PK-PD Modeling and Optimal Dosing Regimen of Acetylkitasamycin against Streptococcus suis in Piglets

Streptococcus suis (S. suis) causes severe respiratory diseases in pigs and is also an important pathogen causing hidden dangers to public health and safety. Acetylkitasamycin is a new macrolide agent that has shown good activity to Gram-positive cocci such as Streptococcus. The purpose of this study was to perform pharmacokinetic–pharmacodynamic (PK-PD) modeling to formulate a dosing regimen of acetylkitasamycin for treatment of S. suis and to decrease the emergence of acetylkitasamycin-resistant S. suis. The minimal inhibitory concentration (MIC) of 110 S. suis isolates was determined by broth micro dilution method. The MIC50 of the 55 sensitive S. suis isolates was 1.21 μg/mL. The strain HB1607 with MIC close to MIC50 and high pathogenicity was used for the PK-PD experiments. The MIC and MBC of HB1607 in both MH broth and pulmonary epithelial lining fluid (PELF) was 1 and 2 μg/mL, respectively. The liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was used to determine the concentration change of acetylkitasamycin in piglet plasma and PELF after intragastric administration of a single dose of 50 mg/kg b.w. acetylkitasamycin. The PK parameters were calculated by WinNolin software. The PK data showed that the maximum concentration (Cmax), peak time (Tmax), and area under the concentration–time curve (AUC) were 9.84 ± 0.39 μg/mL, 4.27 ± 0.19 h and 248.58 ± 21.17 h·μg/mL, respectively. Integration of the in vivo PK data and ex vivo PD data, an inhibition sigmoid Emax equation was established. The dosing regimen of acetylkitasamycin for the treatment S. suis infection established as 33.12 mg/kg b.w. every 12 h for 3 days. This study provided a reasonable dosing regimen for a new drug used in clinical treatment, which can effectively be used to treat S. suis infection and slow down the generation of drug resistance.


Introduction
Streptococcus suis (S. suis) is an important pathogen in swine, and can cause serious respiratory disease, resulting in great economic losses to the swine industry worldwide each year [1]. As a zoonotic pathogen S. suis can even cause human death [2]. Every year, a large number of antibiotics, including macrolides, are used for the treatment of respiratory disease. However, drug resistance has emerged as a result of the unreasonable use of drugs [3].
Macrolides are widely used in veterinary medicine to prevent and treat respiratory diseases and necrotic enteritis [4]. Acetylkitasamycin is a product of kitasamycin acetylation [5]. It has a similar spectrum of antibiotic activity to kitasamycin, but has superior The minimal inhibitory concentration (MIC) of acetylkitasamycin and its five main components against S. suis strains were determined in MH broth and PELF by broth micro dilution method according to the CLSI 2007 [26,27]. The S. suis ATCC 49619 and E. coli ATCC 25922 strains were used as the quality control strain for antibiotic susceptibility determination. According to the MIC 50 values of sensitive strains, an S. suis HB1607 strain with its MIC similar with MIC 50 , was used for PD study of acetylkitasamycin.
The supernatant was sucked up from the MIC determination wells to the TSA in order to determine the minimal bactericidal concentration (MBC) [28]. The agar dilution method was used to determine the mutant prevention concentration (MPC) of acetylkitasamycin [28]. The 10 10 CFU/mL bacterial were inoculated on the agar plates containing continuous concentrations of acetylkitasamycin (1MIC, 2MIC, 4MIC, 8MIC, 16MIC, 32MIC) and cultured at 37 • C for 72 h, and the lowest concentration without bacterial growth was MPC.
Post-antibiotic effect (PAE) was estimated by incubating bacteria with drug for a period of time and then removal of drug [29]. First, the bacteria were incubated with 1MIC, 2MIC, 4MIC of drugs for 1 and 2 h; second, the drugs were removed by washing with new medium; third, 100 µL new incubation was sucked up at different time points and counted by plating on TSA. Then the recovery growth kinetic curves were established for computing the PAE. The PAE was calculated as follows: PAE = T − C, where T is the time required for viable counts of bacteria to increase by 1 − log10 CFU in drug removal phase, respectively; C is the time for untreated control.

