Pharmacokinetic/pharmacodynamic profiles of baicalin against Mycoplasma gallisepticum in an in vivo infection model

Mycoplasma gallisepticum (M. gallisepticum), a devastating avian pathogen that commonly causes chronic respiratory disease in chicken, is responsible for tremendous economic losses to the poultry industry. Baicalin is the main constituent of Scutellaria baicalensis that shows potential therapeutic effects against M. gallisepticum. However, the pharmacokinetic/pharmacodynamics (PK/PD) profiles of baicalin against M. gallisepticum are not well understood. The main objective of the present study was to determine the relationship between the PK/PD index and efficacy of baicalin in the M. gallisepticum infection model in chickens. The experiments were carried out on 10-day-old chickens that were challenged with M. gallisepticum in the bilateral air sacs. While, baicalin was orally administrated once in a day for 3 consecutive days, started from d 3 postinfection. Ultra-performance liquid chromatography (UPLC) was used to evaluate the PK parameters of baicalin at doses of 200, 400, and 600 mg/kg in M. gallisepticum-infected chickens. Real-time PCR (RT-PCR) was used for the quantitative detection of M. gallisepticum in lungs. The PK and PD data were fitted to WinNonlin software to evaluate the PK/PD profiles of baicalin against M. gallisepticum. The minimum inhibitory concentration (MIC) of baicalin against M. gallisepticum strain Rlow was 31.25 µg/mL. The in vivo data suggested that baicalin concentration in the lung tissues was higher than plasma (1.21–1.73 times higher). The ratios of AUC24h/MIC of baicalin against bacteriostatic, bactericidal, and eradication were 0.62, 1.33, and 1.49 h, respectively. In conclusion, these results provided potential reference for future clinical dose selection of baicalin and evaluation of susceptibility breakpoints.


INTRODUCTION
Mycoplasma gallisepticum (M. gallisepticum) is one of the serious pathogens that harm the global poultry industry. M. gallisepticum is considered one of the primary causative agents of chronic respiratory disease in chicken (Stanley et al., 2001;Beaudet et al., 2017). Its primary signs include shedding of tears, sneezing, increased nasal discharge, and other respiratory symptoms. The transmission route of M. gallisepticum includes horizontal route via aerosol and vertical route via eggs. It has been previously demonstrated that M. gallisepticum infection suppressed the host immune response and increased the chances for secondary bacterial infections (Zhiyong et al., 2019;Wu et al., 2020;Wang et al., 2021b). It causes considerable economic losses in poultry industry in terms of reduced egg production, feed conversion rates and high mortality rates . Currently vaccination and antibiotics are used to control M. gallisepticum infection (Kanci Condello et al., 2020). However, it is difficult to eradicate the rapid spread of M. gallisepticum infection. Therefore, there is an urgent need to find better therapeutic agents.
Baicalin, a flavonoid compound possesses potential therapeutic properties, one of the main constituents of Scutellaria baicalensis. Previous studies demonstrated that baicalin has a variety of biological activities, such as antimicrobial, anti-inflammatory, antioxidant, antihepatotoxic, and antitumor properties (de Oliveira et al., 2015;Gong et al., 2017;Fu et al., 2018). Baicalin has the ability to interact with various signaling pathways. Our previous study explained the preventive effects of 1 baicalin against M. gallisepticum-induced inflammation, oxidative stress, and apoptosis by activating the Nrf2 signaling pathway and NF-kB signaling pathway , and baicalin could protect the immune organs from M. gallisepticum infection-mediated structural and functional damage Hu et al., 2021;Ishfaq et al., 2021). However, the lower bioavailability of baicalin limits its clinical uses. Numerous studies have been conducted on the pharmacokinetic profile of baicalin to clarify its in vivo properties (Wei et al., 2016), but it is difficult to obtain the connection between the efficacy and exposure concentration in vivo. These questions may be addressed through simulations from pharmacokinetic and pharmacodynamic models.
Pharmacokinetic-pharmacodynamic (PK/PD) modeling is a better method to understand the underlying mechanisms of drug action and the relationship on the physiologic system compared with single analytical method. It is well-known that PK/PD models, which have been used extensively in clinical trial design and the selection of dose regimens, provides useful information in optimizing the clinical dosage, improving the therapeutic efficacy and prohibiting resistance emergence (Nielsen and Friberg, 2013). In this study, we evaluated the PK/PD profiles of baicalin using M. gallisepticum infection model in chickens. Furthermore, we determined the better PK/PD index that correlated with the efficacy of baicalin. It was expected that our investigation would illustrate the relationship between dosing regimens and efficacy of baicalin, and assist further clinical development of optimal dosing strategies.

