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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Vallé, M., Schneider, M., Galland, D., Giboin, H., Woehrlé, F. Pharmacokinetic and pharmacodynamic testing of marbofloxacin administered as a single injection for the treatment of bovine respiratory disease. J. vet. Pharmacol. Therap. 35, 519–528.

New approaches in Pharmacokinetic/Pharmacodynamic (PK/PD) integration suggested that marbofloxacin, a fluoroquinolone already licensed for the treatment of bovine respiratory disease at a daily dosage of 2 mg/kg for 3–5 days, would be equally clinically effective at 10 mg/kg once (Forcyl®), whilst also reducing the risk of resistance. This marbofloxacin dosage regimen was studied using mutant prevention concentration (MPC), PK simulation, PK/PD integration and an in vitro dynamic system. This system simulated the concentration–time profile of marbofloxacin in bovine plasma established in vivo after a single 10 mg/kg intramuscular dose and killing curves of field isolated Pasteurellaceae strains of high (minimum inhibitory concentration (MIC) MIC ≤0.03 μg/mL), average (MIC of 0.12–0.25 μg/mL) and low (MIC of 1 μg/mL) susceptibility to marbofloxacin. The marbofloxacin MPC values were 2- to 4-fold the MIC values for all Mannheimia haemolytica, Pasteurella multocida tested. Marbofloxacin demonstrated a concentration-dependant killing profile with bactericidal activity observed within 1 h for most strains. No resistance development (MIC ≥4 μg/mL) was detected in the dynamic tests. Target values for risk of resistance PK/PD surrogates (area under the curve (AUC) AUC24 h/MPC and T>MPC/TMSW ratio) were achieved for all clinically susceptible pathogens. The new proposed dosing regimen was validated in vitro and by PK/PD integration confirming the single-injection short-acting antibiotic concept.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Marbofloxacin is a synthetic third-generation fluoroquinolone, specifically developed for individual veterinary treatment (Schneider et al., 1996; Aliabadi & Lees, 2002; Meunier et al., 2004). With a broad spectrum of activity against many pathogens of veterinary importance, marbofloxacin acts primarily as a bactericidal concentration-dependant antibiotic for Gram-negative bacteria (Aliabadi & Lees, 2002; Martinez et al., 2006; Pellet et al., 2006). As Marbocyl® 10% (Vétoquinol, Lure, France), marbofloxacin has been authorized in Europe since 1997 for the treatment of bovine infections at a daily dosage of 2 mg/kg for 3–5 days using various routes of administration. For this approved dosing regimen, PK/PD integration and PK/PD modelling using the two main PK/PD surrogate markers area under the curve (AUC)/ minimum inhibitory concentration (MIC) ratio ≥125 h and Cmax/MIC ratio ≥10 (maximal concentration/minimum inhibitory concentration) correlated very well with clinical efficacy in the field (Thomas et al., 2001; Aliabadi & Lees, 2002; Sidhu et al., 2011).

The use of fluoroquinolone agents in veterinary medicine has recently raised concerns regarding the emergence of bacterial resistance. This has lead to efforts to limit the emergence of resistance whilst also treating the diseased individual following the guidance of the International Medicine’s Agencies with respect to the registration of antibiotic drugs for food-producing animals: EMEA/CVMP/627/01 (EMEA, 2002). Thus, a novel risk assessment approach to susceptibility/resistance has been developed based on the concept that, plasma concentrations of drug which exceed the threshold for spontaneous drug-resistant mutant concentration mutant prevention concentration (MPC), will prevent the emergence of resistance as all target pathogens, including the potential mutants, will be killed (Dong et al., 1999; Zhao & Drlica, 2001). When the drug concentration falls between the MIC and the MPC, susceptible bacteria are inhibited, whereas resistant subpopulations may be selectively amplified; therefore, this concentration range is termed the mutant selection window (MSW). Below the MIC, no mutant is selected because the selection pressure is insufficient as neither susceptible nor first-step-resistant bacteria will be inhibited. No mutant should grow above the MPC because the bacteria would require two mutations which are rare (Blondeau et al., 2001; Firsov et al., 2003; Campion et al., 2004; Zhao & Drlica, 2008). Subsequently, during therapy, if the serum/tissue drug concentration is maintained above the MPC concentration (T>MPC), very few or no resistant mutants will be selectively amplified. Thus, both the MSW and MPC may be very important parameters when treating infections with antimicrobial agents against organisms with the potential to become resistant.

The optimal treatment should therefore quickly reach concentrations above the MPC and then decrease to levels below the MIC where the selective pressure is least to limit the time within the MSW. Theoretically, a single administration of a high dose of a short-acting antimicrobial should provide this optimal treatment (single-injection short-acting antibiotic (SISAAB) SISAAB concept). To date, this approach has been primarily developed and applied to fluoroquinolones used in human medicine such as ciprofloxacin, gatifloxacin, levofloxacin or moxifloxacin (Croisier et al., 2004; Cui et al., 2006; Zhao & Drlica, 2008). The scientific rationale for the use of fluoroquinolones as a SISAAB for the treatment of bovine respiratory disease (BRD) clearly differentiate from the simple convenience of commonly used single-injection long-acting antibiotics such as macrolide derivatives for the treatment and prevention of BRD (Forbes et al., 2011; Grandemange, E., Fournel, S., Giboin, H. & Woehrle, F., personnel communication).

