10658 39.5 >12800 NA Mean 1490 31.2 5039 39.4 6775 42.0 Median 940 31.4 3847 41.1 6942 44.9 SD 1438 6.99 3921 7.53 4700 8.11 SP, Streptococcus pneumoniae; PA, Pseudomonas aeruginosa; NA, not achieved 243

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A. B.

C. D.

Figure 1. Panel A: Dose-response relationship for the 6 hourly dosing of ceftolozane against 6 S. pneumoniae isolates. The dose is expressed as mg/kg/24 h. Each symbol

represents the mean log10 cfu/thigh for 2 mice (4 thighs) and the error bar represents the standard deviation. Black symbols represent an MIC of 0.125 mg/L, green 0.25 mg/L,

blue 8 mg/L, and red 16 mg/L. The dashed horizontal line represents the burden of organisms in the thighs at the start of therapy. Panel B: Dose-response relationship for the

6 hourly dosing of ceftolozane against 14 P. aeruginosa isolates. The dose is expressed as mg/kg/24 h. Each symbol represents the mean log10 cfu/thigh for 2 mice (4 thighs)

and the error bar represents the standard deviation. Black Symbols represent an MIC of 2 mg/L, green 4 mg/L, blue 8mg/L, and red 16 mg/L. The dashed horizontal line

represents the burden of organisms in the thighs at the start of therapy. Panel C: Relationship between the percent time above MIC and change in cfu/thigh over 24h of

treatment with ceftolozane against 6 S. pneumoniae isolates. Each symbol represents the mean log10 cfu/thigh for 2 mice (4 thighs). Black symbols represent an MIC of 0.125

mg/L, green 0.25 mg/L, blue 8 mg/L, and red 16 mg/L to ceftolozane. R2 represents the coefficient of determination. The Emax, ED50, N represent the maximal effect, 50%

effect, and slope of the relationship resulting from the Sigmoid Emax model, respectively. The dashed horizontal line represents the burden of organisms in the thighs at the

start of therapy. The solid sigmoid shaped line represent the best fit line using the Sigmoid Emax model, respectively. Panel D: Relationship between the percent time above

MIC and change in cfu/thigh over 24h of treatment with ceftolozane against 14 P. aeruginosa isolates. Each symbol represents the mean log10 cfu/thigh for 2 mice (4 thighs).

Black symbols represent an MIC of 0.125 mg/L, green 0.25 mg/L, blue 8 mg/L, and red 16 mg/L to ceftolozane. R2 represents the coefficient of determination. The Emax,

ED50, N represent the maximal effect, 50% effect, and slope of the relationship resulting from the Sigmoid Emax model, respectively. The dashed horizontal line represents the

burden of organisms in the thighs at the start of therapy. The solid sigmoid shaped line represent the best fit line using the Sigmoid Emax model. Black symbols represent an

MIC of 2 mg/L, green 4 mg/L, blue 8 mg/L, and red 16 mg/L to ceftolozane.