Pharmacokinetics, pharmacodynamics and PK/PD integration
Integration of in vitro generated potency estimates with in vivo PK data has been used extensively to generate three indices to predict clinical outcome, namely the ratios, Cmax/MIC and AUC24h/MIC and T > MIC, the time for which concentration exceeds MIC. Integration of pharmacokinetic and pharmacodynamic data for MPC are presented in the Additional file 1. All MPC ratios were much lower than the MIC ratios. From previous marbofloxacin studies, Cmax/MIC and AUC24h/MIC ratios provided good correlation with bacteriological cure in human patients [30, 31]. For fluoroquinolones used in veterinary medicine, a Cmax/MIC of 8–10 and an AUC24h/MIC greater than 100–125 h have been proposed . However, other studies have suggested that a ratio of AUC24h/MIC of 35–50 is effective for Gram-positive bacteria . Many authors have proposed achieving numerical values of AUC24h/MIC of 125:1 or 250:1 for gram negative organisms, corresponding to average concentrations over the dosing interval of 5.2 to 10.4, respectively, as a multiple of MIC.
For marbofloxacin, de Jong et al.  reported identical MIC50 values for both A. pleuropneumoniae and P. multocida of European pig origin of 0.03 μg/mL and identical MIC90 values of 0.06 μg/mL. Schneider et al.  reported on marbofloxacin PK in healthy pigs, aged 12, 16 and 27 weeks, administered intramuscularly at three dose rates of 4, 8 and 16 mg/kg. PK/PD integration of data from these studies is presented in a Additional file 1 to this paper. Briefly, for both bacterial species and pigs aged 27 weeks, Cmax/MIC90 ratios were 56, 105 and 258, respectively, for marbofloxacin doses of 4, 8 and 16 mg/kg. Even average concentrations over the 92 h period after dosing provided Caverage/MIC90 ratios of 9.6, 19.7 and 39.1 for these dose rates. Therefore, a preliminary prediction of likely successful clinical outcome for doses of marbofloxacin in the range 4–16 mg/kg can be made for pig pneumonia caused by the pathogens, A. pleuropneumoniae and P. multocida.
PK/PD modelling and breakpoint determination
PK/PD integration is not a precise tool; it should be regarded as a first initial step in predicting efficacy in clinical use. It is especially useful when correlated with outcome in clinical trials. However, the next essential step is to define PK/PD breakpoints for each antimicrobial drug acting against representative isolates of each pathogenic species. PK/PD modelling describes the whole sweep of the concentration-effect relationship. Therefore, any pre-determined level of activity, ranging from bacteriostasis to virtual eradication, indicated by the breakpoint AUC24h/MIC index, can be determined. Applying PK/PD breakpoints for indices such as AUC24h/MIC, derived from PK/PD modelling, with MCS provides an approach to dose prediction, which takes account of animal species based PK, wild-type MIC distributions, protein binding and breakpoints for specific bacterial species. From such time-kill studies, numerical values of PK/PD breakpoints have been determined by PK/PD modelling by previous workers [7–10, 12, 13, 21, 25, 33].
In this study, breakpoint values for each level of growth inhibition, 0log10, 3log10 and 4log10 reductions in count, were broadly similar for the two growth matrices. This is not unexpected because, although MICs in broth and serum differed, the breakpoint values are based on MIC multiples. AUC24h/MIC ratios were similar for broth and serum for each level of kill, being based on MIC values separately for each matrix. Moreover, inter-isolate variability in PK/PD breakpoint values was small to moderate.
