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MONTE CARLO SIMULATION TO ESTIMATE IN VITRO SUSCEPTIBILITY BREAKPOINTS FOR MOXIFLOXACIN, GATIFLOXACIN, AND LEVOFLOXACIN AGAINST STAPHYLOCOCCUS AUREUS

AK MEAGHER1, WA CRAIG2, RN JONES3, A DALHOFF4, H STASS4, PG AMBROSE1 1Cognigen Corp, Buffalo, NY; 2Univ of Wisc, Madison, WI; 3The JONES Group, N. Liberty, IA; 4Bayer HealthCare, Wuppertal, Germany

Author Contact Information – Presenting Author: Alison K. Meagher, PharmD Assistant Director, Division of Infectious Disease Cognigen Corporation 395 Youngs Road Buffalo, NY 14221-5831 Phone: 716-633-3463 ext 277 E-mail: ameagher@cognigencorp.com

ABSTRACT

Background: Emerging resistance with staphylococci has called for a change in treatment paradigms and review of breakpoints. Current

NCCLS breakpoints Staphylococcus spp.

for are

gatifloxacin 2 µg/mL for

and levofloxacin against susceptible; breakpoints for

moxifloxacin have not been established. Breakpoints using PK-PD models to predict in vivo efficacy.

can

be

evaluated

Methods: Monte Carlo simulation was used to identify the probability of attaining PK-PD targets associated with efficacy using standard dosing regimens: moxifloxacin 400 mg QD, gatifloxacin 400 mg QD, and levofloxacin 500 mg QD. Single oral dose Phase I AUC0-data for

moxifloxacin was obtained (n=374), FDA submitted PK data were used for gatifloxacin and levofloxacin; all were adjusted for protein binding. PK-PD target was derived from a neutropenic murine-thigh model that identified a static AUC:MIC for reference quinolones. A target free-drug (f) 24hr AUC:MIC value of 30 was evaluated. Clinical isolate MIC distributions, including MSSA and MRSA, were obtained from surveillance studies. 5000 subject simulations were performed using fixed and continuous MIC data.

% of Subjects Achieving PK-PD Target for moxi/gati/levo

0.25

0.5

1

2

Continuous Distribution*

100/100/100

93/100/100

8/23/74

1/1/1

73/66/62

Cumulative % MIC Distribution for moxi/gati/levo *

60/65/60

61/66/62

70/68/63

80/80/64

100/100/100

*N=3,204 strains for moxifloxacin; N=35,528 strains for gatifloxacin; and N=19,296 strains for levofloxacin

Target attainment rates correlated extremely well with S. aureus MIC population statistics; MIC50/MIC90 for moxifloxacin, gatifloxacin, and lev-

ofloxacin are 0.06/4, 0.12/4, and 0.25/8 µg/mL, respectively. Probability of target attainment was 90% for MIC values 0.5 µg/mL for all regi- mens, but approached zero at a MIC value of 2 µg/mL. At a MIC value of 0.5 µg/mL, MRSA accounted for ~10% of susceptible strains.

Results:

PK-PD Target fAUC24:MIC = 30 MIC (µg/mL)

Conclusion: Target attainment was similar for moxifloxacin, gatifloxacin, and levofloxacin. At a PK-PD target (fAUC24:MIC) of 30, these data sug-

gest a breakpoint for moxifloxacin, gatifloxacin, and levofloxacin of 0.5 µg/mL for susceptible. Correlation between in vitro tests and clinical out- come statistics is needed.

INTRODUCTION

  • Resistance rates among Gram-positive organisms have increased dramat- ically over the past decade and staphylococci have emerged as one of the most prevalent pathogens in nosocomial infections [1,2].

  • Inadequate treatment of infections caused by Gram-positive organisms has resulted in increased morbidity and mortality [3].

  • Clinicians are often guided in choosing antimicrobial treatment by suscep- tibility breakpoints [4,5].

