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. 2024 Mar 23;10(3):e1419. doi: 10.1002/vms3.1419

Pharmacokinetic behaviour and pharmacokinetic–pharmacodynamic integration of doxycycline in rainbow trout (Oncorhynchus mykiss) after intravascular, intramuscular and oral administrations

Feray Altan 1,✉, Orhan Corum 2, Duygu Durna Corum 2, Kamil Uney 3, Ertugrul Terzi 4, Soner Bilen 5, Adem Yavuz Sonmez 5, Muammer Elmas 3
PMCID: PMC10960609  PMID: 38520701

Abstract

Objective

Doxycycline (DO) has been used in fish for a long time, but there are some factors that have not yet been clarified regarding its pharmacokinetic (PK) and pharmacodynamic (PD) properties. Therefore, the aim of this study was to investigate the PK and PK/PD targets of DO after 20 mg/kg intravascular (IV), intramuscular (IM) and oral (OR) gavage administration in rainbow trout (Oncorhynchus mykiss).

Methods

Plasma samples were collected at specific time points and subsequently analysed by HPLC‐ultraviolet. The PK/PD indices were calculated based on the MIC90 (Aeromonas hydrophila and Aeromonas sobria) values obtained for the respective bacteria and the PK parameters obtained for DO following both IM and OR administration.

Results

After IV administration, the elimination half‐life (t 1/2 ʎz ), area under the concentration vs. time curve (AUC), apparent volume of distribution at steady‐state and total body clearance of DO were 34.81 h, 723.82 h µg/mL, 1.24 L/kg and 0.03 L/kg/h, respectively. The t 1/2λz of the DO was found to be 37.39 and 39.78 h after IM, and OR administration, respectively. The bioavailability was calculated 57.02% and 32.29%, respectively, after IM and OR administration. The MIC90 of DO against A. hydrophila and A. sobria was 4 µg/mL. The PK/PD integration showed that DO (20 mg/kg dose) for A. hydrophila and A. sobria with MIC90 ≤4 µg/mL achieved target AUC/MIC value after IM administration.

Conclusions

These results suggest that when rainbow trout was treated with 20 mg/kg IV and IM administered DO, therapeutically effective concentrations were reached in the control of infections caused by A. hydrophila and A. sobria.

Keywords: Aeromonas hydrophila, Aeromonas sobria, doxycycline, pharmacokinetics, PK–PD integration, rainbow trout

  • Doxycycline exhibited a long t 1/2 ʎz and a large volume of distribution in rainbow trout.

  • Oral and IM bioavailability of doxycycline were 57.02% and 32.29% in rainbow trout, respectively.

  • The MIC90 of doxycycline against Aeromonas hydrophila and Aeromonas sobria at 13°C was 4 µg/mL.

  • The PK/PD integration showed that DO (20 mg/kg dose) for A. hydrophila and A. sobria with MIC90 ≤4 µg/mL achieved target AUC/MIC value after IM administration.

  • The scaling factors (for 192 h) of DC administered at a dose of 20 mg/kg by the IM, and oral routes of administration were found to be 0.54 and 0.30, respectively, for A. hydrophila and A. sobria.

graphic file with name VMS3-10-e1419-g001.jpg

1. INTRODUCTION

Rainbow trout, classified as Oncorhynchus mykiss, is a freshwater fish belonging to the Salmonidae family. It is a freshwater fish species with various characteristics, such as easy spawning, rapid growth and tolerance of wide water temperature range (Molony, 2001). Rainbow trout, which lack intramuscular (IM) spines in its muscles and generally contains meat suitable for consumption by humans and animals, the dominant species in the cultivation of products as an important economic value throughout the world (Khalili Tilami & Sampels, 2018; Lu & Luo, 2020). The ever‐increasing demands of consumers have led to an increase in rainbow trout production (Bostock et al., 2010; D'Agaro et al., 2022). Because of this rapid growth in the fish industry, rainbow trout are exposed to bacterial diseases such as motile Aeromonas septicaemia, enteric red mouth or yersiniosis and pseudomoniasis caused by some pathogens, such as Aeromonas hydrophila, Yersinia ruckeri and Pseudomonas putida (Balcı et al., 2023; Capkin et al., 2015; Duman et al., 2018; Kumar et al., 2015). It is known that the bacteria causing these infections cause serious economic losses and deaths for the trout industry (D'Agaro et al., 2022; Ibrahim et al., 2020).