Animal Experiment and Sample Collection for PK Study
Acetylkitasamycin was administrated in six piglets with a single dose of 50 mg/kg b.w. by intragastric administration. After administration, 5 mL blood samples were collected through the anterior vena cava at 0. 5,1,2,4,6,8,12,24,36,48,72 and 96 h. Plasma was separated from blood by centrifugation at 3500 rpm for 10 min.

Assay of Acetylkitasamycin and Kitasamycin Every Component in Plasma and PELF
The urea dilution method was used to determine the volume of PELF, as described previously [33]. To determine the urea concentration in lavage fluid samples, a biochemical analyzer machine (SYNCHRON CX4 PRO) was used. Estimation of the volume of PELF was done by the urea dilution method. The final concentration of acetylkitasamycin in PELF (C PELF ) was derived from the following equation: Quantitation of acetylkitasamycin and kitasamycin in every component of piglet plasma and PELF was conducted using the sensitive and selective high-performance liquid chromatographic mass spectrometry (LC-MS/MS) method [34]. The plasma specimens (0.25 mL) and BAL specimens (0.25 mL) were thawed and added to a 10 mL centrifuge tube with 0.75 mL acetonitrile, and vortexed for 3 min (12,000 r/10 min); 1 mL ethylacetate was added to the supernatant and vortexed for 3 min (12,000 r/10 min), and then the supernatant was taken out. The above operation process was repeated. The two supernatants were merged and blown dry with nitrogen. Then the residue was reconstituted in 0.25 mL solution (0.1% Formic acid water (60): Acetonitrile (40)).
All PK parameters of plasma and PELF were performed using WinNonlin software (version 5.2.1, Pharsight Corporation, Mountain View, CA, USA). Each piglet's drug concentrations were depicted on semilogarithmic graphs to choose appropriate PK compartmental models.
Taking into account that acetylkitasamycin has several components, the weight coefficient was introduced in this study. Weight coefficient (w j ) can be customized for each component AUC 0-∞j in consideration of the total AUC 0-∞t ratio. Every monomer composition of acetylkitasamycin in the concentration of PELF was given their own weight coefficient, and then the total concentration was calculated (C T ).

PK-PD Integration and Modeling
All the parameters were calculated using WinNonlin 5.2 software. The surrogate parameters (C max /MIC, AUC 24h /MIC, T > MIC) of PELF were determined after intragastric administration of acetylkitasamycin, used for in established vitro MIC and in vivo PK relationships.
The inhibitory sigmoid E max model was used to establish the relationship between ex vivo AUC 24h /MIC ratio and the bacteria decrease in the PELF of piglets [16]. The model formula can be described as follows: . E is the summary PD endpoint, and E 0 is the effect representing the value of the PD endpoint without drug treatment (i.e., the value of the summary endpoint when the PK-PD index is 0). C is one of the three PK-PD indices, as defined above, and PD max is the maximum effect (in relation to E 0 ) indicated by the plateau where further exposure does not result in further killing. EC 50 is the magnitude of C that is needed to achieve 50% of E max − E 0 , and N is the sigmoidicity factor. The PD target under different efficiencies (E = 0, −3 and −4 (bacteriostasis, bactericidal and eradication)) was determined using the Sigmoid E max equation [35].

Dosage Designation
Daily dose was calculated using the dosage equation: Dose = CL×(AUC 24h /MIC)×MIC F×f u , where CL is the clearance, AUC 24h /MIC is the targeted endpoint for optimal efficacy, f u is free fraction of drug in plasma, F is the bioavailability factor(from 0 to 1) [36]. The f u in epithelial lining fluid can be ignored, because of the low albumin levels in epithelial lining fluid [33]. To investigate the effect of different dosing regimens, the PD model indicates that the bacterial growth rate in the function of acetylkitasamycin concentration is combined with PK model, and simulations were performed with MlxPlore software (version-1.1.1, Lixoft, Orsay, France).