  • Susceptibility breakpoints can be estimated using Monte Carlo simulation to integrate pharmacokinetic-pharmacodynamic (PK-PD) animal infection models, human PK, and in vitro microbiological activity data to predict clin- ical and microbiological response [6,7].

  • For this analysis, Monte Carlo simulation was used to estimate susceptibil- ity breakpoints for moxifloxacin, gatifloxacin, and levofloxacin against S. aureus.

INTRODUCTION (CONT.)

  • At the time these analyses were conducted, the National Committee on Central Laboratory Standards (NCCLS) breakpoint for gatifloxacin and lev- ofloxacin against Staphylococcus spp. was 2 µg/mL; a breakpoint for moxifloxacin had not yet been established [4,5].

  • These data were presented to the NCCLS in June 2004 as decision-sup- port for the establishment of staphylococcal susceptibility breakpoints for moxifloxacin and the re-evaluation of those breakpoints for other fluoro- quinolones.

METHODS

PK-PD TARGET EXPOSURE

  • A neutropenic murine thigh infection model was used.

  • Five strains of S. aureus were evaluated and MIC values were determined by the broth microdilution method described by the NCCLS [4,5].

  • Mice were inoculated with 106 to 107 CFU/mL 2 hours prior to moxifloxacin administration by injection of 0.1 mL of inoculum into each posterior thigh.

  • Single-dose serum pharmacokinetic studies were performed in thigh-infect- ed mice given subcutaneous doses of moxifloxacin (0.293 to 75 mg/kg every 12 hours).

  • For each of the examined doses, three mice were sampled at 24 hours after the start of treatment. Control mice were sampled at 0-hour and at 24 hours. Serial dilutions of thigh homogenates were plated for CFU determi- nations.

  • Serum moxifloxacin concentrations were determined by standard microbi- ologic assays with S. aureus ATCC 6538p as the test organism and antibi- otic medium 1 as the agar diffusion medium.

  • The lower limit of detection for assays was 0.1 µg/mL with an intraday vari- ation less than 14%.

  • PK parameters were calculated using standard non-compartmental tech-

niques.

  • Serum protein binding in infected neutropenic mice was performed with

ultra-filtration methods [8].

• Efficacy was calculated by subtracting the log mouse at the end of therapy from the mean log 10 10

CFU/thigh of each treated CFU/thigh of control mice

just prior to treatment (0 hour) and at the end of therapy (24 hour).

  • A sigmoid dose-effect model was used to analyze the data:

  • E = (Emax • DN)/(ED50N + DN), where E is the effect, Emax is the maxi-

mal effect, D is the 24 hour total dose, ED50

is the dose required to

achieve 50% Emax, and N is the slope of the dose-effect curve.

  • The correlation between efficacy and three PK-PD indices (fAUC0-24

:MIC

ratio, fpeak:MIC ratio, and T>MIC) were examined by using nonlinear least- squares multivariate regression.

PHARMACOKINETIC PARAMETERS

  • Moxifloxacin PK data (AUC0-) were from single-dose Phase I studies of

the oral administration of moxifloxacin 400 mg to normal adult volunteers

(n=374) (Table 1).

  • Phase I single-dose AUC0-

values for orally administered gatifloxacin 400

mg daily and levofloxacin 500 mg were obtained from each product’s label

(Table 1) [9,10].

  • Sample distribution of the moxifloxacin AUC0-

distribution.

data was fit to a lognormal

  • A lognormal distribution was also employed for gatifloxacin and lev- ofloxacin PK data.

  • The free fraction (f) of moxifloxacin, gatifloxacin, and levofloxacin were fixed at 0.61, 0.8, and 0.69, respectively.

METHODS (CONT.)

Moxifloxacin

400 mg q.d.

36.1 ± 9.1

37-50%b

Gatifloxacin

400 mg q.d.

33.0 ± 6.2

20%

Levofloxacin

500 mg q.d.