Ethical Statement
The present study was conducted under the approval of the Laboratory Animal Ethics Committee of Northeast Agricultural University (Heilongjiang province, China) by Laboratory animal-Guideline for ethical review of animal welfare (GB/T 35892-2018, National Standards of the People's Republic of China).

Mycoplasma Strains and Chemicals
The M. gallisepticum strain R low was obtained from Harbin Institute of Veterinary Medicine, Chinese Academy of Agricultural Sciences. The culture conditions for growing M. gallisepticum were kept the same as mentioned in our previous study . Briefly, M. gallisepticum was grown at 37°C in modified Hayflicks medium containing 0.05% Penicillins, 0.1% Nicotinamide adenine dinucleotide (NAD), 10% freshly prepared yeast extract, 20% fetal bovine serum and 0.05% thallium acetate. At mid-exponential phase of M. gallisepticum, a color change was observed from phenol red to orange. Its concentration was adjusted at a density of 5 £ 10 9 color change unit per milliliter (CCU/ mL) and kept at°C for use within 2 wk.
The reference standards of baicalin with a purity of above 98%, was purchased from the National Institute for the Food and Drug Control (Beijing, China). Methanol and Acetonitrile was of CHROMASOLV gradient grade for HPLC and obtained from Merck. LC/MSgrade Concentrated phosphoric acid and triethylamine were supplied by VWR.

Susceptibility Determination
The in vitro susceptibility of baicalin against M. gallisepticum R low was determined by the Microdilution method: 10 mL samples of M. gallisepticum culture (5 £ 10 9 CCU/mL) were inoculated onto 96-well plates containing 2-fold serial dilutions of baicalin. Growth control (M. gallisepticum inoculum without baicalin) and sterility control (blank medium) were also included in the MIC determination. The MIC was determined as the minimal concentration of the antibacterial agent that resulted in no growth on the 96-well plates after 7 d.

Animals and Inoculation
Three hundred 1-day-old Hyline variety of White Leghorn chickens were bought from the Harbin Yinong farming Co., Ltd. (Harbin, China). Chickens were housed in a positive-pressure fiberglass isolator, and provided with antibacterial-free balanced feed and fresh drinking water ad libitum. The antibacterial-free chicken feed was purchased from Lenong Feed Co., Ltd. (Harbin, Heilongjiang, China). The chickens were randomly divided into 4 groups: 1) A group of 50 chickens were used as negative control group; 2) 20 chickens were used as a M. gallisepticum-infected group and challenged with M. gallisepticum strain R low (1 £ 10 9 CCU/mL) in the bilateral air sacs of the thoracic region on d 10. The M. gallisepticuminfected group and negative control group were administrated by oral gavage with 0.85% NaCl started on d 13, once daily for 3 d; (3) A group of 180 chickens were used to study the pharmacokinetics of baicalin in infected chickens (infection route is same as described above, and the baicalin treatment started on day 13 in 3 different doses for 3 d consecutively, and 60 chickens were orally administrated for each dose once in a day). 4) A group of 50 chickens were used to study the pharmacodynamics of baicalin in infected chickens (the baicalin treatment was administered on d 13 in 8 different doses for 3 consecutive days, and 6 chickens were orally administrated once in a day for each dose separately). At the 16th d, all chickens were humanely sacrificed to avoid pain and suffering of chickens. Plasma and lung samples in each group were collected for further analysis.

M. Gallisepticum Quantitation by Real-Time qRT-PCR
Quantitative real-time polymerase chain reaction (qRT-PCR) was used to identify DNA copies of M. gallisepticum in different samples using a Roche LightCycler instrument (Shanghai, China) with M. gallisepticum-specific mgc2 gene. The primers are: mgc2-F: 5'-TTGGGTTTAGGGATTGGGATT; mgc2-R: 5'-CCAAGGGATTCAACCATCTT, as described in a previous study (Raviv and Kleven, 2009). Lungs were collected, homogenized in 2 mL PBS and centrifuged at 500 rpm for 5 min. An aliquot of 0.5 mL supernatant was used for DNA extraction with a bacterial DNA kit (Omega Bio-tek, Inc., Norcross, GA). The in vitro standard DNA curve was plotted by the numbers of M. gallisepticum derived from the culture method and cycle threshold (Ct) values obtained using qRT-PCR results as previously described .