PK/PD breakpoints were used with wild type MIC distributions of susceptible pathogens and literature PK data, to conduct MCS to predict doses providing a range of pre-determined levels of kill. The deterministic approach provided an estimate of once daily doses at steady state. It is based on MIC90 for each pathogen and average values for other variables. It provides an initial indication of likely effective dosage, but does not take account either of variability or incidence of each input variable and in this study. Predicted daily doses were less than 0.5 mg/kg for a bactericidal kill against both pathogens. Nevertheless, the deterministic approach comprises an initial indication, prior to estimation of population doses for each selected TAR. The latter is a dose encompassing a given percentile of the target population, for example, 50 or 90% and for three pre-determined levels of bacterial kill and for both a single dose and a daily dose at steady state. Monte Carlo simulations predict doses which allow for incidence within MIC distributions and encompass best, worst and all intermediate values for distributions of Cl/F and breakpoint AUC24h/MIC ratios. Furthermore, basing potency estimates on serum as a growth matrix, as in this study, has greater relevance to disease conditions than MICs determined in broths. Nevertheless, it is recognised that serum, although preferred to broth for MIC determination, is similar but not identical in composition to the biophase at infection sites, for example pulmonary epithelial lining fluid.
As discussed by Martinez et al.  it is the exposure achieved after the first dose, which is most relevant in determining therapeutic outcome. In this study, low marbofloxacin doses were predicted for a greater than bactericidal action, with 90% TAR in both species; for 72 h and a 4log10 reduction in count, the predicted doses were 2.08 and 1.14 mg/kg, respectively, for P. multocida and A. pleuropneumoniae. Both dosages are less than the dosages of 4, 8 and 16 mg/kg studied by Schneider et al. . To achieve a bactericidal action (3log10 reduction in count) for P. multocida for 90% TAR, once daily doses at steady state were even lower, 0.43 mg/kg for P. multocida and for A. pleuropneumoniae 0.29 mg/kg for pigs aged 27 weeks.
These predicted doses are lower than those of 2.5 mg/kg and 8 mg/kg investigated by Ding et al.  and Ferran et al.  as well as the 2 mg/kg recommended dose for several licensed marbofloxacin products. Ferren et al.  suggested that even lower doses of marbofloxacin could potentially eradicate low counts (105 CFU/mL) in the lung, while having a minimal impact on the microbiota of the large intestine. On the other hand, Vallé et al.  validated for the bovine pneumonia pathogens, P. multocida and Mannheimia haemolytica, the concept of a single high dose of marbofloxacin compared to a daily dose of 2 mg/kg for 3–5 days. A bactericidal effect against bovine P. multocida was achieved within one hour, when marbofloxacin was administered at five times the recommended daily dose (10 mg/kg).
The present study illustrates the principles of using MCS to predict dosages of marbofloxacin for the treatment of pneumonia in the young pig. The proposed dosage regimen is for A. pleuropneumoniae and P.multocida induced pneumonias only. For other organisms, independent PK/PD studies will be required. However, in future studies it will be important to extend the present findings for A. pleuropneumoniae and P.multocida also. Whilst the inter-isolate variability in PK/PD breakpoint values for bacteriostatic and bactericidal levels of kill was small in the present study, estimates were based on only six isolates for each species. Moreover, the time-kill studies generating the PK/PD breakpoints used fixed drug concentrations (eight multiples of MIC) for a fixed time period. In clinical use, on the other hand, plasma drug concentrations first increase and then decrease after intramuscular dosing, exposing organisms to a continuously variable concentration. A third consideration is the relatively small number of isolates in the report of de Jong et al. .
In future studies, these concerns could be addressed by increasing numbers of isolates in field distribution studies and in PK/PD breakpoint estimation studies. Moreover, exposure of organisms to varying drug concentrations could be addressed by use for example of hollow fibre methods to simulate in vivo patterns of change in concentration with time. Furthermore, marbofloxacin PK data were used as mean and standard deviation values from the literature. In future studies, it will be useful to incorporate individual animal PK data in the MCSs, and, in particular it will be helpful to use population PK data obtained in clinically ill pigs. Finally, the methodology in this study did not consider the contribution to pathogen elimination by the body’s natural defence mechanisms, which are known to be important in immune competent clinical subjects. In addition, potentially beneficial properties of antimicrobial drugs, such as immunomodulatory and anti-inflammatory actions are of importance for some drug classes. Finally, dose prediction studies, as reported in this manuscript, should always be correlated with clinical and bacteriological outcomes in animal disease models and clinical trials [7, 11–13].