47.9 ± 6.8

24-38%c

Table 1: Pharmacokinetic parameter values for moxifloxacin, gatifloxacin, and levofloxacin

a

b c

Mean and standard deviation (SD) values are from single oral dose healthy adult Phase I studies Moxifloxacin protein binding was fixed at 39% Levofloxacin protein binding was fixed at 31%

Agent

Dose (mg)

AUC0-

a

(mg · hr/L)

Protein binding

(%)

MICROBIOLOGICAL SUSCEPTIBILITY DATA

  • Moxifloxacin, gatifloxacin, and levofloxacin susceptibility data were obtained from the SENTRY Antimicrobial Surveillance Program (2000-

    • 2002)

      .

  • Isolates were primarily from patients with documented pneumonia or bloodstream infections.

  • MIC values were determined by the broth microdilution method described by the NCCLS [4,5].

MONTE CARLO SIMULATION AND SUSCEPTIBILITY BREAKPOINT ESTIMATION

  • PK-PD target attainment analyses were performed using Monte Carlo simulation.

  • Five thousand random subject simulations were performed using the fol- lowing structural model:

    • fAUC0-24:MIC = f · AUC0-:MIC

  • The MIC susceptible breakpoint was determined as the highest clinically rel- evant MIC value with a probability of PK-PD target attainment of 0.9.

RESULTS

PK-PD TARGET EXPOSURE

  • MIC values of moxifloxacin for the five strains of S. aureus ranged from

    • 0.03

      to 0.06 µg/mL:

      • Three strains were oxacillin-susceptible (MIC 2 µg/mL)

      • Two strains were oxacillin-resistant (MIC 4 µg/mL)

  • The PK of moxifloxacin in infected neutropenic mice at doses of 4.68, 18.8, and 75 mg/kg was linear:

    • The elimination half-life was 0.6 to 0.8 hours

    • Mean peak serum concentration/dose ratio was 0.5 to 0.6

    • Mean AUC0-24/dose ratio was 0.7 to 0.8

  • Serum protein binding was 50 to 55% at concentrations ranging from 1.0

to 5.0 µg/mL

  • Mice had 6.79 ± 0.29 log10

CFU/thigh of S. aureus at the initiation of ther-

apy and organisms grew 1.75 ± 0.34 log10 mice.

CFU/thigh in untreated control

  • Increasing moxifloxacin doses resulted in concentration-dependent killing.

  • The highest dose studied reduced the bacterial burden at 0-hour by 3.12 ±

    • 0.37

      log10 CFU/thigh.

  • A strong relationship was seen when results were correlated with the fAUC0-24:MIC ratio, with an r2 value of 89% (Figure 1).

  • An fAUC0-24:MIC ratio of approximately 30 is required for stasis.

RESULTS (CONT.)

Figure 1: Relationship between fAUC0-24: MIC ratio and change in bacterial density of five strains of S. aureus in the thighs of neutropenic mice after 24 hours of moxifloxacin therapy

T h e d a s h e d l i n e r e p r e s e n t s t h e n u m b e r o f b a c t e r i a a t t h e b e g i n n i n g o f t h e r a p y P o s i t i v e v a l u e s o n t h e y - a x s r e f l e c t b a c t e r i a l g r o w t h w h i l e n e g a t i v e v a u e s r e f l e c t b a c t e r i a l d e a t h A n f A U C 0 - 2 4 : M I C r a t i o o f a p p r o x i m a t e l y 3 0 i s r e q u i r e d f o r s t a s i s .

MICROBIOLOGICAL SUSCEPTIBILITY DATA

  • Figures 2, 3, and 4 show the MIC distribution for the three fluoro- quinolones against all S. aureus isolates, oxacillin-susceptible S. aureus, and oxacillin-resistant S. aureus, respectively.

  • The total number of isolates tested for moxifloxacin, gatifloxacin, and lev- ofloxacin were 3,204, 35,528, and 19,296, respectively.

  • The rank order of in vitro potency (most active to least) was as follows:

moxifloxacin (MIC50/90 and levofloxacin (MIC50/90 : 0.06/4 µg/mL); gatifloxacin (MIC50/90 : 0.25/8 µg/mL).

