1. Introduction
Yellow catfish (
Pelteobagrus fulvidraco) is a predominant cultured fish species in China with a total production of more than 0.48 million tons per year [
1]. To pursue a high yield, the culture density of yellow catfish per 1 m
3 is increased continuously, which makes it easy to cause an outbreak of bacterial diseases, especially
Edwardsiella ictaluri infection. Clinical manifestations of
Edwardsiellosis are mainly classified as an acute type and a chronic type [
2,
3]. The acute type has a higher mortality that is infected from the digestive tract to blood and various organs to cause organ hyperemia, hemorrhage, inflammation, denaturation, necrosis, and ulceration. The typical symptom is a sick fish hanging in the water with head up, tail down, sometimes in spasmodically spiral swimming, and leading to death. The chronic type has a longer course than the acute type. The pathogen invades the olfactory bulb through the nasal cavity, then travels to the brain, and finally reaches the skull through the meninges and the skin of head. The typical symptoms are skin necrosis and ulceration and the formation of an open ulcer on the head, known as “head hole disease” [
3]. Due to the widespread infection in the fish body of
E. ictaluri, an aquatic drug with a high permeability is needed to cure the disease. In clinical therapy, the first selected drug is sulfadiazine, which can penetrate the blood–brain barrier to reach the brain, but its therapeutic efficacy is rapidly decreasing because of serious drug resistance [
4]. Fortunately, it has been found that doxycycline (DC) is an ideal choice among the approved drugs due to its good penetration properties in the tissues [
5,
6,
7].
Doxycycline (DC), a member of second-generation tetracyclines, has been extensively used in global aquaculture due to better chemical properties of plasma half-lives, lipid solubility, and antibiotic activity than its analogs [
8,
9]. DC is also approved in aquaculture against
Aeromonas hydrophila,
E. ictaluri,
Fibrobacter columnaris,
Pseudomonas fluorescens, and
Vibrio vulnificus in China [
2,
10,
11,
12,
13]. Currently, multiple pharmacokinetic (PK) and residue depletion studies of DC are available in tilapia [
14] and grass carp [
15,
16]. These studies reported that DC had a plasma elimination half-life of >20 h in grass carp following a single oral dose at 20 mg/kg, and of 39 h in tilapia following a single intravenous dose at 20 mg/kg; these relatively long plasma half-lives were in part caused by enterohepatic recycling [
14,
15,
16]. For the purpose of fish health, it is important to establish an efficient therapeutic regimen for specific fish species based on pharmacokinetic–pharmacodynamic (PK/PD) studies. Some PK/PD studies have been performed in veterinary animals for optimizing DC’s therapeutic regimen. For example, a PK/PD study of DC was carried out in
Mycoplasma gallisepticum, which causes chronic respiratory disease in chickens using an in vitro dynamic model [
17]. The estimated %T > MIC values for 0log10 (CFU/mL), 2log10 (CFU/mL) reduction, and 3log10 (CFU/mL) reduction were 32.48%, 45.68%, and 54.36%, respectively. This study showed good effectiveness and time-dependent characteristics of DC against
M. gallisepticum in vitro [
17]. Zhang and colleagues reported DC’s optimum dosage regime against
Haemophilus parasuis in pigs based on PK/PD integration modeling [
18]. According to values of AUC
0–24 h/MIC, the doses predicted to obtain bacteriostatic, bactericidal, and elimination effects for
H. parasuis over 24 h were 5.25, 8.55, and 10.37 mg/kg for the 50% target attainment rate (TAR), and 7.26, 13.82, and 18.17 mg/kg for 90% TAR, respectively [
18]. However, there are no PK/PD studies reported in any specific fish species. Furthermore, limited PK/PD information on DC concerning
E. ictaluri is available in yellow catfish.
The objective of this study was to investigate the pharmacokinetics of DC in yellow catfish at different oral doses and to calculate related PK/PD parameters of DC against E. ictaluri. The results will provide useful information to optimize the dosing regimen of DC against E. ictaluri in yellow catfish.
3. Discussion
E. ictaluri is an important pathogen in global aquaculture, particularly in the culture of yellow catfish and channel catfish, and it causes a great economical loss every year. However, the therapeutic information of the concerned drug based on PK/PD indices is scarce. In this study, we evaluated the PK/PD parameters of a candidate drug of DC against E. ictaluri in yellow catfish based on the MIC value and PK parameters following different single oral doses at 10, 20, and 40 mg/kg, respectively. This study provides useful information for the effective use of DC in yellow catfish against E. ictaluri.