Tetracyclines, which have a bacteriostatic effect by inhibiting the protein synthesis of bacteria, are one of the most widely used antibiotic groups for therapeutic and prophylactic purposes in aquaculture (Ahmed et al., 2020; Morshdy et al., 2022). Doxycycline (DO), a second‐generation, semi‐synthetic tetracycline derivative antibacterial drug, has a broad‐spectrum bacteriostatic activity against a wide range of gram‐positive and gram‐negative bacteria as well as many protozoa (Bilen & Elbeshti, 2019; del Castillo, 2013; Holmes & Charles, 2009). In addition to its antibacterial properties, it is recommended to be used in the treatment of many diseases due to its anti‐inflammatory, antineoplastic, neuroprotective and matrix metalloproteinase inhibitor effects (Di Caprio et al., 2015; Park et al., 2020). It has been approved for use in the treatment of various fish diseases in some countries, such as Japan, India, China and the Philippines, at a dose of 20 mg/kg/day for 3–5 days on some freshwater fish species weighing 120–150 g (Fauzi et al., 2021; Xu et al., 2020a, 2021).

Pharmacokinetic/pharmacodynamic (PK/PD) analysis is a crucial tool as it integrates all the necessary information to achieve clinical improvement and minimize the emergence of antimicrobial drug resistance (Rodríguez‐Gascón et al., 2021). The PK/PD index for antibiotics with concentration‐dependent effects is defined by the ratio of the area under the plasma concentration–time curve (AUC) to the minimum inhibitory concentration (MIC) (AUC/MIC), whereas for time‐dependent antibiotics, it is related to the time the drug concentration remains above the MIC (Chapuis et al., 2021; Toutain et al., 2021). In studies showing the relationship between the antimicrobial activity of tetracycline and its clinical results through PK and PD indices, it has been determined that the best parameter associated with a successful therapeutic effect is AUC/MIC (De Lucas et al., 2021; Dorey et al., 2017). The PK/PD studies have been performed in various fish and animal species to optimize the therapeutic regimen of DO, but not in rainbow trout (Prats et al., 2005; Xu et al., 2019b, 2021; Zhang et al., 2016).

There are a few studies on the PKs of DO in aquatic animals, including grass crap (Xu et al., 2020b), crayfish (Xu et al., 2022), catfish (Xu et al., 2021) and tilapia (Yang et al., 2014), and other animal species, such as goat. In these studies, it was determined that DO has advantages such as a wide volume of distribution and long elimination half‐life in fish. Although there is a study on the effect of temperature on the oral (OR) PKs of DO in trout, no study has been found on the PKs, bioavailability and PK/PD relationship of DO administered by different administration routes in rainbow trout. The objectives of this research were (i) to determine the plasma concentration–time profile and derive PK data for DO in rainbow trout after OR, IM and intravascular (IV) administration at the dose of 20 mg/kg; (ii) to determine the MIC of DO, of each of the two bacterial species, A. hydrophila and A. sorbia; (iii) to integrate the in vivo PK and in vitro PD data for DO.

2. MATERIALS AND METHODS

2.1. Drugs and reagents

The standards of DO hyclate (HPLC purity >97%) were commercial products of the Tokyo Chemical Industry. Chemicals were purchased from Merck Chemicals Co., Ltd. Ultrapure water was purified by Milli‐Q system from Millipore.