Results
3.1. PD Study of Acetylkitasamycin on S. suis 3.1.1. MIC of Acetylkitasamycin against S. suis Isolates All the MIC of acetylkitasamycin against 110 S. suis were in the range of 0.25~128 µg/mL. MIC distribution of the acetylkitasamycin against 110 S. suis is shown in Figure 1. Nonlinear least squares regression was used to fit a series distribution of log 2 -transformed MIC data to a range of symmetrical 'bell-shaped' theoretical population distributions which was conducted in GraphPad Prism 5 software (Tables 1 and 2). The results manifested the smallest difference between the estimated and true number of isolates in the subset of 16 µg/mL (Table 2), so 16 µg/mL was set as the wild-type cutoff value. To select a sensitive strain, the wild-type cutoff value was set as interpretive criteria. The MIC 50 and MIC 90 of 110 strains were 9.10 µg/mL and 100.31 µg/mL, respectively. According to the interpretation criteria, which were set as described above, a total of 55 strains were sensitive strains, and the MIC 50 and MIC 90 were 1.21 µg/mL and 6.94 µg/mL, respectively (Table 3).

Dosage Designation
Daily dose was calculated using the dosage equation where CL is the clearance, AUC24h/MIC is the targeted endpoint for optimal efficacy, free fraction of drug in plasma, F is the bioavailability factor(from 0 to 1) [36]. The epithelial lining fluid can be ignored, because of the low albumin levels in epithelial li fluid [33]. To investigate the effect of different dosing regimens, the PD model indicates tha bacterial growth rate in the function of acetylkitasamycin concentration is combined PK model, and simulations were performed with MlxPlore software (version-1.1.1, Li Orsay, France).

MIC of Acetylkitasamycin against S. suis Isolates
All the MIC of acetylkitasamycin against 110 S. suis were in the range of 0.25 μg/mL. MIC distribution of the acetylkitasamycin against 110 S. suis is shown in Figu Non-linear least squares regression was used to fit a series distribution of log2-tr formed MIC data to a range of symmetrical 'bell-shaped' theoretical population distr tions which was conducted in GraphPad Prism 5 software (Tables 1 and 2). The res manifested the smallest difference between the estimated and true number of isolate the subset of 16 μg/mL (Table 2), so 16 μg/mL was set as the wild-type cutoff value select a sensitive strain, the wild-type cutoff value was set as interpretive criteria. MIC50 and MIC90 of 110 strains were 9.10 μg/mL and 100.31 μg/mL, respectively. Acc ing to the interpretation criteria, which were set as described above, a total of 55 str were sensitive strains, and the MIC50 and MIC90 were 1.21 μg/mL and 6.94 μg/mL, res tively (Table 3).    Through the toxicity experiment in mice the strain number HB1607 with serotype 2 which MIC close to MIC 50 was used to study the antimicrobial activity of acetylkitasamycin in vitro and ex vivo.
MICs of acetylkitasamycin in MHB and PELF against S. suis HB1607 were 1 µg/mL, MBC were 2 µg/mL and 4 µg/mL, respectively. The MICs of single components of acetylkitasamycin against S. suis HB1607 in MHB were both 1 µg/mL, and MBCs were both 2 µg/mL. In addition, the MICs of metabolites kitasamycin against S. suis HB1607 were both 2 µg/mL, MBCs were both 8 µg/mL. The MPC of acetylkitasamycin against S. suis HB1607 in MHB was 5 µg/mL. The PAE of acetylkitasamycin against S. suis HB1607 is shown in Table 4. According to the MIC values, a series of concentrations of acetylkitasamycin was prepared to describe the killing curve. The curves were characteristically time dependent with significant PAE (Figure 2). Along with the extended time, the bacteria number decreased slowly, but the bactericidal activity was enhanced. In addition, when exposed to the higher concentrations (≥1 µg/mL) of acetylkitasamycin for 4 h, the bacteria decreased, but not to an undetectable level (<30 CFU). The ex vivo killing curve results showed that acetylkitasamycin was also time-dependent (Figure 3), and was consistent with the in vitro killing curve. Along with time prolongation and the increase in concentration, the number of bacteria decreased sharply.  The ex vivo killing curve results showed that acetylkitasamycin was also time-dependent (Figure 3), and was consistent with the in vitro killing curve. Along with time prolongation and the increase in concentration, the number of bacteria decreased sharply.