For bovine bacterial respiratory disease, recent field surveys have shown that the MIC50 range for marbofloxacin against Mannheimia haemolytica (M. haemolytica), Pasteurella multocida (P. multocida) and Histophilus somni was between 0.008 and 0.022 μg/mL (MIC ≤0.03 μg/mL) and the MIC90 range was between 0.029 and 0.560 μg/mL (MIC ≤1 μg/mL) (Meunier et al., 2004; Kroemer et al., 2011). The MIC of the modal classes of the first-step mutant Pasteurellaceae strains were considered to be 0.12–0.25 μg/mL. Using PK data generated following the administration of 2 mg/kg (AUC24 h = 10–15 μg·h/mL) and assuming that the pharmacokinetics of marbofloxacin are proportional to the dose, we calculated the AUC and Cmax required to reach the theoretically optimal AUC/MIC and Cmax/MIC ratio for the least susceptible strains (Toutain et al., 2002). The maximum marbofloxacin MIC90 observed over several years of survey was 0.560 μg/mL; hence, to achieve an AUC/MIC ≥125 h and a Cmax/MIC ≥8 for this MIC90, an AUC of 125 × 0.560 = 65 μg·h/mL and a Cmax of 8 × 0.560 = 4.48 μg/mL would be required. To reach an AUC and Cmax in the order of 60 μg·h/mL and 5 μg/mL, a dose of 10 mg/kg was proposed. For injection-site tolerance and more convenient administration, a more concentrated solution of 16% marbofloxacin has been developed.

The aim of the presented work was to validate these theoretical calculations by using MPC determination and an in vitro dynamic system simulating a concentration–time profile of marbofloxacin in bovine plasma established in vivo, using field isolated Pasteurellaceae strains. Further, the one-dose regimen optimized based on AUC/MPC threshold was compared by PK simulations and PK/PD integration to the multiple-dose regimen optimized based on AUC/MIC threshold.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

In vivo concentration–time profile in ruminating calves following a single intramuscular injection of 10 mg/kg marbofloxacin

Animals.  Ten ruminant Prim’Holstein and Charolais calves (five males and five females) weighing between 235 and 355 kg were used. The animals were individually tethered in a large animal facility to which they had been acclimatized for about 1 week before study start. The calves were fed hay ad libitum and ground barleycorn supplemented with minerals. Water was available ad libitum.

Treatments, plasma sample collection and analysis.  Marbofloxacin was administered as a 16% solution for injection (Forcyl®; Vetoquinol) by the intramuscular route into the neck. After treatment, heparinized blood samples of approximately 7 mL were taken by puncture of the jugular vein, at the following time points: 0, 15, 30 and 45 min, 1, 1.5, 2, 3, 4, 6, 10, 24, 32, 48, 56, 72, 80 and 96 h. The blood samples were centrifuged at 1945 g for 10 min at 5 °C. The harvested plasma from each sample was divided into three aliquots (>1 mL) and stored in labelled polypropylene tubes at –75 °C until analysis. A high-performance liquid chromatography (HPLC) assay method for marbofloxacin as previously described (Schneider et al., 1996) was used with the following minor modifications. Before extracting 1 mL of plasma sample with dichloromethane, proteins were precipitated by the addition of 100 μL of acetonitrile and eliminated by centrifugation at 15875 g for 10 min. The detection method was switched from UV to fluorimetric detection with an excitation wavelength of 295 nm and an emission wavelength of 500 nm. The limit of quantification of the method was 0.002 μg/mL, and the linearity range was from 0.002 to 5 μg/mL. Intra-assay and interassay coefficients of variation were 3% and 3.5%, respectively, at a concentration of 0.004 μg/mL and 2.3% and 3.1%, respectively, at a concentration of 2.5 μg/mL.

Pharmacokinetic analysis.  The PK parameters were determined using noncompartmental analysis with WinNonlin software version 5.0.1 (Pharsight Corporation, St Louis, MO, USA). The area under the plasma concentration–time curve until 24 h after administration (AUC24 h) was calculated using the log-linear trapezoidal method from time 0–24 h. The maximum plasma concentration (Cmax) and the occurrence time of Cmax (Tmax) for each animal were taken directly from the concentration data. The rate constant associated with the slope of the terminal elimination phase (λz) was determined using linear regression. The terminal plasma half-life (T½λz) was calculated by the formula: T½λz = ln(2)/λz.