2.2. Experimental design and animals

The experiment was approved (2019/01) and carried out in accordance with the European Directive (2010/63/EU). The research was carried out on a total of 306 healthy rainbow trout, weighing 150–170 g. After visually inspecting the fish for signs of disease, trauma or poor body condition, the apparently healthy rainbow trout were randomly distributed into the aquarium recirculation system with 100 L of aerated tap water in each and six fish in each and acclimated to the laboratory conditions for 2 weeks before the experiments. The water flow from the reserve tank, which is constantly filled with tap water, was set to 30 L/h for renewal of water. Water temperature, dissolved oxygen concentration, pH and combined ammonia and nitrite concentrations in the aquarium were 13 ± 0.5°C, 8.10 ± 0.35 mg/L, 7.4 ± 0.2, 0.001, 45 ± 3.4 ng/L and 35 ± 2.3 µg/L, respectively. During acclimation and all experimental tests, fishes were fed daily by commercial fish feed (Sibal Yem).

2.3. Experimental methods

In the study, a total of 306 fish were equally and randomly assigned into three groups, and these groups were then divided equally into 17 subgroups. The analytical standard of DO hyclate diluted to 10 mg/mL with sterile pure water was used as drug for OR, IM and IV administrations, respectively. Fish were fasted for 12 h before and after OR drug administration. The fish received a single IV (caudal vessel, n = 102), IM (right epaxial muscle, n = 102) and OR (gastric gavage, n = 102) route of DO at a dose of 20 mg/kg. The drug administration and blood samples were performed after the fish were anaesthetized in water containing 200 mg/L tricaine methanesulphonate (MS‐222). Blood samples (1.5 mL) were taken from the caudal vessel into heparin tubes using a 26‐ga needle just prior to administration (control), and 0.08, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168 and 192 h post‐injection. The collected blood samples (six fish per sampling time) were centrifuged at 4000 g for 10 min to obtain plasma and then stored at −80°C until analysis. All plasma samples were analysed for DO concentration within 3 months after the experiments ended.

2.4. HPLC‐UV analysis

The plasma concentration of DO was determined using HPLC‐ultraviolet (UV) system by minor modifying the previously defined method (Tekeli et al., 2020; Turk et al., 2020). Plasma samples (200 µL) were thawed at room temperature and added to a 1.5 mL centrifuge tube. Subsequently, 400 µL of buffer/EDTA (0.1 M disodium EDTA, containing 0.1 M sodium phosphate) and 100 µL of perchloric acid (60%) were added and mixed with plasma using a vortex. After vortexing 2 min, the samples were centrifugation at 12,000 g for 15 min, and the supernatant was transferred to a vial. Then, the 50 µL supernatant was injected into the HPLC.

The HPLC‐UV system was a Shimadzu (Shimadzu, Co., Ltd.) system consisting of a degasser (DGU‐20A), a pump (LC‐20AT controlled by the CBM‐20A), an autosampler (SIL20A), a column oven (CTO‐10A) and detector (SPD‐20A UV–VIS) set at 350 nm. The LC solution software programme (Shimadzu) was used for data acquisition and processing. Chromatographic separation was performed on ODS‐3 (5 µm, 250 × 4.6 mm2 I.D. column, GL SCI) column. The column and autosampler temperatures were set at 30 and 22°C, respectively. The mobile phase consisted of trifluoroacetic acid in water (0.01 mmol, 70%) and acetonitrile (30%). The flow rate was set at 1 mL/min.

The chromatographic method was validated according to EMA (2011) guidelines. The stock solution of DO was prepared in purified water to obtain a concentration of 1 mg/mL. Working standard solutions of different concentrations (0.04–100 µg/mL) were obtained by further diluting the stock solution with purified water. The selectivity of the method was evaluated by extracting the blank plasma samples of fish for the interference from plasma. Calibration standards (0.04, 0.1, 0.4, 1, 4, 10, 40 and 100 µg/mL) and quality control samples (0.1, 4 and 40 µg/mL) were prepared by adding working standard solutions of DO into blank plasma.

