Pharmacokinetic/pharmacodynamic integration of amphenmulin: a novel pleuromutilin derivative against Mycoplasma gallisepticum

ABSTRACT Amphenmulin is a novel pleuromutilin derivative with great anti-mycoplasma potential. The present study evaluated the action characteristics of amphenmulin against Mycoplasma gallisepticum using pharmacokinetic/pharmacodynamic (PK/PD) modeling approaches. Following intravenous administration, amphenmulin exhibited an elimination half-life of 2.13 h and an apparent volume of distribution of 3.64 L/kg in healthy broiler chickens, demonstrating PK profiles of extensive distribution and rapid elimination. The minimum inhibitory concentration (MIC) of amphenmulin against M. gallisepticum was determined to be 0.0039 µg/mL using the broth microdilution method, and the analysis of the static time–kill curves through the sigmoid Emax model showed a highly correlated relationship (R ≥ 0.9649) between the kill rate and drug concentrations (1–64 MIC). A one-compartment open model with first-order elimination was implemented to simulate the in vivo anti-mycoplasma effect of amphenmulin, and it was found that bactericidal levels were reached with continuous administration for 3 days at doses exceeding 0.8 µg/mL. Furthermore, the area under the concentration–time curve divided by MIC (AUC/MIC) correlated well with the anti-mycoplasma effect of amphenmulin within 24 h after each administration, with a target value of 904.05 h for predicting a reduction of M. gallisepticum by 1 Log10CFU/mL. These investigations broadened the antibacterial spectrum of amphenmulin and revealed its characteristics of action against M. gallisepticum, providing a theoretical basis for further clinical development. IMPORTANCE Mycoplasma has long been recognized as a significant pathogen causing global livestock production losses and public health concerns, and the use of antimicrobial agents is currently one of the mainstream strategies for its prevention and control. Amphenmulin is a promising candidate pleuromutilin derivative that was designed, synthesized, and screened by our laboratory in previous studies. Moreover, this study further confirms the excellent antibacterial activity of amphenmulin against Mycoplasma gallisepticum and reveals its action characteristics and model targets on M. gallisepticum by establishing an in vitro pharmacokinetic/pharmacodynamic synchronization model. These findings can further broaden the pharmacological theoretical basis of amphenmulin and serve as data support for its clinical development, which is of great significance for the discovery of new antimicrobial drugs and the control of bacterial diseases in humans and animals.

and researchers.Its infections frequently present predominantly as a chronic respira tory condition, but it also commonly gives rise to secondary infections by other pathogens like Newcastle disease virus, infectious bursal disease virus, and Escherichia coli that worsen the disease course (2).Moreover, there is considerable variability in the clinical symptoms resulting from different isolated strains (3), complicating epidemio logical monitoring and control.The presently available commercial vaccines for M. gallisepticum principally comprise live attenuated forms as well as inactivated vaccines (4).However, owing to the complexity of strains and their capacity for immune escape, concerns remain over the potential for reversion to virulence and adverse effects with live vaccines, while inactivated vaccines are costly and involve intricate vaccination protocols (5,6).Additionally, Mycoplasmas lack cell walls, conferring intrinsic resistance to cell wall synthesis inhibitors like β-lactams.Other mainstream effective drugs, such as quinolones and macrolides, have also shown a significant decrease in susceptibility to M. gallisepticum over long-term application (7).Therefore, the discovery and evaluation of new antimicrobial agents continue to be a sustained and crucial effort for researchers.
Pleuromutilin (Fig. 1 ) has attracted the investment of pharmaceutical workers due to its unique antimicrobial mechanism of action, targeting the peptidyl transferase center of the ribosome (8).Thus far, just four pleuromutilin derivatives have reached commerci alization as final drugs-lefamulin (Fig. 1 and 2), retapamulin (Fig. 1 and 3), valnemulin (Fig. 1 and 4), and tiamulin (Fig. 1 and 5), the last two being restricted to veterinary applications.In recent years, numerous pleuromutilin derivatives have been designed globally using pleuromutilin as precursor structures (9).These derivatives are often reported to have excellent in vitro antimicrobial activities, but there have been limited publicly available results on their subsequent pharmaceutical development research and clinical evaluations.Amphenmulin (Fig. 1 and 6) was independently designed and synthesized in our laboratory.Previous studies have determined that it has better antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) than tiamulin, and the acute toxicity tests have also proven it to be practically non-toxic (10,11).Its pharmacological properties and application prospects deserve further explora tion and development.
In the preclinical stage of innovative drugs, pharmacokinetic (PK) and pharmacody namic (PD) studies are indispensable, serving as the cornerstone for subsequent clinical trials.PK/PD modeling integrates drug exposure levels and pharmacological responses, standing as a powerful tool for investigating dosing regimens and mitigating antimicro bial resistance risks (12).The European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) have both released PK/PD modeling guidance, underscoring its importance in new drug research and development (R&D) (13,14).In this study, we carried out pharmacokinetics of amphenmulin in broiler chickens, its pharmacodynamics against M. gallisepticum, and established in vitro dynamic modeling to obtain the PK/PD target indices, which further extended the antimicrobial spectrum of amphenmulin and enriched the theoretical basis of its action characteristics.