Marbofloxacin MIC and MPC determination for representative bovine respiratory disease pathogens

Strain selection.  Pathogenic strains were selected to provide a representation of the range of susceptibilities to marbofloxacin observed in the field. Three strains each of P. multocida and M. haemolytica with MIC ranging from 0.015 to 1 μg/mL were selected from the library of field isolates collected between 2002 and 2008 during natural outbreaks of bovine respiratory disease (Meunier et al., 2004; Kroemer et al., 2011). Thus, strains with either a high (MIC of 0.015–0.03 μg/mL), average (MIC of 0.12–0.25 μg/mL) or low (MIC of 1 μg/mL) susceptibility to marbofloxacin were included for each pathogen (Table 1). A resistant strain of M. haemolytica was also included (MIC of 8 μg/mL).

Table 1.   Marbofloxacin minimum inhibitory concentration (MIC) and classification of selected pathogens relative to recent European survey data
Susceptibility level Pasteurella multocida MIC (μg/mL) Mannheimia haemolytica MIC (μg/mL)
  1. *MIC epidemiology data according to Kroemer et al., 2011;. According to epidemiological cut-off value from CA-SFM and CLSI guidelines. The resistant strain of Mannheimia haemolytica (MIC = 8 μg/mL) was not included in mutant prevention concentration determination part of the experiments.

High
 MICmin 2002–2008*0.004 (n = 751)0.008 (n = 514)
 MIC50 overall (min–max) 2002–2008*0.015 (0.015–0.015)0.030 (0.30–0.030)
 Selected strain MIC0.0150.030
Average
 MIC90 overall (min–max) 2002–2008*0.120 (0.030–0.500)0.250 (0.120–0.500)
 Selected strain MIC0.030.250
Low
 Clinical breakpoint1.01.0
 Selected strain MIC1.01.0
Resistant
 Clinical breakpoint44.0
 Selected strain MICNA8.0

MIC and MPC determination.  The MIC of each of the six selected strains was verified using a broth microdilution method according to CLSI guideline (M31-A3 2006). The MPC is the first concentration of antibiotic drug able to eradicate a bioburden ≥1010 cfu/mL (Smith et al., 2003). Practically, it is determined by measuring the growth on plates containing a range of antibiotic concentrations with an inoculum covering the known mutation frequency: as the mutation frequency typically is between 10−7 and 10−9 (Wolfson & Hooper, 1985; Olliver et al., 2005; Van Bambeke et al., 2005), starting inoculums of 109 or 1010 colony-forming units are used (Dong et al., 1999; Ferran et al., 2007). The MPC is usually higher than the MIC because the bacterial population tested encompasses a larger tail of the MIC distribution and thus will depend on the shape and extent of that tail (Mouton et al., 2005). Simply, a MPC is an MIC determination with a large inoculum (Mouton et al., 2005). To determine the MPC, a method adapted from Blondeau et al. (2001) was used. Each strain was cultured to provide a bacterial suspension in sterile Mueller-Hinton broth (MHB) supplemented with 5% sterile horse serum (M. haemolytica strains only). Duplicate Petri dishes (150 mm) were inoculated with 100 μL of broth containing enough bacteria to provide a final bacterial concentration of at least 1010 cfu/mL in doubling dilutions of marbofloxacin (0.008–32 μg/mL). A noninoculated dish was included as a negative control, and an inoculated dish without marbofloxacin was included as a positive control. Dishes were then incubated at 35 ±2 °C for 24 h and eventually up to 72 h to obtain visible count bacteria for certain dishes. The MPC was reported as the lowest concentration preventing the growth of bacterial colonies and was expressed as a multiple of the MIC.

Dynamic in vitro PK/PD testing.  The pharmacokinetics and pharmacodynamics of 10 mg/kg marbofloxacin administered by the intramuscular route were simulated in sterile glass flasks using a push-pull draw-pull device maintained in a secure flow hood (Fig. 1). Flasks contained pooled sterile bovine plasma supplemented with 10% MHB and inoculated with 106–107 cfu/mL of one of each selected BRD pathogen. The inoculum size was intermediate between MIC and MPC inoculums sizes to be representative of the pathogenic bioburden in natural infections with a significant chance to observe mutant strains (Ferran et al., 2007, 2009, 2011). The flasks were maintained at 35 ± 2 °C, continuously agitated (both a sterile magnetic stirrer and a reciprocating horizontal agitator) and were fitted with a three-way cap to the push-pull draw-pull device (Z106 KsScientific). The ‘entrance’ port was used to add either untreated plasma or plasma spiked with 64 μg/mL marbofloxacin to provide a dynamic concentration over 24 h, which replicated the concentration–time kinetics observed in vivo. The ‘exit’ port was positioned to allow excess plasma to drain from the flask and maintain a constant volume (20 mL) of plasma. The third port was used to collect samples (1 mL) from the inoculated plasma before and at 0.5, 1, 4, 8, 12 and 24 h following the start of the simulation. A replicate of the experiment was performed in parallel with untreated serum only as a control for bacterial growth. The simulation was repeated with each strain as described in Table 1.

image

Figure 1.  PK/PD system reconstituting marbofloxacin exposure profile in cattle in supplemented bovine plasma containing the pathogenic strain. Up till Tmax, the incoming serum to the PK/PD flask is spiked with 64 μg/mL marbofloxacin and is blank for the control; after Tmax, both flasks receive blank serum.