Efficacy of Baicalin Against M. Gallisepticum in Chicken Infection Model
To evaluate the efficacy of baicalin at 24 h post 3-d infection, the infected chickens were administrated baicalin orally by gavage with either 0.85% NaCl (controls) or baicalin at 100, 150, 200, 250, 300, 400, 500, or 600 mg/kg once daily for 3 consecutive days (6 chickens/dose). After 24 h of the last drug administration, the amounts of DNA copies of M. gallisepticum in each chicken were calculated using the method as described above.

Baicalin Pharmacokinetics in In Vivo Infection Model
Groups of infected chickens were orally administrated baicalin by gavage at a dose of 200 mg/kg, 400 mg/kg, 600 mg/kg once daily for 3 d, and they were euthanized at 0.17, 0.25, 0.5, 0.75, 1, 2, 3, 6, 8, 12, and 24 h after the first oral gavage of baicalin. Blood and lung tissues were collected from 5 chickens at each sampling time point per treatment group. Blood samples were centrifuged at 3,000 £ g for 10 min at°C, and then plasma was collected. The samples of plasma and lung tissues were stored at À20°C until analyzed by UPLC within 2 wk.
Baicalin concentrations in plasma and lung tissues were determined by Ultra-Performance Liquid Chromatography (UPLC) (Waters Technologies, Shanghai, China). The UPLC was equipped with a Waters BEH-C18 column (2.1 mm £ 50 mm, 1.7 mm) using a mobile phase of triethylamine phosphate (pH 2.4): Methanol (47:53, v/v) and a flow rate of 0.8 mL/min. Injection volume was 6 mL. The calibration curve was established with seven baicalin concentrations in plasma and lung tissues respectively.
Plasma and lung tissues were treated with three times the volume of methanol-acetonitrile mixed solution, vortexed for 2 min and incubated at 45°C in a water bath for 10 min to precipitate the proteins. Then, the samples were centrifuged at 12,000 g for 10 min. The supernatant was collected and the residue was extracted again. The extracts were combined and evaporated to dryness under a gentle stream of nitrogen at 40°C. The residue was dissolved in 200-mL mobile phase, and filtered through a 0.22 mm syringe filter prior to UPLC analysis. The recovery and precision were calculated by analysis of spiked samples at 3 concentration levels (5 replicates of each concentration).

Pharmacokinetics and Pharmacodynamics Analysis
The PK profiles of baicalin were analyzed by the noncompartmental model with uniform weighting using the WinNonlin software (version 6.1; Pharsight, CA). The surrogate index of antibacterial activity, AUC 24h /MIC and C max /MIC were calculated using in vitro MIC value and PK parameters obtained from 3 doses of baicalin. The bacterial load for each animal was calculated based on Ct values and the in vitro standard DNA curve. The efficacy of baicalin was evaluated by the reduction of M. gallisepticum load after treatment compared with the initial bacterial count before treatment. The PK/PD relationship of baicalin against M. gallisepticum was described by WinNonlin software (version 6.1; Pharsight). Linear, E max and Sigmoid E max models were chosen as candidate models. A dose-response relationship for baicalin was detected by multiple contrast tests, and then we choose the Sigmoid E max model as the best-fit model in terms with the following equation: where E is the change in Log 10 CCU/mL for different dosage regimens, E 0 is the change in Log 10 CCU/mL in the control sample (absence of baicalin), E max is the difference in effect between the greatest amount of growth (as seen for the growth control, E 0 ) and the greatest amount of kill, Ce is the AUC 24h /MIC in the effect compartment, EC 50 is the AUC 24h /MIC value producing a 50% reduction in bacterial counts, and N is the Hill coefficient that describes the steepness of the curve.

Susceptibility Testing
The MIC of baicalin against the studied strain was 31.25 mg/mL.

M. Gallisepticum Infection Model
The model was successfully developed after 3 d of infection. These signs were observed including coughing, sneezing, nasal exudation and tracheitis in infected birds. Necropsy examination revealed cloudiness of air sacs and inflammation of the lungs. On the 13th day, the morbidity and mortality rates were 96 and 20%, respectively. While, the clinical signs of disease or M. gallisepticum-induced antibody were not observed in the negative control group.