2.5. Pharmacokinetic analysis

The plasma concentration–time curves of DO were plotted using the WinNonlin 6.1.0.173 software (Pharsight Corporation, Scientific Consulting Inc.). Plasma concentrations were presented as mean ± standard deviation values. PKs of DO were determined by the non‐compartmental analysis using mean plasma concentration values collected after IV, IM and OR administrations. The AUC, AUC extrapolated from t last to ∞ in % of the total AUC (AUCextrap %), terminal elimination half‐life (t 1/2λz), mean residence time (MRT), total clearance (ClT = dose/AUC) and volume of distribution at steady state (V dss = ClT × MRT) were calculated. AUCIV and AUCIM,OR were calculated using the linear/log trapezoidal method and the linear up/log down method, respectively. The peak plasma concentration (C max) and the time to reach C max (T max) were determined directly from the data on the plasma concentration–time curve. Bioavailability (F) after IM and OR administrations was determined using the following formula: F = (AUClastIM,OR/AUClastIv) × 100.

2.6. Pharmacodynamic analysis

Broth microdilution method of the Clinical and Laboratory Standards Institute (CLSI, 2020) was used to determine the MICs of DO for A. hydrophila (n = 13) and Aeromonas sobria (n = 13) isolated from the fish farms (kindly provided from Prof. Dr. Sevki KAYIŞ). For this purpose, eight different concentrations of DO (from 4 to 0.031 µg/mL) were prepared in Mueller Hinton Broth, and a volume of 150 µL of each concentration per well was put into the 96‐well plate and used for the MIC test. The lowest concentration of DO that visibly inhibited bacterial growth was accepted as MIC. Escherichia coli ATCC 25922 was used as the quality control in the MIC test. Antibiotic‐free cultures were used as positive controls, and bacteria‐free cultures were used as negative controls. The MIC50 and MIC90 represent the MIC value, at which at least 50% and 90% of the isolates, respectively, are inhibited. The MIC50 and MIC90 of DO were calculated using ‘n × 0.5’ and ‘n × 0.9’, respectively, in which n was the number of test strains (Schwarz et al., 2010).

2.7. Pharmacokinetic/Pharmacodynamic index

The PK/PD indices (AUC/MIC) were calculated based on the MIC90 values obtained for the A. hydrophila and A. sobria species and the PK parameters of DO following IM or OR administration.

3. RESULTS

3.1. HPLC‐UV analyses

The calibration curve of DO was linear (R 2 > 0.9992) between 0.04 and 100 µg/mL. The quality control samples, which were prepared in six replicate analyses of each level at the concentration of 0.1, 4 and 40 µg/mL within 1 day or on 6 consecutive days, were used to determine the recovery, precision and accuracy. The recoveries of DO ranged from 87% to 97%. The lower limit of quantification was 0.04 µg/mL for DO in plasma with the coefficient of variation less than 20% and the bias of ±15%. The intra‐ and interday coefficients of variation were ≤6.6% and ≤8.4%, respectively. The intra‐ and interday biases were ±7.2% and ±8.0%, respectively.

3.2. Pharmacokinetic analysis

All fish during the experimental period tolerated DO administered by IV, IM or OR injection, and no adverse effects were observed. The semi‐logarithmic plasma concentration–time curves were plotted according to the concentration of DO following IV, IM and OR administrations in rainbow trout (Figure 1).

FIGURE 1.

FIGURE 1

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Semi‐logarithmic plasma concentration–time curves following intravenous (IV), intramuscular (IM) and oral administrations of doxycycline (DO) in rainbow trout at a single dose of 20 mg/kg (L) at 13 ± 0.5°C (mean ± SD, n = 6).