Bacterial strain, reagents, and animals
The M. gallisepticum standard strain S6 was obtained from the National Center for Veterinary Culture Collection (Beijing, China).Amphenmulin (98%) was synthesized by our laboratory according to the published methods (10).Acetonitrile and all other liquid chemicals used were purchased from Macklin Biochemical Technology Co., Ltd (Shang hai, China).M. gallisepticum in this study was cultured under standard conditions of 37°C with the following nutrients: swine serum, M. gallisepticum artificial medium base from Qingdao Hope Biological Technology Co., Ltd (Qingdao, China), nicotinamide adenine dinucleotide (NADH) and cysteine from Aladdin Biochemical Technology Co., Ltd (Shanghai, China).Thirty healthy adult yellow-feathered broilers weighing 1.58 to 2.04 kg were used in this study, caged, fed without antibiotics and anticoccidial drugs, and watered and fed ad libitum.All animal experimental procedures were approved by the Animal Ethics Committee of South China Agricultural University (Approval number: 2023a015).
After the plasma samples were restored to room temperature, 200 µL was taken and vortexed with 800 µL of acetonitrile, centrifuged at 12,000 rpm for 10 min.The supernatant was filtered through a 0.22-µm filter and analyzed using a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) system (Agilent 1200 HPLC and Applied Biosystem API4000 mass spectrometer, ABI Sciex, USA).Amphenmulin was separated on a C18 column (150 mm × 2 mm, 5 µm) at a flow rate of 0.5 mL/min, with a mobile phase of 0.1% aqueous formic acid and acetonitrile (40:60, vol/vol).The electrospray ionization (ESI) source of the mass spectrometer was operated in positive ion mode for multi-reaction monitoring, with the following ion source parameters: ion spray voltage 4,500 V, collision gas 5 psi, ion source gas1 50 psi, ion source gas2 55 psi, curtain gas 20 psi, source temperature 400°C.The precursor ion m/z of amphenmulin was 486.2, and the product ions m/z were 184.1 (quantifier) and 124.0, corresponding to collision energies of 8 and 48 V, respectively, with a declustering potential of 64 V, entrance potential of 10 V, and collision cell exit potential of 14 V. Importantly, the validation of the above method showed that the lower limit of quantification and limit of detection of amphenmulin in chicken plasma were 0.005 and 0.001 µg/mL, respectively, and the linearity was good in the concentration range of 0.005-0.5 μg/mL, with the correlation coefficients exceeding 0.99.Plasma spiked recoveries ranged from 86.00% to 103.58%, with intra-and inter-day variations of 0.75%-8.46%and 3.15%-10.63%,respectively.
Additionally, the plasma concentration-time data of amphenmulin in each individual were further analyzed using the non-compartmental method in Phoenix 8.1 (Certara, USA) to calculate pharmacokinetic parameters, in which the area under the curve (AUC) was calculated using the log-linear trapezoidal method, and the results were reported as mean ± standard deviation (SD).