UPLC Analyses
The selectivity of the method was obtained by comparing the control samples before administration with the samples after addition of the baicalin standard. Figure 1 shows that UPLC method had a good selectivity. The linear regression equation is showed in Table 1, and baicalin in the plasma and lung homogenates all had good linearity within the concentration range. The intra and interday precision and accuracy were showed in Table 2. The precision of baicalin calculated as the relative standard deviation (RSD) at various concentrations. It has been noted that RSD was lower than 14% for intraand interday experiment. The results showed that the precision was acceptable. The recovery of baicalin was higher than 85%, the LOD was 0.2 mg/ mL and the LOQ was 0.05 mg/mL in the samples. These results suggested that excellent sensitivity and reproducibility were achieved under the above condition.

PK Profiles of Baicalin in Infection Model
The time-concentration curves of baicalin in plasma and lung tissues after 3 oral administrations at a dose of 200, 400, and 600 mg/kg are shown in Figures 2 and 3, respectively. The main PK parameters obtained from plasma and lung tissues are presented in Tables 3 and 4, respectively. The T max was 0.75 h in plasma and lung tissues for 3 different doses. A second peak was observed for all the doses administered at 7 to 9 h in plasma. The mean half-life (T 1/2 ) was 14.45 § 3.44 h in plasma and 8.75 § 1.93 h in lung tissues. There were significant differences in T 1/2 between lung and plasma. Besides, the PK parameters were dose-dependent, a significant correlation between dose and AUC 24h or C max was observed (R 2 = 0.996 and 0.997 for dose-to-AUC 24h and dose-to-C max ratios, respectively; Figures 4). The AUC 24h values at different doses were calculated according to the linear relationship within the dose range from 100 to 600 mg/kg.

PD of Baicalin in Infected Chicken model
The effects of baicalin against M. gallisepticum in lung tissues with different regimens are shown in Figures 5. The data indicated that as the baicalin concentration increased from 100 to 600 mg/kg results in decrease in the bacterial load, implying that antimycoplasmal activity of baicalin was increased.

PK and PD Analysis
The PK/PD indices AUC 24h /MIC and C max /MIC were integrated using the PK parameters, dose proportionality, and MIC data. The effect (E) was calculated as the reduction of M. gallisepticum using the unit of Log 10 CCU/mL. The relationship between the ratio of AUC 24h /MIC and the ratio of C max /MIC with efficacy was described using the Sigmoid E max model ( Figure 6). The efficacy of baicalin correlated best with the AUC 24h /MIC ratio with an R 2 value of 0.959, followed by the C max /MIC (R 2 = 0.898). The model parameters of Hill coefficient, N, E max and AUC 24h /MIC are presented in Table 5. The values of the AUC 24h /MIC ratio required for bacteriostatic activity (E = 0), bactericidal activity (E = À2), and bacterial elimination (E = À3) were 0.62, 1.33, and 1.49 h, respectively.