After IV, IM or OR administration, at the first observational time point (0.08 h), the DO concentrations were 75.72 ± 14.67, 4.93 ± 0.76 and 1.19 ± 0.25 µg/mL, respectively. Then, the plasma concentration of DO following the IV, IM or OR administration dropped to 0.27 ± 0.05, 0.21 ± 0.03 and 0.15 ± 0.02 µg/mL, respectively, at 192 h. Non‐compartmental PK parameters of DO are represented in Table 1. The t 1/2λz was 34.81, 37.39 and 39.78 h after the IV, IM and OR administrations, respectively. After the IV injection, total ClT and Vdss were 0.03 L/kg/h and 1.24 L/kg, respectively. The AUClast after IV, IM and OR administrations was 723.82, 412.69 and 233.73 h µg/mL, respectively. The C max after IM and OR administrations reached to 10.42 and 5.06 µg/mL, respectively. The F after IM and OR administrations was 57.02% and 32.29%, respectively.

TABLE 1.

Plasma pharmacokinetic parameters following intravenous (IV), intramuscular (IM) and oral (OR) administrations of doxycycline (DO) in rainbow trout at a single dose of 20 mg/kg (L) at 13 ± 0.5°C (n = 6).

Parameters Unit IV IM OR
t 1/2 ʎz H 34.81 37.39 39.78
AUC0‐last h µg/mL 723.82 412.69 233.73
AUC0‐∞ h µg/mL 737.19 424.02 242.25
MRT0‐∞ H 45.62 53.63 59.02
ClT L/h/kg 0.03
Vdss L/kg 1.24
VZ L/kg 1.36 2.54 4.74
T max H 2.00 4.00
C max µg/mL 75.72 10.42 5.06
F % 57.02 32.29
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Abbreviations: AUC, area under the concentration vs. time curve; ClT, total clearance; C max, the peak plasma concentration; F, bioavailability; MRT, mean residence time; t1/2 ʎz , the elimination half‐life; T max, the time to reach C max; Vdss, volume of distribution at steady state; Vz, volume of distribution.

3.3. Pharmacodynamic analysis

The MICs for DO against A. hydrophila and A. sobria are presented in Table 2. The MIC values of DO were 0.25–4 µg/mL for A. hydrophila and 0.5–4 µg/mL for A. sobria. In this study, the MIC50 values of DO for A. hydrophila and A. sobria were found to be 1 and 2 µg/mL, respectively. The MIC90 values of DO for A. hydrophila and A. sobria were found 4 µg/mL, respectively.

TABLE 2.

The Minimum inhibitory concentration of doxycycline (DO) at 13°C for Aeromonas hydrophila and Aeromonas sobria isolated from fish farm.

Distribution (numbers of isolates) of doxycycline MIC (µg/mL)
Bacterial species N 0.031 0.063 0.125 0.25 0.5 1 2 4 8
A. hydrophila 13 0 0 0 1 3 6 1 2 0
A. sobria 13 0 0 0 0 5 1 3 4 0
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Abbreviation: MIC, minimum inhibitory concentration.

3.4. Pharmacokinetic/pharmacodynamic integration

The PK/PD integration parameters of DO for IM and OR routes at a dose of 20 mg/kg dose of DO against A. hydrophila and A. sobria are presented in Table 3. The AUC0‐last/MIC90 estimated using the in vitro MIC90 of DO for IM and OR administration were 103.17 and 58.43, respectively, against A. hydrophila and A. sobria (MIC90: 4 µg/mL).

TABLE 3.

The pharmacokinetic‐pharmacodynamic of doxycycline (DO) tested against Aeromonas hydrophila and Aeromonas sobria isolated from fish farm.

A. hydrophila and A. sobria
Administration route MIC90 AUC0‐192/MIC90
IM 4 103.17
OR 4 58.43
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Abbreviations: AUC/MIC, the ratio of the area under the free plasma concentration‐time curve to MIC; IM, intramuscular; MIC, minimum inhibitory concentration; OR, oral administration; PK/PD, pharmacokinetic–pharmacodynamic.