: 0.12/4 µg/mL);

  • Approximately 8 to 10% of oxacillin-resistant isolates had fluoroquinolone MIC values of 0.5 µg/mL and 6 to 7% of oxacillin-susceptible isolates had MIC values of 1 µg/mL.

Figure 2: Fractional probability of PK-PD target attainment (fAUC24:MIC = 30) for moxi- floxacin, gatifloxacin, and levofloxacin against Staphylococcus aureus

MIC Probability

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

Moxifloxacin 400mg Gatifloxacin 400mg Levofloxacin 500mg

<0.03 0.06

0.12

0 2 5 0 5 M I C ( mg

/L)

1

2

4

8

Probability of Target Attainment

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

T h e g r a y l i n e , b l a c k l i n e s h o r t - d a s h e d a n d l o n g - d a s h e d l i n e s r e p r e s e n t t h e f r a c t i o n a l p r o b a b i l i t y o f t a r g e t a t t a i n m e n t f o r m o x i f l o x a c n g a t i f l o x a c n a n d l e v o f l o x a c n r e s p e c t i v e y T h e g r a y b a r s b l a c k b a r s a n d w h i t e b a r s r e p r e s e n t m o x i f l o x a c n g a t i f l o x a c n a n d e v o f l o x a c i n M I C d i s t r i b u t i n s , r e s p e c t i v e y .

RESULTS (CONT.)

Figure 3: Fractional probability of PK-PD target attainment (fAUC24:MIC = 30) for moxi- floxacin, gatifloxacin, and levofloxacin against oxacillin-sensitive Staphylococcus aureus

MIC Probability

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

Moxifloxacin 400mg Gatifloxacin 400mg Levofloxacin 500mg

<0.03 0.06

0.12

0 2 5 0 5 M I C ( mg

/L)

1

2

4

8

Probability of Target Attainment

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

T h e g r a y l i n e b l a c k l i n e s h o r t - d a s h e d a n d l o n g - d a s h e d l i n e s r e p r e s e n t t h e f r a c t i o n a l p r o b a b i l i t y o f t a r g e t a t t a i n m e n t f o r m o x i f l o x a c i n g a t i f l o x a c i n a n d l e v o f l o x a c i n r e s p e c t i v e l y T h e g r a y b a r s b l a c k b a r s a n d w h i t e b a r s r e p r e s e n m o x i f l o x a c i n g a t i f l o x a c i n a n d l e v o f l o x a c i n M I C d i s t r i b u t i o n s , r e s p e c t i v e l y .

Figure 4: Fractional probability of PK-PD target attainment (fAUC24:MIC = 30) for moxi- floxacin, gatifloxacin, and levofloxacin against oxacillin-resistant Staphylococcus aureus

MIC Probability

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

Moxifloxacin 400mg Gatifloxacin 400mg Levofloxacin 500mg

<0.03 0.06

0.12

0 2 5 0 5 M I C ( mg

/L)

1

2

4

8

Probability of Target Attainment

1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0

T h e g r a y l i n e b l a c k l i n e s h o r t - d a s h e d a n d l o n g - d a s h e d l i n e s r e p r e s e n t t h e f r a c t i o n a l p r o b a b i l i t y o f t a r g e t a t t a i n m e n t f o r m o x i f l o x a c i n g a t i f l o x a c i n a n d l e v o f l o x a c i n r e s p e c t i v e l y T h e g r a y b a r s b l a c k b a r s a n d w h i t e b a r s r e p r e s e n m o x i f l o x a c i n g a t i f l o x a c i n a n d l e v o f l o x a c i n M I C d i s t r i b u t i o n s , r e s p e c t i v e l y .

RESULTS (CONT.)

MONTE CARLO SIMULATION AND SUSCEPTIBILITY BREAKPOINT ESTIMATION

  • The forecast AUC distributions (mean ± SD) for moxifloxacin, gatifloxacin,

and levofloxacin were 35.9 ± 9.04, 33.0 ± 6.1, and 48.1 ± 6.77 mg•hr/L,

respectively.