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Sample analysis.  Each 1 mL sample was used to determine the bacterial count (2 × 100 μL aliquots) and to determine the marbofloxacin concentration (remaining aliquot filtered on 0.2 μm to remove bacteria).

The bacteria count was established both following marbofloxacin depletion (viable bacteria count) and in the presence of 4 μg/mL marbofloxacin (clinical resistant viable bacterial count). Two dilutions at 100 and 10 000 were prepared in sterile aqueous solution (NaCl, 0.9% and Tween 80, 0.02%). Each dilution was performed in duplicate, and 25 μL spot samples of the pure culture and the two dilutions were cultured on Mueller-Hinton agar dishes supplemented with 5% sheep serum, 1% active charcoal and 1% MgSO4 7H2O at 36 ± 2 °C, with 6% CO2 for 18–24 h (up to 72 h to obtain visible count bacteria for certain dishes). After incubation, colonies were counted (cfu/mL) and results were expressed as Log10 cfu/mL plasma with a limit of detection of 1.90 Log10 cfu/mL. The mean Log10 cfu/mL plasma was calculated for both the viable and resistant viable counts. The percentage of surviving bacteria (PBS) was calculated as the surviving bacterial population (cfu/mL) detected at each time of sampling divided by the initial bacterial population (cfu/mL). (Pellet et al., 2006). Bacteriostatic and bactericidal activities are defined as an inoculum reduction of at least 1.0% and 99.9%, respectively in the value of the percentage of surviving bacteria (PSB).

Marbofloxacin concentrations were determined by HPLC, as described earlier.

Pharmacokinetic/pharmacodynamic integration.  The marbofloxacin concentration data obtained for each bacteria strain were analysed separately using WinNonlin version 5.0.1 (Pharsight Corporation, Mountain View, CA, USA). Marbofloxacin pharmacokinetic parameters obtained in vivo were used to estimate and validate the data obtained for each in vitro PK/PD testing.

The AUC24 h was obtained for each PK/PD test by noncompartmental analysis. The AUC24 h/MIC, AUC24 h/MPC and Cmax/MIC were calculated. The T>MPC (the time when concentrations are above the MPC), TMSW (the time that the concentrations are within the MSW) and the ratio T>MPC/TMSW were also determined over 24 h. As the level of plasma protein binding of marbofloxacin is low in cattle, approximately 30% (Ismail & El-Kattan, 2007), the PK/PD indices were determined from total plasma concentrations. To compare the one-dose regimen (10 mg/kg once) to the multiple-dose regimen (2 mg/kg per day for 3 days), the mean in vivo PK parameters were used to simulate mean profiles. For the one-dose regimen, the mean parameters were determined in the present study, and for the multiple-dose regimen, published parameters were used (Aliabadi & Lees, 2002). The same PK/PD indices were calculated from 0 to 72 h.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

Confirmation of the in vivo concentration–time profile in ruminating calves following a single intramuscular injection of 10 mg/kg marbofloxacin

The observed (total) plasma concentrations of marbofloxacin following single intramuscular dose of 10 mg/kg in ruminant cattle are shown in Fig. 2. The values for marbofloxacin pharmacokinetic parameters are shown in Table 2.

image

Figure 2.  Individual and mean concentration–time profiles of marbofloxacin in ruminant calves after a single intramuscular injection of a 16% solution at a dose of 10 mg/kg.

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Table 2.   Mean plasma pharmacokinetic parameters of marbofloxacin in ruminant calves after a single intramuscular injection of a 16% solution at a dose of 10 mg/kg (noncompartmental modelling)
ParameterMeanSD
  1. Tlag, lag time before absorption; λz, elimination rate constant calculated by linear regression with the last measured concentration–time points; T½λz, elimination half-life; Tmax, observed occurrence time of the maximum plasma concentration; Cmax, observed maximum plasma concentration; AUClast, area under the concentration–time curve until the last measurable time point; AUCINF, area under the concentration–time curve extrapolated to infinity; AUC24 h, area under the concentration–time curve extrapolated to 24 h; MRTlast, mean residence time until the last measurable time point; MRTINF, mean residence time extrapolated to infinity; SD, standard deviation.