To obtain sufficient pharmacological information on DC, PK studies of DC were performed in yellow catfish at different single oral doses. According to observed results, an obvious multiple-peak phenomenon was found in DC concentration vs. time curves in plasma and tissues, which was consistent with the results in grass carp (
Ctenopharyngodon idella) and tilapia (
Oreochromis aureus × Oreochromis niloticus) following a single oral dose at 20 mg/kg at 24 °C [
14,
16]. In addition, DC displayed multiple peaks in PK profiles in ducks [
19], pigs [
20], and humans [
21]. This multiple-peak feature could be partly due to the impact of enterohepatic recycling because DC might form stable complexes with bile and enter the intestine via the biliary excretion to be reabsorbed into liver after digestion [
8].
The PK parameter of T
1/2λz ranged from 16.27 to 56.55 h in gill, from 29.60 to 143.26 h in kidney, from 16.49 to 142.52 h in liver, from 82.31 to 147.18 h, and from 80.81 to 106.38 h in plasma after a single oral dose at different levels (10, 20, or 40 mg/kg). These data did not present an apparent dose-dependence of T
1/2λz with the increased dose of DC but showed a large difference in the same tissue among different given dose levels. These discrepancies may be possibly due to the calculation method used in the software Phoenix. The T
1/2λz was calculated using the equation of T
1/2λz = 0.693/λ
z. The value of λ
z is a linear slope of the kinetic profile at the terminal elimination phase. In Phoenix, there are two calculation approaches for λ
z, one is the best slope identified automatically by the Phoenix software, and another is to manually choose three or more time points to perform the calculation [
16]. In the present study, the authors chose the former method to calculate λ
z without manual adjustments. In addition, due to the multiple-peak phenomenon, the selected time points for calculation in each tissue were different, which, in part, caused the differences in values of λ
z. Consequently, T
1/2λz in the same tissue under disparate doses presented different values.
In addition to the increase of oral dose, the C
max also presented an increasing trend in all tissues except in liver. The exact reason for the lack of a dose-dependent increase in the Cmax of liver is unknown. Only the value of C
max in gill was higher than grass carp, but the values in other tissues and plasma were all smaller than grass carp by oral administration at the same dose at 24 °C [
16]. The calculated T
max values ranged from 0.5 to 24 h following different single DC doses of 10, 20, and 40 mg/kg, which did not exhibit apparent regularities in each tissue as the rise of the dose. At the dose of 20 mg/kg, T
max values ranged from 0.5 to 24 h in plasma and tissues except for gill. These values were longer than the corresponding values in grass carp receiving the same dose at 24 °C [
16]. Moreover, there was also no obvious dose-dependence in V
z_F values and CL_F values accompanying the increase of administration dose. The V
z_F value (7.47 L/kg) at the dose of 20 mg/kg in yellow catfish was notably higher than that in tilapia (2.32 L/kg) [
14] and grass carp (0.87 L/kg) [
16] following the same oral dose at identical water temperature, suggesting that the distribution of DC in yellow catfish was more widely than tilapia and grass carp. The CL_F value in yellow catfish (0.06 L/h/kg) was larger than the corresponding values in tilapia (0.04 L/h/kg) [
14] and grass carp (0.03 L/h/kg) [
16]. Finally, the values of AUC
0-96 exhibited an increasing trend with the rise of given dose in gill, kidney, muscle and skin, and plasma, but its value was firstly increased (at a dose from 10 to 20 mg/kg) and then decreased (at a dose from 20 to 40 mg/kg) in liver. The exact reasons for this phenomenon are not known. The AUC_%extrap values were consistently higher than 20% in the plasma and muscle and skin for all dose groups and in all tissues in the 10 mg/kg dose group. This is a limitation of this study and these results suggest that the sensitivity of the analytical method was not good enough and/or the sampling duration was not long enough; thus, the calculation of the half-life values could be inaccurate. Future studies using more sensitive detection methods with longer sampling duration are needed to more accurately calculate the half-life of DC in yellow catfish.