Susceptibility assay
The minimum inhibitory concentration (MIC) of amphenmulin against M. gallisepticum S6 strain was determined with reference to the protocols previously reported (16,17).The broth microdilution method was performed using 96-well plates to achieve a final titer of approximately 10 7 colony-forming units (CFU)/mL of M. gallisepticum in each well.The final concentrations tested of amphenmulin ranged from 0.488 × 10 −3 to 0.125 µg/mL.Concurrently, a growth control (without drug), an end-point control (blank medium), and a sterile control (sterile broth) were also implemented.The minimum concentration that did not produce a color change in the wells after incubation was considered as the MIC value.For the agar dilution method, M. gallisepticum was inoculated and cultured on agar plates containing a twofold dilution series of amphen mulin concentrations, and the MIC was the lowest drug concentration that inhibited growth.Additionally, following the procedure of broth microdilution, the initial culture before incubation and the samples from wells where the color remained unchanged after 48 h of incubation were subjected to 10-fold serial dilutions.Each dilution sample (10 µL) was then plated on drug-free agar for colony counting.The lowest concentration that resulted in a bacterial reduction of greater than 3 Log 10 CFU/mL was considered the minimum bactericidal concentration (MBC).
We also determined the minimum concentration inhibiting colony formation by 99% (MIC 99 ) of amphenmulin against the S6 strain by slightly modifying the existing methods (18,19).The MIC measured by agar dilution was used as a baseline, and the concentration of the drug was linearly decreased by 10% to 0.5 MIC.By counting the colony growth of M. gallisepticum inoculated on these drug-containing agar plates and performing linear regression with drug concentration, we calculated the amphenmulin concentration that inhibits 99% growth of the S6 strain.
Furthermore, we determined the mutant prevention concentration (MPC) of amphenmulin against the S6 strain by agar dilution method (20).Briefly, M. gallisepticum at exponential phase was enriched to ≥10 9 CFU/mL by centrifugation and cultured as described above.The lowest drug concentration that inhibited mycoplasma growth in the agar plate was determined as the primary MPC (MPCpr), and the determination was repeated after linearly reducing the MPCpr to 0.5 MPCpr at 10% drug concentration.The final mycoplasma-free growth concentration was the MPC.All the experiments of the above pharmacodynamic studies were conducted in triplicate.

Static time-kill curves
Based on the MIC value from broth microdilution method, 7 mL blank medium, 0.2 mL amphenmulin solution, and 0.8 mL M. gallisepticum suspension were taken into 10-mL Celine bottles to make the final mycoplasma count of 10 7 CFU/mL, as well as final concentrations of amphenmulin of 1, 2, 4, 6, 8, 16, 32, and 64 MIC, respectively.Growth control (without drug) and negative control (blank medium) were also set up for culture.Then at 0, 6, 9, 12, 24, 36, and 48 h, 100 µL of each culture was taken for counting using the aforementioned method, with a lower limit of 100 CFU/mL.The experiment was repeated three times.
The efficacy of amphenmulin against M. gallisepticum was measured by the kill rate per unit of time.Considering the growth of M. gallisepticum observed in preliminary experiments, this study selected seven time periods of 0-24, 0-36, 0-48, 6-24, 6-36, 6-48, and 12-48 h to calculate the kill rates of amphenmulin.The Sigmoid E max model in Phoenix was used to fit the relationship between mean kill rate and drug concentration, which is shown below: E, kill rate of amphenmulin against M. gallisepticum within a period; E 0 , change rate of M. gallisepticum in the control group (without drug); E max , maximum kill rate within a period; C e , concentration of amphenmulin; EC 50 , drug concentration at half-maximal kill rate; N, Hill coefficient, reflecting the slope of the kill rate-concentration curve.