T lag (h)0.030.05
λz (per h)0.0400.0054
T ½λz (h)17.502.49
T max (h)1.281.04
C max (μg/mL)7.922.24
AUC24 h (μg·h/mL)50.6911.55
AUClast (μg·h/mL)52.5812.35
AUCINF (μg·h/mL)52.6612.39
MRTlast (h)6.431.19
MRTINF (h)6.591.21

After administration of the intramuscular dose of 10 mg/kg, the absorption was quite fast with a mean observed occurrence time of the maximum plasma concentration (Tmax) of 1.28 h (SD = 1.04 h). The mean observed maximum plasma concentration (Cmax) was 7.915 μg/mL (SD = 2.242 μg/mL). The mean terminal elimination half-life, T½λz, was 17.50 h (SD = 2.49 h). The mean area under the concentration–time curve until 24 h after administration, AUC24 h, was 50.69 μg·h/mL (SD = 11.55 μg·h/mL). The mean area under the concentration–time curve extrapolated to infinity, AUCINF, was 52.66 μg·h/mL (SD = 12.39 μg·h/mL). Other parameters are presented in Table 1. Both dose-dependent parameters, Cmax and AUC, are about five times higher than the parameters obtained at a dose of 2 mg/kg (Aliabadi & Lees, 2002). Subsequently, it can be assumed that the kinetics of marbofloxacin are proportional to the dose within a range of 2–10 mg/kg.

The Determination of MIC and MPC of representative bovine respiratory disease pathogens

The marbofloxacin MICs and MPCs for the panel of selected pathogens are presented in Table 3. The MPC values were higher than the MIC values for all strains tested. The ratio of MPC/MIC remained between two and four, with only the highly susceptible strain of M. haemolytica having the higher ratio of four.

Table 3.   Marbofloxacin minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) determination against Mannheimia haemolytica and Pasteurella multocida selected strains
StrainMarbofloxacin MIC (μg/mL)Marbofloxacin MPC (μg/mL)Ratio MPC/MICInoculum size cfu/mL
M. haemolytica 0.030.1241.38 1010
M. haemolytica 0.250.521.18 1010
M. haemolytica 1221.18 1010
P. multocida 0.0150.0324.30 1010
P. multocida 0.030.0621.48 1010
P. multocida 1228.40 1010

Dynamic in vitro PK/PD testing

System validation.  Viable bacteria counts confirmed an inoculum size of between 6.06 and 7.30 Log10 cfu/mL before the introduction of marbofloxacin into the PK/PD incubator, indicating adequate inoculation to allow mutant detection. Increasing viable counts were obtained in the parallel experiment without marbofloxacin confirming that the effect of dilution on the bacterial media during the first hour was minimal. Increasing viable and resistant viable counts were obtained for the marbofloxacin-resistant strain of M. haemolytica (MIC = 8 μg/mL) confirming that resistant mutants could multiply in the model system. The marbofloxacin pharmacokinetic plasma profile was reliably repeated in all the experiments (data not shown). The AUC24 h calculated for each run was between 46.0 and 55.7 μg/mL, which is representative of the value obtained in vivo (Table 2). Appropriate Cmax (between 7.1 and 9.8 μg/mL) and Tmax (1 h) representative of the in vivo situation were also consistently achieved (data not shown). These data collectively confirm that the test system provides an in vitro representation of the in vivo situation.

In vitrodynamic bactericidal killing curves and PSB.  The in vitro dynamic killing curves against the M. haemolytica and P. multocida selected strains are presented in Fig. 3. for M. haemolytica strains and Fig. 4. for P. multocida strains. The killing profile of marbofloxacin was concentration dependant, such that the bacteria with the lowest MICs were killed more rapidly than those with higher MICs. Bactericidal activity against both M. haemolytica and P. multocida was observed by the 0.5 or 1 h time point for most strains except for the P. multocida strain with the lowest susceptibility to marbofloxacin (MIC = 1 μg/mL) for which it was observed at the next sampling time (4 h). By 24 h following the start of the marbofloxacin treatment, the PSB was <0.01% for all but one of the selected pathogens (Table 4). Bacterial regrowth did occur after the 12-h time point for the M. haemolytica strain with marbofloxacin MIC of 1 μg/mL, although, for this strain, the PSB was <0.001% at 8 h following marbofloxacin treatment. Further, no resistant mutants (MIC ≥4 μg/mL) were detected during the resistant viable bacterial count at any sampling time confirming that the regrowth was not because of the development of resistance. No killing was observed for the clinically resistant M. haemolytica tested.

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Figure 3.  Total bacterial counts in each in vitro dynamic test (Log10 cfu/mL) and mean marbofloxacin pharmacokinetic profile obtained in the PK/PD experiment with strains of Mannheimia haemolytica with varying minimum inhibitory concentration (0.03, 0.25, 1 and 8 μg/mL).

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image

Figure 4.  Total bacterial counts in each in vitro dynamic test (Log10 cfu/mL) and mean marbofloxacin pharmacokinetic profile obtained in the PK/PD experiment with strains of Pasteurella multocida with varying minimum inhibitory concentration (0.015, 0.03 and 1 μg/mL).

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Table 4.   Percentage of surviving bacteria after the simulation of a single 10 mg/kg intramuscular administration in bovine plasma supplemented with Mueller-Hinton broth (90/10 v/v), against Mannheimia haemolytica and Pasteurella multocida field strains
Bacteria Mannheimia haemolytica Pasteurella multocida
MIC (μg/mL)0.030.25180.0150.031
Time (h)PSB: Percentage of surviving bacteria (%)
  1. MIC, minimum inhibitory concentration.