  • Figure 2 shows the fractional PK-PD target (fAUC0-24

:MIC ratio 30)

attainment over the S. aureus MIC distributions for each drug. Figures 3 and 4 show the fractional target attainment over the oxacillin-sensitive and

  • -

    resistant S. aureus distributions, respectively.

  • The probability of target attainment was greater than 0.9 for MIC values 0.5 µg/mL for moxifloxacin, gatifloxacin, and levofloxacin regimens.

  • Target attainment rapidly degraded for MIC values > 0.5 µg/mL and approached zero for MIC values 2 µg/mL.

  • These data suggest susceptible MIC breakpoints of 0.5 µg/mL for moxi- floxacin, gatifloxacin and levofloxacin.

  • The estimated susceptibility breakpoints correlate extremely well with the SENTRY Antimicrobial Surveillance Program MIC distributions of S. aureus, including both oxacillin-susceptible and -resistant strains (Figures 2, 3, and 4) [2].

    • Each fluoroquinolone displayed a bimodal distribution of MIC values, with a natural cleave or breakpoint at 0.5 µg/mL for all three agents.

    • This breakpoint effectively differentiated the oxacillin-susceptible and oxacillin-resistant S. aureus subpopulation as well as the quinolone-sus- ceptible and -resistant subpopulations, the latter with QRDR mutations.

CONCLUSIONS

  • The probability of target attainment at an MIC of 2 µg/mL, the previously establish NCCLS breakpoints, approached zero for all three studied fluoro- quinolones using traditional dosing regimens.

  • Results from this PK-PD analysis were presented to the NCCLS in June 2004 to establish a susceptibility breakpoint for moxifloxacin at 0.5 µg/mL, and to lower the susceptibility breakpoints for gatifloxacin and lev- ofloxacin to 0.5 µg/mL and 1 µg/mL, respectively.

  • The revised breakpoints resulted in nearly identical perceived spectrums of anti-staphylococcal activity as measured by percentage susceptible rates for all evaluated fluoroquinolones, and a clear lack of potency for the three agents against oxacillin-resistant S. aureus isolates.

REFERENCES

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    Jones RN. Contemporary antimicrobial susceptibility patterns of bacterial pathogens commonly asso- ciated with febrile patients with neutropenia. Clin Infect Dis 1999; 29:495-502.

  • 2.

    Jones RN. Global Epidemiology of Antimicrobial Resistance Among Community-Acquired and Nosocomial Pathogens: A Five-Year Summary From the SENTRY Antimicrobial Surveillance Program (1997-2001). Semin Respir Crit Care Med 2003; 24:121-134.

  • 3.

    Ibrahim EH, Sherman G, Ward S, Fraser VJ, Kollef MH. The influence of inadequate antimicrobial treatment of bloodstream infections on patient outcomes in the ICU setting. Chest 2000; 118:146-155.

  • 4.

    NCCLS. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard M7- A6. 6th edition. Wayne, PA: NCCLS, 2003.

  • 5.

    NCCLS. Performance standards for antimicrobial susceptibility testing; 14th informational supplement M100-S14. Wayne, PA: NCCLS, 2004.

  • 6.

    Dudley MN, Ambrose PG. Pharmacodynamics in the study of drug resistance and establishing in vitro susceptibility breakpoints: ready for prime time. Curr Opin Microbiol 2000; 3:515-521.

  • 7.

    Drusano GL, Preston SL, Hardalo C, Hare R, Banfield C, Andes D et al. Use of preclinical data for selection of a phase II/III dose for evernimicin and identification of a preclinical MIC breakpoint. Antimicrob Agents Chemother 2001; 45:13-22.

  • 8.

    Craig WA, Suh B. Protein binding and the antimicrobial effects. Methods for the determination of pro- tein binding. In: Lorian V, editor. Antibiotics in Laboratory Medicine. Baltimore, MD: Williams & Wilkins, 1991: 367-402.

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