For the purpose of reducing the number of experimental animals, this study used a non-parametric superposition approach with the Phoenix software to simulate the PK profiles after multiple oral doses with different time intervals based on the PK parameters from a single oral dose [
22]. Phoenix’s non-parametric superposition object is based on non-compartmental results describing single-dose data to predict drug concentrations after multiple doses at a steady state. The predictions are on the basis of an accumulation ratio calculated from the terminal slope, which can be used for simple (the same dose was given in a constant interval) or complicated dosing schedules (based on Phoenix WinNonlin User’s Guide). The simulated results can help design optimal dosage regimes or predict outcomes of clinical trials when used in conjunction with the semi-compartmental modeling function. In actual PK studies, the non-parametric superposition approach has been extensively used [
23,
24,
25]. Its assumptions are typically as follows: (a) Application of linear PK to accommodate a change in dose during the multiple dosing regimen; (b) each dose of a drug acts independently of every other dose; (c) the rate of absorption and the average systemic clearance are consistent for each dosing interval [
25].
Regarding the pharmacodynamic component of this study, the parameter of MIC for DC against
E. ictaluri was measured in yellow catfish plasma. It has been reported that the MIC value measured in the broth was conspicuously different from that measured in plasma [
26,
27]. A study found that the MIC value of enrofloxacin in plasma countering
A. hydrophila was remarkably higher than that in broth [
28]. The authors proposed that, if different MICs were found in broth and plasma, the corresponding adjustment should be performed by a scaling factor when the PK/PD breakpoint indices were used to optimize dosages. Furthermore, the in vitro susceptibility of macrolides and ketolides also manifested a marked enhancement of antibiotic activity against
Pseudomonas aeruginosa in RPMI 1640 medium [
29]. Therefore, the matrix between broth and plasma may influence antimicrobial activity, and it is better to use plasma for dilution and incubation of bacteria to determine the MIC because the composition of plasma is the closest to the in vivo environment.
DC possesses a high lipophilicity and permeability that can result in high concentrations in various tissues after oral administration [
8]. This feature is beneficial for treating infectious diseases. Generally, DC is considered a time-dependent drug. A previous study showed that DC presented time-dependent killing for
M. gallisepticum in an in vitro model [
17]. Cunha and co-workers also reported that DC exhibited a time-dependent killing at low concentrations of 2–4 times the MIC, but a concentration-dependent killing at high concentrations of 8–16 times the MIC against
Staphylococcus aureus,
Streptococcus pneumoniae,
Escherichia coli, and
Pasteurella multocida [
30]. However, a PK/PD study of DC against
H. parasuis directly showed a dose-dependent property [
18]. These discrepancies may be caused by different target pathogens. This viewpoint has been proven in the PK/PD study of gentamicin, which displayed a time-dependent kinetic profile for countering
S. aureus, but a concentration-dependent kinetic profile against
Pseudomonas aeruginosa [
31]. In this study, one limitation was that the in vitro killing curve was not determined. As a result, we were unable to establish the PK/PD correlation based on the sigmoid inhibitory
Emax model. Nevertheless, the present study provides valuable information on AUC/MIC and %T > MIC using the non-parametric superstition approach based on PK characteristics at different single oral doses.
AUC/MIC and %T > MIC are important PK/PD indices for establishing or optimizing the dosage regimen. In this study, with the increase of the given dose from 10 to 40 mg/kg, AUC/MIC values were considerably increased in plasma and each tissue except for liver. When the given dose was increased from 10 to 20 mg/kg, %T > MIC values were notably increased in plasma and tissues (e.g., gill, increased from 43.99% to 100.0%). However, when the dose was increased from 20 to 40 mg/kg, no obvious changes for %T > MIC occurred in plasma and tissues (e.g., gill, from 100.0% to 100.0%). From these results, the AUC/MIC have a concentration-dependent effect along with the increase of DC dose in plasma and tissues except in liver, but the %T > MIC was only increased moderately at certain dose levels. If the dosage was over a threshold (e.g., 20 mg/kg), it would remain at a constant. Previous studies have demonstrated that the AUC/MIC ratio of 100–125 is recommended to achieve a higher therapeutic efficacy [
32,
33,
34]. In this study, the ratios of AUC
0–96/MIC were more than 173.03 in plasma and tissues after oral administration at a dose of 20 mg/kg with the time interval of 96 h. In addition, with the increase of frequency of the given dose from every 96 h to every 12 h, AUC
0–96/MIC values were increased by 409.7%–563.7% in plasma and tissues. At the given dose of 20 mg/kg with a time interval of 96 h, %T > MIC values were greater than 99.0%. However, along with the increase of administration frequency, %T > MIC values exhibited a declined tendency. These results indicate that the antimicrobial activity of DC is not necessarily proportional to the frequency of administration. Therefore, we speculate that DC presents time-dependence and %T > MIC is a more suitable PK/PD index for DC against
E. ictaluri in yellow catfish.