In vitro dynamic model
According to previous reports (21)(22)(23), an in vitro model compatible with M. gallisep ticum growth and amphenmulin pharmacokinetics was developed (24).It comprised a reservoir chamber containing fresh, drug-free medium; a reaction chamber with a three-necked flask where amphenmulin interacts with M. gallisepticum, containing 300 mL of medium in the outer chamber (external compartment, EC) and 10 mL of M. gallisepticum culture (10 7 CFU/mL) in a semipermeable membrane chamber (internal compartment, IC); and a waste chamber collecting effluent, which were connected via tubing and controlled by a peristaltic pump (Fig. 2).In this model, the flow rate was calculated as 1.63 mL/min from the reaction chamber volume and amphenmu lin's elimination half-life (T 1/2Ke ) after intravascular administration in chickens, enabling dynamic changes in drug concentrations that simulate amphenmulin elimination in chickens and its activity against M. gallisepticum.The in vitro elimination of amphenmulin followed first-order kinetics: where C 0 is the initial amphenmulin concentration, C is the amphenmulin concentra tion at time t, k is the elimination rate constant, and t is the time after dosing.Based on the maximum concentration reached in chickens after oral administration of amphen mulin, six dosing regimens of 0.1, 0.2, 0.5, 0.8, 1.0, and 1.5 µg/mL were designed, administered every 24 h for three times.By injecting the drug into both sides of the dialysis membrane simultaneously, the drug concentrations inside and outside the membrane reached equilibrium rapidly.Samples from the reaction chamber were collected at 0.083, 0.167, 0.25, 0.50, 0.75, 1, 2, 3, 4, 6, 9, 12, 24, 24.08, 25, 30, 36, 48.08, 54, 60, and 72 h after dosing for determination of amphenmulin concentration.Meanwhile, cultures from the IC were collected at 0, 3, 6, 9, 12, 24, 30, 36, 48, 54, 60, and 72 h after dosing for mycoplasma counting.Each sample was performed in triplicate.
The determination of amphenmulin in the medium referenced our laboratory's method for extracting tiamulin (25) and was also detected by the HPLC-MS/MS method mentioned in the PK section.The method validation results demonstrated that amphen mulin showed good linearity within the concentration range of 0.005-0.5 μg/mL (R > 0.99), blank medium spiked recoveries ranging from 86.54% to 100.55%, with intraand inter-day variations of 1.58%-11.49%and 2.89%-10.49%,respectively, indicating the feasibility of the method.

Integration and modeling of pharmacokinetics/pharmacodynamics
Phoenix software was used to analyze the in vitro concentration-time data of amphen mulin and calculate the AUC and peak concentration (C max ), which were combined with MIC to obtain PK/PD parameters.Concurrently, the anti-mycoplasma effect was derived from the dynamic time-kill curve, which was analyzed with the PK/PD index using an inhibitory Sigmoid E max model as follows: N + C e N E, anti-mycoplasma effect, the change in M. gallisepticum counts during 24 h of cultivation; E 0 , M. gallisepticum change in control (without drug); E max , maximal effect produced by amphenmulin against M. gallisepticum during 24 h; C e , PK/PD parameters (AUC/MIC or C max /MIC); EC 50 , the C e value needed to produce 50% of maximal effect; N, Hill coefficient, the slope of the fitted curve.Bacterial reduction ≥3 Log 10 CFU/mL was defined as bactericidal effect; <3 Log 10 CFU/mL was defined as bacteriostatic effect.

In vivo pharmacokinetics
The blood concentration-time curves of amphenmulin in chickens after intravenous, intramuscular, and oral administrations are shown in Fig. 3.The two extravascular routes reached peak levels before 30 min after administration, with C max of 0.73 ± 0.36 and 1.93 ± 0.48 µg/mL for oral and intramuscular administrations, respectively.The main pharmacokinetic parameters of amphenmulin in chickens are summarized in Table 1.From the results, the T 1/2Ke of amphenmulin after intravenous and oral administrations was significantly lower than that of intramuscular injection.Based on the AUC of the three routes, the absolute bioavailability of oral and intramuscular injections was calculated to be 5.88% and 52.14%, respectively.

Susceptibility assay
At an inoculum of 10 7 CFU/mL, the MIC of amphenmulin for M. gallisepticum was determined to be 0.0039 and 0.0078 µg/mL using the microbroth dilution method  and agar dilution method, respectively.In addition, the MIC 99 and MBC were 0.0077 and 0.0156 µg/mL, respectively.For an inoculum of 10 9 CFU/mL, the MPC value was 0.0500 mL, and a mutation selection window (MSW) of 0.77 × 10 −2 -0.05 µg/mL can be derived for amphenmulin, which exhibits a degree of potential resistance risk.