PK Study of Acetylkitasamycin in Piglets
The concentrations of total and every component of acetylkitasamycin in plasma and PELF were best fit first order two compartment model and the PK parameters were calculated by WinNonlin 5.2 (Table 5). After intragastric administration, the concentrations of total and every component of acetylkitasamycin in plasma were below the 1 μg/mL, and only A5′ and A4A5 were detected in plasma with continuous concentrations. Cmax-PELF/Cmaxplasma of A5′ was 31.54, AUCPELF/AUCplasma was 141.04; CmaxPELF/Cmaxplasma of A4A5 was 44.38, AUCPELF/AUCplasma was 241.65. In addition, the AUC, Cmax, Tmax in PELF were 248.58 h·μg/mL, 9.85 μg/mL and 4.27 h, respectively.

PK Study of Acetylkitasamycin in Piglets
The concentrations of total and every component of acetylkitasamycin in plasma and PELF were best fit first order two compartment model and the PK parameters were calculated by WinNonlin 5.2 (Table 5). After intragastric administration, the concentrations of total and every component of acetylkitasamycin in plasma were below the 1 µg/mL, and only A 5 and A 4 A 5 were detected in plasma with continuous concentrations. C maxPELF /C maxplasma of A 5 was 31.54, AUC PELF /AUC plasma was 141.04; C maxPELF /C maxplasma of A 4 A 5 was 44.38, AUC PELF /AUC plasma was 241.65. In addition, the AUC, C max , T max in PELF were 248.58 h·µg/mL, 9.85 µg/mL and 4.27 h, respectively.
The concentrations of every component of acetylkitasamycin and its main metabolite kitasamycin in PELF were determined by LC-MS/MS after intragastric administration is shown in Figure 4.

PK-PD Model Integration
The PK-PD parameters AUC 24h /MIC, AUC 24h /MPC, and T > MIC, T > MPC, integrating the PK-PD of acetylkitasamycin against S. suis, were 139.54 ± 5.30 h, 27.91 ± 1.06 h, and 45.54 h, 11.08 h, respectively. The inhibitory sigmoid E max model flawless expressed the relationship between antimicrobial efficacy of acetylkitasamycin and the PK-PD parameter of AUC 24h /MIC ratio in PELF ( Table 6). The parameters acquired were the values of N, E 0 , PD max , EC 50 and AUC 24h /MIC, which represent different levels of antibacterial activity ( Table 6).