0100100100100100100100
0.5<0.0021.2701.89710.51<0.0020.56636
1<0.0020.0120.0986.159<0.0020.2581.946
4<0.002<0.0060.014>100<0.0020.0710.013
8<0.002<0.0060.007>100<0.002<0.0020.001
12<0.002<0.006<0.007>100<0.002<0.002<0.001
240.004<0.006>100>100<0.002<0.002<0.001

PK/PD integration.  The PK/PD parameters obtained by integrating the in vitro PK/PD testing data and in vitro MIC values or MPC values for the test strains are presented in Table 5. As a similar pharmacokinetic profile was reconstituted for each PK/PD test, the PK/PD parameters were mainly dependent upon the pharmacodynamic data of each pathogen tested. For strains of P. multocida and M. haemolytica of low or average susceptibility (Table 1), PK/PD surrogates for treatment efficacy AUC24 h/MIC and Cmax/MIC exceeded or were within the target of 125–250 h for AUC24 h/MIC and 8–10 for Cmax/MIC (Schentag, 1999; Toutain et al., 2002; Sidhu et al., 2011). For strains with a MIC corresponding to the susceptibility breakpoint (1 μg/mL), only the Cmax/MIC target ratio was obtained. For PK/PD surrogates of risk of emergence of resistance, the target of 20–70 h for AUC24 h/MPC (Zhao & Drlica, 2008) and the target of T>MPC/TMSW ratio superior to 0.5–1 (Kesteman et al., 2009) were also achieved for all clinically susceptible pathogens (MIC ≤1 μg/mL).

Table 5.   PK/PD indices of antimicrobial efficacy and risk of resistance selection amongst the respiratory pathogenic strains following in vitro testing of a single i.m. administration of marbofloxacin at 10 mg/kg
StrainMIC (μg/mL)MPC (μg/mL)AUC24 h/MIC (h)AUC24 h/MPC (h) C max/MIC T >MPC (%) T MSW (%) T >MPC/TMSW
  1. AUC, area under the curve; MIC, minimum inhibitory concentration; MPC, mutant prevention concentration.

M. haemolytica 0.030.121546387264.21000Not calculable
0.250.522311132.683810.38
1254279.836261.38
P. multocida 0.0150.0330631532470.21000Not calculable
0.030.061646823262.61000Not calculable
1254278.137.5291.28

Table 6 summarizes the comparison between the PK and PK/PD simulations in WinNonlin, obtained following the single-dose 10 mg/kg administration or a multiple-dosing regimen at 2 mg/kg every 24 h for 3 days (Aliabadi & Lees, 2002), matched to Pasteurellaceae of varying susceptibility (Fig. 5.). For pathogens of high susceptibility, the multiple-dose regimen achieved high surrogate AUC72 h/MIC or AUC72 h/MPC with more time above the MPC than the single-dose protocol that translated in a higher T>MPC/TMSW ratio (1.92 for single dose compared with 2.17 for multiple doses, both largely superior to one). The fractionated dosage regimen achieved plasma concentrations above the MSW after each administration for pathogens of high susceptibility (Fig. 5.). However, the very high exposure after the single-dose regimen is very likely to kill all target pathogens including potential first-step mutants (T>MPC>23 h). For pathogens of average susceptibility and in particular for pathogens of low susceptibility, the single-dose protocol achieved higher T>MPC/TMSW ratio than the multiple-dose regimen of, respectively, 3.83 and 2.08 compared with 2.17 and 0.0. The 2 mg/kg dosing regimen Cmax of 1.5 μg/mL indicates that such a dosing regimen would not achieve concentrations greater than the MPC for a Pasteurellaceae strain with an MIC of 1 μg/mL and an MPC of 2 μg/mL.

Table 6.   PK/PD indices of antimicrobial efficacy and risk of resistance selection amongst Mannheimia haemolytica of various mean or minimum inhibitory concentration (MIC) over the complete dosing interval following PK simulations of a single i.m. administration of marbofloxacin at 10 mg/kg or multiple doses of 2 mg/kg administered every 24 h
Dosing regimenAUC24 h/AUC72 hMIC (μg/mL)MPC (μg/mL)AUC72 h/MIC (h)AUC72 h/MPC (h) T >MPC 72 h (%) T MSW 72 h (%) T >MPC/TMSW
  1. AUC, area under the curve; MPC, mutant prevention concentration.