Analysis of static time-kill curves
The static time-kill curve of amphenmulin against M. gallisepticum strain S6 at 0-64 MIC drug concentrations are shown in Fig. 4a.Within 48 h of exposure of M. gallisepti cum to amphenmulin, the mycoplasma counts only decreased by 0.44 Log 10 CFU/mL at 1 MIC.At 2-32 MIC, the mycoplasma counts decreased by 0.57-2.7 Log 10 CFU/mL, indicating a bacteriostatic effect of amphenmulin within this concentration range.However, when the concentration reached 64 MIC, the mycoplasma count decreased by 3.85 Log 10 CFU/mL, exhibiting a bactericidal effect.These results indicated that the anti-mycoplasma effect of amphenmulin is enhanced with increasing static concentra tion in vitro (≤64 MIC).
The mean kill rates and amphenmulin concentrations were fitted to the Sigmoid E max model to obtain parameters like E max and EC 50 .The results in Table 2 indicate that the kill rates at different periods were well correlated with amphenmulin concentrations, with the goodness of fit (R) ranging from 0.9649 to 0.9936 and the maximum kill rate being 0.3277 1 /h.The model fitting within 0-24 h was optimal, and the relationship curve is visualized in Fig. 4b.

In vitro pharmacokinetics and the effects on M. gallisepticum
In the one-compartment intravenous administration model simulated in this work, at doses of 0.1-1.5 μg/mL, the AUC at 0-24 h, 24-48 h, and 48-72 h were 0.34-6.19,0.57-7.69,and 0.62-8.23 h•μg/mL, respectively, with measured C max after each administration of 0.12-1.52,0.12-1.58,and 0.12-1.50μg/mL, respectively.Throughout the experiment, the changes in drug concentration were higher than the MIC (0.0039 µg/mL) of amphenmulin against M. gallisepticum strain S6.The concentration-time curves of amphenmulin under different dosages are displayed in Fig. 5a.
The growth of M. gallisepticum exposed to dynamic drug concentrations can be seen in Fig. 5b.The decrease in M. gallisepticum counts within 24 h after each administration ranged from 0.15 to 2.08 Log 10 CFU/mL, which was seen as a significant inhibition of proliferation.Within 0-72 h, the M. gallisepticum counts decreased by a maximum of 2.82 Log 10 CFU/mL at doses of 0.1-0.5 μg/mL administered.When the dosage reached 0.8 µg/mL or above, amphenmulin exhibited bactericidal effects, and myco plasma numbers at the highest dose fell below the counting limit (100 CFU/mL).The results demonstrated that the anti-mycoplasma effect of amphenmulin was positively correlated with the dosage.

PK/PD modeling and analysis
Combining the in vitro PK data with the MIC value, the AUC 24 h /MIC and C max /MIC parameters of amphenmulin against M. gallisepticum can be obtained, and their relationship with the anti-mycoplasma effect is shown in Fig. 6, with AUC 24 h /MIC demonstrating a higher correlation (R = 0.9657).The findings suggests that the effect of amphenmulin against M. gallisepticum is likely to be related to both the level and duration of drug exposure.The results of the inhibitory E max model can be seen in Table 3, which can be further calculated that the AUC 24 h /MIC and C max /MIC target values for a decrease of 1 Log 10 CFU/mL of M. gallisepticum are 904.05h and 190.11, respectively.