Estimation and Assessment of Dose
According to the dosage equation, the optimal dose was calculated. CL/F was 202.49 ± 15.69 mL/h/kg, calculated by WinNonlin s oftware. The MIC 50 was 1 µg/mL, and the fu was ignored. When E = 0 (bacteriostatic action), the AUC 24h /MIC was 57.65 h, the dosage calculated for bacteriostatic was 11.67 mg/kg. When E = −3 (bactericidal action), the AUC 24h /MIC was 163.56 h, the dosage calculated for bactericidal was 33.12 mg/kg. When E = −4 (eradication action), the AUC 24h /MIC was 407.12 h, the dosage calculated for eradication was 82.44 mg/kg. Table 6. PK-PD integration parameters for acetylkitasamycin in PELF after intragastric administration at a dose of 50 mg/kg b.w. (n = 6). MlxPlore software was used to simulate the effects of different doses (11.67, 33.12, 82.44 mg/kg) in vivo ( Figure 5). On the basis of Figure 5, higher doses (33.12, 82.44 mg/kg) possessed bactericidal or eradication action during 0-12 h, but the bacterial regrowth occurred under the lower dose (11.67 mg/kg) treatment. Different dosing regimens (11.67 mg/kg every 12 h, 33.12 mg/kg every 12 h, 82.44 mg/kg every 12 h) were employed, simulating 3 days for treatment ( Figure 5). At least 33.12 mg/kg every 12 h was sufficient to achieve bactericidal activity in PELF. Therefore, the dosing regimen of acetylkitasamycin for the treatment S. suis infection established as 33.12 mg/kg b.w. every 12 h for 3 days.

Discussion
There is a substantial lack of preclinical and clinical PK data for acetylkitasamycin, and only the PK study in the ileum content has been studied [10]. The previous results indicated that the concentrations of every component of acetylkitasamycin in plasma were much lower than the MIC (1 μg/mL). On the contrary, the concentrations in PELF (9.84 μg/mL) were more than 10 times that of plasma. This result is the same as that of other macrolides; the concentrations of azithromycin and clarithromycin in foal PELF were

Discussion
There is a substantial lack of preclinical and clinical PK data for acetylkitasamycin, and only the PK study in the ileum content has been studied [10]. The previous results indicated that the concentrations of every component of acetylkitasamycin in plasma were much lower than the MIC (1 µg/mL). On the contrary, the concentrations in PELF (9.84 µg/mL) were more than 10 times that of plasma. This result is the same as that of other macrolides; the concentrations of azithromycin and clarithromycin in foal PELF were more than 10 times that in foal plasma [8].
Recent evidence from this experiment and other studies (including animals and folks) have highlighted the importance of drug concentrations at the infection site in predicting the appropriate dosage for therapy. In addition to the macrolide drugs, other drugs such as doxycycline [37], moxifloxacin [38], linezolid [39] also have a higher concentrations in PELF. Therefore, the PK-PD study using the drug concentrations at the infection site can better reflect the actual clinical administration situation than using the drug concentrations in the plasma [8].
The T max values of the five main components A 6 A 7 , A 5 , A 4 A 5 , A 1 A 3 and A 13 of acetylkitasamycin were 4.54 h, 3.81 h, 3.90 h, 4.34 h and 3.74 h, respectively. Analysis shows that there is no significant difference from the total T max value 4.27 h. Meanwhile, there was a significant difference between C max and AUC, due to the content of each component is different in pharmaceutical ingredients. Just like acetylkitasamycin, bitespiramycin has many components (Bitespiramycin I, II, III). After giving 80 mg/kg bitespiramycin to rats, the three main components reaching T max in plasma were 2.37 h, 2.69 h and 2.84 h, respectively, with no significant difference. However, there were significant differences in C max and AUC for the three main components [40].
To reveal the influence of every component in the total concentrations, weight coefficient (wj) was introduced [41]. Every monomer composition of acetylkitasamycin in PELF concentrations was given its own weight coefficient, in order to calculate the total concentration (C T ). This was superior to simply summing the concentrations (enrofloxacin + ciprofloxacin) at the different time points [42]. Through the software WinNonlin 5.2, the values of C max , T max and AUC were 9.84 µg/mL, 4.27 h and 248.58 h·µg/mL, respectively.
A very limited veterinary breakpoints has been properly established, and even most of testing laboratories continue routinely use human breakpoints [43]. Zafar's research demonstrated that S. suis resistance to macrolides increased steeply from 2002 to 2009, from 13% to 29.7% [44]. It is necessary to establish the breakpoint of acetylkitasamycin against S. suis. In this study, the wild-type cutoff value, one of the three cutoff values necessary to establish a breakpoint, was established as 16 µg/mL. There were 55 S. suis strains, where MIC ≤ 16 µg/mL, and MIC 50 and MIC 90 were 1.21 µg/mL and 6.94 µg/mL, respectively.
The MIC of the clinical separation bacteria S. suis HB1607 in MHB was not significantly greater than in PELF. It is worth noting that the total protein concentration in MHB used in this research was 3.78 g/L [45], and the corresponding concentrations in PELF were 0.25~0.62 g/L [46]. Therefore, the drug in PELF were perceived to be in free form when it comes to protein binding rates in PELF [33].
The PD target of AUC 24h /MIC ≥ 30 was an indicator for the success of therapy and preventing emergence of resistance of macrolide [47]. In this study, the AUC 24h /MIC obtained for bactericidal action in PELF was 137.71, which was much larger than 30. This may be because the concentrations in PELF were much higher than that in plasma. Zhanel [48] simulated the effect of azithromycin against S. pneumoniae in vitro, and determined the PD target in serum, AUC 24h /MIC ≥ 36.7, to be bactericidal. Schentag [49] believed that AUC 24h /MIC was a good predictor of the effect of erythromycin in human plasma, with reported PD target AUC 24h /MIC = 53. Furthermore, some studies in the literature show that the parameter of AUC/MIC indicates that the effect is better than T > MIC, such as macrolides, azalides, ketolides and clindamycin [17,50].
In the past, dosing regimens for clinically used antimicrobials were generally developed on the basis of related PK data obtained in in vitro measurements of antibacterial activity or the empirical clinical treatment, which are not based on solid PK-PD data. However, overuse and misuse of drugs is considered to be the primary factor that increases bacteria resistance in both humans and animals. PK-PD integration is regarded as a complimentary approach to PK-PD modeling for predicting the adequacy of the regimen in clinical subjects. According to the PK and PD data in this experiment, the dosages required to obtain different effects (bacteriostatic, bactericidal, eradication) were 11.67 mg/kg, 33.12 mg/kg, 82.44 mg/kg, respectively. After the professional software simulation, different dosages showed that 33.12 mg/ kg treatment every 12 h for 3 days was sufficient to cure S. suis (HB1607). Therefore, the dosing regimen of acetylkitasamycin to treat S. suis was established as 33.12 mg/kg b.w. every 12 h for 3 days.