10 mg/kg one dose42.9 μg·h/mL/24 h, 44.1 μg·h/mL/72 h0.030.12147036832.516.91.92
0.250.51768820.75.43.83
1244.2210.45.02.08
2 mg/kg multiple dosing9.85 μg·h/mL/24 h, 30.0 μg·h/mL/72 h0.030.12100025068.531.52.17
0.250.51206031.418.11.73
123015013.30
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Figure 5.  Simulation of mean PK profiles after one-dose 10 mg/kg marbofloxacin and classical multiple-dose regimen of 2 mg/kg (Aliabadi & Lees, 2002), presented with the Pasteurellaceae minimum inhibitory concentration (MIC90) (Meunier et al., 2004 and Kroemer et al., 2011) and the corresponding mutant prevention concentration equal to twice the MIC showing the putative mutant selection window.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References

In these experiments, we found that a single dose of 10 mg/kg marbofloxacin was effective against representative field clinical susceptible isolates of BRD. The bacterial respiratory pathogens (M. haemolytica and P. multocida) were obtained from field outbreaks of BRD. Bacterial bovine respiratory pathogens with a marbofloxacin MIC ≤1 μg/mL were all rapidly killed when exposed to the concentration–time curve obtained in vivo following the administration of 10 mg/mL, with no emergence of resistance. A range of susceptibility to marbofloxacin (high, average and low) was chosen to evaluate the different scenarios and cattle, which may be encountered in the field situation. Pathogens with a high susceptibility (MIC ≤0.03 μg/mL) considered as wild type are representative of the MIC50 observed in field surveys conducted between 2002 and 2008, whilst the pathogens of average susceptibility (MIC ≤0.25 μg/mL) were representative of the MIC90 in the same surveys (Meunier et al., 2004; Kroemer et al., 2011). Thus, the pathogens of high susceptibility represent half the infected cattle population, whilst the pathogens of average susceptibility represent 90% of the cattle population with BRD. Essentially, these pathogens of high and average susceptibility collectively represent the herd situation. The pathogens of low susceptibility (MIC ≤1 μg/mL), corresponding to the clinical susceptibility breakpoint for marbofloxacin, represent the worst-case scenario for the remaining diseased cattle that are likely individual BRD cases. Nonsusceptible bacteria (MIC > 1 μg/mL) are likely to lead to therapeutic treatment failure.

The MPC for the same field isolates was also determined. The MPC/MIC ratio for bovine respiratory Pasteurellaceae (2–4) was much lower than the MPC/MIC ratio for Escherichia coli and Klebsiella pneumoniae of eight or more (Ferran et al., 2007; Kesteman et al., 2009). Clearly, the MPC/MIC ratio is a feature dependent upon the bacteria strain/antibiotic couple and associated with the genetic drug target inducing mutation (Blondeau et al., 2001). Therefore, it is possible that the frequency of mutation to marbofloxacin in Enterobacteriaceae may be higher than in bovine respiratory pathogenic Pasteurellaceae (Wolfson & Hooper, 1985). Interestingly, for bovine Pasteurellaceae pathogens, the marbofloxacin MPC and the MBC values are the same (Aliabadi & Lees, 2002; Sidhu et al., 2011). This very low MPC/MIC ratio of marbofloxacin against bovine respiratory Pasteurellaceae is an ideal situation for preventing first-step mutation.

In the spirit of the 3 Rs (refine, reduce and replace), the system presented here provides an in vitro ethical alternative to previously used in vivo model systems such as the tissue cage (Aliabadi & Lees, 2002; Sidhu et al., 2011). In contrast to tissue cages, this system allows the dynamic testing of the antimicrobial–bacteria interaction, without the interference of other innate antimicrobial mechanisms such as the immune system or inflammation. Further, optimal conditions can be set for maximal bacterial growth (worst-case scenario), and fairly large volumes of sample can be collected. The dynamic test system gives the flexibility to test for a wide range of dosing regimens and bacterial strains (Gebru et al., 2011). Whilst this system allows the testing of different bacterial field strains, it does not replace in vivo field trials that still must be conducted to confirm the clinical relevance efficacy and safety of the selected dose or dose range.

In the dynamic experiment that tested the M. haemolytica of low susceptibility (MIC = 1 μg/mL), whilst the bacteria count fell below the limit of quantification at the 8- and 12-h time points with <0.01% of surviving bacteria, the strain grew back between the 12- and the 24-h time point. The marbofloxacin concentration was above the MPC until the 8-h time point and above the MIC until the 12-h time point. As no resistance emergence was observed, the post-12-h bacterial regrowth is unlikely to be due to the selection of a resistance mechanism. Rather, it is probable that sufficient bacteria survived the high marbofloxacin concentration to resume exponential growth when concentrations had fallen to below the MIC. Indeed <8 h of exponential growth would allow a few remaining bacteria cells to reach a 108 cfu/mL population. In vivo, the innate and adaptive immune system of the lung would likely clear those few bacteria (Ackermann et al., 2010). Although this has not been investigated, it could be that for the surviving pathogenic bacteria, called ‘persister cells’, an ‘Eagle effect’ was observed. The persister cells are tolerant but not resistant to the antibiotic (Eagle & Musselman, 1948; Lewis, 2010).