DISCUSSION
Currently, in veterinary medicine, antimicrobial drugs are confronted with the issues of increasing drug resistance, reduced efficacy caused by inappropriate use, and safety risks (26).Pleuromutilin mainly exerts antibacterial effects by inhibiting bacterial ribosomal protein synthesis and has low cross-resistance with other drugs; the resistance rates remain low after more than 30 years of use (27).In the development progress of this class of drugs, the structure-activity relationship determined that the optimization of pharmacological properties mainly focuses on the structural modification of the C14 side chain (28,29), and amphenmulin was screened on this basis.A promising antimicrobial agent should possess desirable pharmacokinetic and pharmacodynamic properties, which are the focus of preclinical research.
In our findings, rapid peak concentrations of amphenmulin were achieved in chickens after extravascular administration.However, in the intramuscular injection group, the T 1/2Ke of amphenmulin was significantly longer than that in the intravenous and oral groups, exhibiting flip-flop kinetics, likely due to prolonged retention and slow release absorption from the injection site.As a typical pleuromutilin drug, high lipophilicity is a common trait (30,31).This results in poor solubility of amphenmulin in the hydro philic internal environment of organisms, making it difficult to efficiently penetrate biological membranes, leading to a low absorption rate.Similarly, the oral bioavailability of amphenmulin in chickens was only 5.88%, and 13.65% in mice (11).The complex gastrointestinal environment of animals means that only drugs with effective dissolution can be absorbed by the small intestine.Currently, the two pleuromutilin veterinary drugs are mainly administered as strong acid salts, such as tiamulin fumarate and valnemulin hydrochloride, which show bioavailability advantages among similar drugs (32)(33)(34).Additionally, the apparent volume of distribution (V ss ) of amphenmulin reached 3.64 L/kg, indicating extensive distribution in vivo (35), which coincides with the results obtained by Fu et al. in a tissue distribution study of another similar drug (36).Zeitlin ger et al. also proved that after intravenous injection, the free drug concentration of lefamulin in the pulmonary epithelial lining fluid was 5.7 times higher than that in plasma, exhibiting good tissue penetration (37).Extensive tissue distribution has been validated among analogous agents, which is significant for systemic infectious disease treatment.
In pharmacodynamics, the MIC of amphenmulin against M. gallisepticum S6 strain was determined to be 0.0039 µg/mL by broth microdilution, half of the agar dilution result.This may be because agar dilution judges the MIC based on colony growth, while a small amount of M. gallisepticum may not noticeably change the broth color, causing misjudgment.However, this result is lower than the 0.03 µg/mL MIC of tiamulin against the same strain reported by Xiao et al. (38), indicating that S6 is more susceptible to amphenmulin.Additionally, we preliminarily evaluated the MSW of amphenmulin against M. gallisepticum in vitro.The MSW is generally between the MIC 99 and MPC.Bacteria exposed to drug concentrations within this window for extended periods have an increased risk of enriched resistant populations (39).The MPC of amphenmulin was about 6.5 times the MIC 99 for M. gallisepticum, and fortunately, due to the sufficiently low value of MPC, the concentration of amphenmulin in chickens can easily exceed MPC for most of the time.Studies by Blondeau et al. showed that antibacterial effects at the MIC level are slow and incomplete (40).The MPC is a more valuable parameter than the MIC for preventing acquired resistance and optimizing dosing regimens (41,42).Inadequately, the present study was conducted only with the standard strain of M. gallisepticum, and the susceptibility distribution of amphenmulin against different isolates remains to be further broadened and clarified.
Furthermore, static kill curves can more reliably demonstrate antibacterial effects compared to MIC measurement, as mathematical relationships are introduced to analyze PD parameters (43).Our results preliminarily proved that amphenmulin has significant inhibitory effects on M. gallisepticum growth, with a good correlation between kill rates and concentration in the 1-64 MIC range.However, this differs from the static kill curve of amphenmulin against MRSA, which shows more time-dependent tendency and bacteriostatic effects (11).This closely relates to the growth characteristics of different microorganisms and the action of the antimicrobial agents themselves.Under constant drug concentrations, the confrontation between drug efficacy and microbial regrowth capacity is intuitive, whereas in practical application, the ultimate effect is the combined interactions of the drug, pathogen, and host.Therefore, introducing dynamic PK/PD models is valuable, as it can more comprehensively describe the three-dimensional relationship between amphenmulin concentration, effect, and time against M. gallisepti cum (44), and serve as a reference for in vivo studies, reducing costs of establishing animal models.Initially, we considered oral administration scenarios for amphenmulin, intending to build a one-compartment model with an additional absorption chamber to simulate the absorption process (45).However, we estimated the oral absorption rate constant (K a ) of amphenmulin to be approximately 3.15 1 /h based on the residual method, which would lead to a very small absorption compartment volume.Given the slow in vitro growth of M. gallisepticum, the rapid transient absorption process has little experimental impact, yet may cause errors.Therefore, building a one-compartment elimination model is more straightforward.The doses were referenced to the C max distribution after oral administration, and a multiple dosing strategy was implemented to offset the rapid elimination of amphenmulin, thereby allowing assessment of synchro nous inhibitory effects on M. gallisepticum during the drug elimination phase.
For other similar drugs, Xiao et al. conducted PK/PD studies of tiamulin and val nemulin in an intratracheal infection model of M. gallisepticum, respectively (38,46), and both demonstrated that the parameter most relevant to anti-mycoplasma effect was AUC/MIC, while optimized therapeutic dosages of 45 mg/kg and 6.5 mg/kg were given, respectively.It has also been reported that lefamulin showed time-and concentra tion-dependent activity against Streptococcus pneumoniae and Staphylococcus aureus in neutropenic murine thigh and lung infection model (47).Our study demonstrated that the AUC 24 h /MIC of amphenmulin showed significant correlation with anti-mycoplasma effect, and the target values required to reduce M. gallisepticum by 1 Log 10 CFU/mL were estimated to be 904.05h.Above the dose level of 0.8 µg/mL, amphenmulin could kill M. gallisepticum.These results reflect that the anti-mycoplasma effect of amphenmulin may be driven by both concentration and time, which could provide an important basis for optimizing the antimicrobial efficacy of amphenmulin and screening the appropriate dosage regimens.However, it remains to be determined whether amphenmulin is more time dependent against mycoplasma, as drug concentrations remained above the MIC throughout the multiple dosing in the dynamic model, which was also the main reason we did not examine the parameter %T > MIC.
At present, the anti-mycoplasma activity of amphenmulin has been preliminarily demonstrated to be driven by the amount of drug exposure, which facilitates further assessment of its in vivo antimicrobial activity at a later stage.Although the observed pharmacokinetic profile of the raw amphenmulin in chickens is not ideal, as a highly permeable and poorly soluble compound, its in vivo kinetic processes can be optimized through formulation design, crystal modification, optimal solvent selection, and other strategies.Alternatively, it can be further developed as a lipophilic precursor to enhance its bioavailability (48,49).