Limitations
According to the MIC determination of acetylkitasamycin against 110 S. suis strains, HB1607 with MIC close to MIC 50 and high pathogenicity was selected for the follow-up PD study, which was representative. However, considering the potential PD variability of each strain, this study cannot represent PD studies of acetylkitasamycin against all S. suis.
This study employed post-anesthesia sampling, a procedure necessary for animal welfare. Anesthesia may potentially affect the metabolism of drugs, but there are no reports of anesthetics affecting the metabolism of macrolides.
As shown in Table 5, there were large differences of drug concentrations in plasma and PELF, and only two components were detected in plasma with continuous concentrations. The authors believe that using plasma drug concentrations to calculate the administered dose would increase the dose of drug used during treatment and increase unnecessary risk. The purpose of this study was to determine the optimal dosing regimen of acetylkitasamycin for the treatment of S. suis infection, the PK data in target tissue will be more useful for the precise use of the drug to treatment the pneumonia. Although S. suis may cause systemic infections, the focus here was on pulmonary infections. Therefore, the drug concentrations in the PELF of lung tissue were used to calculate the dosing regimen.

Conclusions
The purpose of this study was to determine the adequacy regimen of acetylkitasamycin that could be effectively used to cure pigs infected with S. suis. The dosage determined was based on PK and PD data analysis. PD data were acquired from analysis of the the static time kill curves obtained from ex vivo experiment. The inhibition E max equation was used to calculate the dosage. The dosing regimen of acetylkitasamycin to treat S. suis wasestablished as 33.12 mg/kg b.w. every 12 h for 3 days.