DISCUSSION
M. gallisepticum is one of the primary pathogens that cause respiratory diseases in poultry, and severely impacts the development of commercial poultry production. Drug resistance to M. gallisepticum has emerged in recent years, due to long-term improper use of antibacterial agents (Morrow et al., 2020). Therefore, there is an urgent need to further explore new treatments and control methods. Our previous study demonstrated that the preventive effects of baicalin against M. gallisepticum-induced inflammation and apoptosis in the lungs of chicken Ishfaq et al., 2020). In the current study, we established an in vivo PK/PD profile of baicalin and evaluated the efficacy of baicalin against M. gallisepticum infection model.
Our choice for inoculation routes and the concentration of M. gallisepticum were based on previous studies. We compared four different M. gallisepticum infection models and found that the direct air sac injection is the faster and more effective method . The concentration of M. gallisepticum for pretest infection was the same as before, 5 £ 10 9 CCU/mL. Additionally, we considered the selection of the PDs calculation method. It is well understood that the isolation rate of M. gallisepticum is low, due to its high requirements on culture medium conditions and susceptibility to external contamination (Garcia et al., 1995). So, it may not be an effective method to evaluate the PD by isolation count.    Studies have confirmed that it is more convenient and efficient to use real-time PCR methodology to detect M. gallisepticum quantitatively, and it can be used to evaluate the PD parameters (Xiao et al., 2015;Xiao et al., 2016). Thus, in this study, we choose to detect M. gallisepticum quantitation by qRT-PCR separately before and after administration, which may provide a more accurate estimation of the difference in PDs.
To obtain a satisfactory analytical method, chromatographic conditions including the composition, pH of mobile phase, and apparatus were all optimized after several trials and referred to some previous studies (Xu et al., 2020;Zhu et al., 2021). We compared the methods and found that the obtained baicalin target peak response value was higher when using 0.4% (v/v) phosphoric acid (A) and methanol (B) as the mobile    phase method. It may be due to the weak acidity of baicalin. A certain amount of phosphoric acid can increase the resolution of baicalin and inhibit its dissociation. Meanwhile, we found that adding triethylamine in the mobile phase can prevent the target peak from tailing and improve the peak shape. In addition, the determination method by HPLC usually took too much time (>40 min), and was not sensitive enough. Taking into account the high protein binding rate of baicalin in vivo, we switched to UPLC for baicalin quantification. It is found that on the basis of the same mobile phase conditions, using UPLC has higher sensitivity and shorter retention time.
To the best of our knowledge, this was the first report about the PK/PD of baicalin in M. gallisepticuminfected chickens. In the present investigation, PK profiles of baicalin ranged from 200 mg/kg to 600 mg/kg has been described using baicalin concentration in the plasma and lung tissues in infected chickens. Baicalin was rapidly absorbed with the peak concentrations achieved at 0.75 h. The half-life was in the range of 10.93 to 17.80 h in plasma and in the range of 6.86 to 10.72 h in the lung, which was lower than the value of previously reported studies (Wei et al., 2017;Li et al., 2018). Hence, it indicated that the elimination rate of baicalin was significantly different among species. The PK profiles revealed that the average baicalin concentration in lung tissues was higher (1.21−1.73 times higher) than the corresponding concentration in plasma. It is consistent with the previously published article that higher concentrations of baicalin in lung tissues of rabbits and rats were noted compared to plasma (Zhu et al., 2013;Wei et al., 2016). The difference in the concentration of baicalin in the lung tissues may be due to transporters (Kalapos-Kov acs et al., 2015). Baicalin can be transferred to the alveolar space as a substrate through a variety of transport proteins.
In this experiment, baicalin was rapidly absorbed in the body, the first absorption peak appeared at 0.75 h, and the second absorption peak appeared at about 8 h. Many studies have given multiple mechanisms to explain this bimodal phenomenon. The previous study showed after oral or intravenous injection of baicalin, it undergoes a glucuronidation reaction in rats, and the hepatoenteric circulation has a great influence on the absorption and metabolism of baicalin (Xing et al., 2005). Some researchers have also proposed that absorption sites and intestinal flora may also be responsible for the multipeak absorption of baicalin. Experiments have shown that the absorption of baicalin was segment dependent (Fong et al., 2012). In addition, the biotransformation and the mechanism of action of baicalin in vivo is closely related to the intestinal flora that produces metabolic enzymes (Noh et al., 2016;Wang et al., 2021a).
The parameters of baicalin (AUC 24h ) showed dose proportionality in the range of 200 to 600 mg/kg following administration allowed us to calculate the AUC 24h for other dosage administrations. We choose AUC 24h / MIC as the PK/PD index of baicalin, since the data from the present multiple dosage studies confirmed the conclusion that baicalin is a concentration-dependent drug (Cheng et al., 2014). Our data showed a stronger correlation between the AUC 24h /MIC and the in vivo antibacterial effects of baicalin against M. gallisepticum (R 2 = 0.959) than C max /MIC (R 2 = 0.898). The AUC 24h /MIC ratios for M. gallisepticum (a reduction of 0 Log 10 CCU/mL), a reduction of 2 Log 10 CCU/mL, and a reduction of 3 Log 10 CCU/mL were 0.62 h, 1.33 h, and 1.49 h, respectively. In addition, a PK population clearance of baicalin should be calculated and the MIC distribution of M. gallisepticum to baicalin should be evaluated. In the clinic, the parameters of this study can be used to formulate dosing schedules in combination with the different immune functions condition.
In conclusion, the present study successfully constructed a synchronous PK/PD model of baicalin in chickens infected with M. gallisepticum. The in vivo data suggested that baicalin concentration in lung tissues was higher than plasma in M. gallisepticum-infected chickens (1.21−1.73 times higher). Taken together, baicalin showed therapeutic potential against M. gallisepticum infection in chickens, and the values of AUC 24h / MIC of baicalin against bacteriostatic, bactericidal, and eradication were 0.62, 1.33, and 1.49 h, respectively. E 0 is the change in Log 10 CCU/mL after 24 h incubation in the control sample (absence of drug) compared with the initial inoculum. E max is the difference in effect of the greatest amount of kill. EC 50 is the AUC 24h /MIC value producing a 50% reduction in bacterial counts from the initial inoculum. AUC 24h /MIC is the 24 h area under concentration-time curve/minimum inhibitory concentration ratios. N is the Hill coefficient that describes the steepness of the AUC 24h /MIC-effect curve.