Conclusion
As a novel pleuromutilin derivative, amphenmulin exhibits rapid elimination kinetics and extensive tissue distribution in broiler chickens.In vitro studies show that M. gallisep ticum is highly susceptible to amphenmulin, and both static and dynamic time-kill curves demonstrate a strong correlation between the effect of amphenmulin against M. gallisepticum and drug exposure.The AUC 24 h /MIC can serve as an important PK/PD target parameter to evaluate the anti-mycoplasma effect of amphenmulin, thus establishing a crucial foundation for subsequent drug efficacy studies.

FIG 3
FIG 3 Plasma concentration-time curves of amphenmulin (20 mg/kg of body weight.) in chickens after intravenous, intramuscular, and oral administrations.Data represent mean ± SD values for 10 chickens (logarithmic scale for the concentration; inset: visualization for 0-2 h).

FIG 5
FIG 5 (a) Concentration-time curves of amphenmulin at different doses in the dynamic model.(b) Dynamic time-kill curves of various concentrations of amphenmulin against Mycoplasma gallisepticum.

FIG 6
FIG 6 Fitting of pharmacokinetic/pharmacodynamic parameters between anti-mycoplasma effects from the inhibitory E max model.(a):24 h area under the curve (AUC) 24 h /minimum inhibitory concentration (MIC).(b): C max /MIC.

TABLE 1 Estimated
pharmacokinetic parameters of amphenmulin [20 mg/kg of body weight (bw.)] in chickens after intravenous (IV), intramuscular (IM), and oral (PO) administrations a a C max , maximal concentration after administration; T max , time to reach maximum concentration; Kel, elimination rate constant; T 1/2Ke , elimination half-life; AUC 0-t , area under the blood concentration-time curve from 0 to last measurable concentration; AUC 0-∞ , area under the blood concentration-time curve from 0 to infinity; MRT, mean residence time; Cl, body clearance; V ss , apparent volume of distribution at steady-state; F, absolute bioavailability.Data represent mean ± SD values (n = 10).

TABLE 2
Estimated parameters of the kill rate of M. gallisepticum by amphenmulin from the E max model

TABLE 3
Estimated PK/PD parameters of amphenmulin against M. gallisepticum from the inhibitory E max model