As we validated the in vitro system for 24-h tests only, we also modelled the current multiple-dose regimen of marbofloxacin to compare with the single-dose regimen tested in vitro. Comparing a single 2 mg/kg dose with a single 10 mg/kg dose in the in vitro dynamic system would have brought little new information on the pharmacodynamics of marbofloxacin. The PK/PD surrogates for a multiple-dose regimen of 2 mg/kg marbofloxacin daily for 3 days were compared with those of the single 10 mg/kg dose over the 72-h treatment duration. The PK parameters used for the simulation of the multiple-dose regimen were as published by Aliabadi & Lees, 2002, although the PK parameters presented here following 10 mg/kg would also have been acceptable because exposure to marbofloxacin is proportional to the dose in that dose range. The simulation was performed over only 3 days because this is the typical duration of treatment reported with marbofloxacin. Further, as there is no accumulation of marbofloxacin with the multiple-dose regimen, there would have been no difference between the T>MPC and TMSW indices if the simulation was conducted over a 5-day treatment period because these parameters are calculated as a percentage of time rather than absolute time in hours.

The AUC in the PK simulations are numerically lower than the mean AUC measured in the PK studies. Therefore, the PK/PD indices obtained by integration of those mean profiles represent a worst-case scenario. For the highly sensitive pathogens, the multiple-dose regimen achieved high surrogate AUC72 h/MIC or AUC72 h/MPC with even more time above the MPC than the single-dose protocol, but split over several days after each administration. This confirms the results of the previous PK/PD experiments in calves, which concluded that the multiple-dose regimen of marbofloxacin at 2 mg/kg was optimized for pathogen eradication (Aliabadi & Lees, 2002; Sidhu et al., 2011). Whilst the multiple-dose regimen may be optimized for highly sensitive pathogens, in contrast, the single-dose protocol achieved higher T>MPC/TMSW ratio for the pathogens of average susceptibility and in particular for pathogens of low susceptibility. Further, with Cmax in the region of 1.5 μg/mL, the multiple-dose regimen could only achieve concentrations greater than the MPC for a Pasteurellaceae strain of low susceptibility for a very short time after each dosing. As pathogens of average and low susceptibility are typically those encountered in second intention, they are also potentially those of greatest resistance risk. Given that the clinical susceptibility breakpoint of 1 μg/mL corresponds to the MIC level of Pasteurellaceae with one or two mechanisms of resistance (Cardenas et al., 2001; Meunier et al., 2004), this means that the single high-dose regimen is more likely to prevent the emergence of resistance amongst the bovine respiratory pathogens, whilst it is still clinically effective.

Interestingly, whilst working with similar in vitro systems and with experimental infections in rodents, others have come to different conclusions regarding marbofloxacin dosing regimens as a single or fractionated dose (Ferran et al., 2007; Kesteman et al., 2009). In comparison with the work presented here, the main differences are because of the interspecies pharmacokinetic differences of marbofloxacin and the MPC/MIC ratio of the bacteria. In rats and mice, marbofloxacin is eliminated very rapidly and although the dose was adjusted in an attempt to reach comparable AUC levels, the slope of the elimination curve remained different as did the time spent over the MPC. Whilst a P. multocida strain was used in later experiments (Ferran et al., 2011), much of their infectious model work used a K. pneumoniae strain relevant for human respiratory infections, for which the MPC was eight times the MIC contrasting with the ratio of two observed for most of the bovine Pasteurellaceae tested here. However, their conclusions that marbofloxacin dose and rapidity of treatment are the main predictors for treatment success, and reduction of resistance emergence is complimented by our results that show an increasing percentage of surviving bacteria with increasing MIC levels. As no resistance emergence was observed in any of our in vitro experiments, the extent of emergence could not be compared between strains of various MICs.

The single-dose regimen described here is optimized for clinical efficacy and to decrease the likelihood of resistance emergence amongst the bovine respiratory pathogens. To complete the picture in terms of broader veterinary and public health, the risk of resistance development on zoonotic and commensal flora especially in the gut should be determined. More studies are required to further understand the effect of veterinary treatments on natural or implanted gut flora, and ways to reduce that impact via dose optimization (Kesteman et al., 2010; Mann et al., 2011). Nevertheless, we can assume that commensal flora is less exposed after a one-dose regimen than after a multiple-dose regimen (exposition being a function of overall dose and time).

In conclusion, the proposed dose of a single administration of 10 mg/kg marbofloxacin for the treatment of BRD was based on the concept that reaching in vivo concentrations above the MPC and reducing the time in the MSW will limit the risk of emergence of resistance. We have studied this dosing regimen in vitro and by PK/PD integration. We demonstrated that clinically susceptible bacterial respiratory pathogens were all rapidly killed when exposed to the concentration–time curve obtained in vivo following the administration of 10 mg/kg, with no emergence of resistance, confirming the single-injection short-acting antibiotic (SISAAB) for the treatment of BRD. The field efficacy and safety of this dosing regimen have also been confirmed in a field trial (Grandemange, E., Fournel, S., Giboin, H. & Woehrle, F., personnel communication).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. References
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