4. DISCUSSION

The route of administration of drugs is one of the main factors affecting the PK properties of drugs (Luo et al., 2019; Toutain & Lees, 2004). There are several advantages and disadvantages of administering drugs by different routes of administration in fish. Although the OR route of administration will make the treatment of large numbers of fish relatively easy, it will prevent the drug from reaching effective concentrations in infected animals due to the loss of appetite that develops in fish suffering from a bacterial infection (Assefa & Abunna, 2018; Samuelsen, 2006). In addition, IV and IM routes of drug administration have some practical advantages over OR administration in fish, such as lower treatment costs and dosage precision. For these reasons, it is necessary to determine the PK properties of different administration routes of the drugs to be used in order to effectively treat infections in fish. In this study, we investigated the PK and PD properties (MIC value) of DO administered by IV, IM and OR administration routes in rainbow trout. The PK/PD integration of DO has been conducted in fish (Xu et al., 2022). However, to our knowledge, this is the first study to determine the PK and PD characteristics of DO in rainbow trout (O. mykiss) after OR, IM and IV administrations.

It has been determined that oxytetracycline applications in fish cause toxicity (Yang et al., 2020), but no reported there were side effects after 20 mg/kg DO application. No general or local adverse effects were observed in rainbow trout treated with a single dose of 20 mg/kg body weight throughout the study period. Previous studies conducted on various fish species have similarly reported that no adverse effects were observed after the administration of 20 mg/kg DO (Yang et al., 2014). Although tetracyclines cause tissue damage at the injection site, they are relatively safe from a toxicological point of view (del Castillo, 2013). Negative reactions after antibiotic treatment in fish vary depending on the antibiotic dosage and the sensitivity of various fish species (Yang et al., 2020).

In this study, the Vdss following a single‐IV injection of DO to rainbow trout was 1.24 L/kg. It has been determined that DO has a variable volume of distribution (0.79–7.47 L/kg) in previous studies in fish, such as yellow catfish (Pelteobagrus fulvidraco), tilapia and grass crap (Holmes & Charles, 2009; Xu et al., 2020b, 2021). In general, although DO is highly bound to plasma proteins in fish (grass crap 97.97% and yellow catfish 99.15%), it has a wide tissue distribution due to its lipophilic nature (Sun et al., 2022; Xu et al., 2019a). Additionally, DO revealed a limited volume of distribution in the vitreous with low protein concentrations (Gilmour et al., 2005). Based on these results, it can be said that the distribution volume of DO shows significant differences among fish species. Several reasons may explain these differences: (i) One reason may be differences in plasma protein concentrations in different fish species. (ii) Adipose tissue differences in fish species may have caused differences in the distribution of this lipophilic antibiotic (Lees & Aliabadi, 2002).

The t 1/2 ʎz and ClT of DO following IV administration were determined to be 34.81 h and 0.03 L/h/kg, respectively, in rainbow trout at 13°C. This value was similar to those previously reported for tilapia (t 1/2 ʎz ; 39 h, ClT 0.04 L/h/kg, 13°C) (Yang et al., 2014), grass carp (t 1/2 ʎz ; 27.75 h, 24°C) (Xu et al., 2020b) and channel catfish (t 1/2 ʎz ; 36.26 h, ClT 0.09 L/h/kg, 24°C) (Xu et al., 2020a).

Compared to IV and IM administrations, t 1/2 ʎz of DO after OR administrations in rainbow trout was prolonged by 5 and 2 h, respectively. The t 1/2 ʎz of DO following OR administration in this study was shorter than the value previously reported in yellow catfish (80.81 h, 24°C, dose of 20 mg/kg) (Xu et al., 2021) and tilapia (77.2 h, 24°C, dose of 20 mg/kg) (Yang et al., 2014) for orally administered DO but longer than those reported for in channel catfish (18.91 h, 24°C, dose of 50 mg/kg) (Xu et al., 2020). Following IM administration, DO reached the maximum concentration (C max 10.42 µg/mL, T max 2 h) faster than OR administration (C max 5.06 µg/mL and T max 4 h) in this study. Additionally, following IM administration, DO exhibited low and slow absorption from the gastrointestinal tract of rainbow trout with an AUC∞ 424.02 h µg/mL and T max 2 h compared to OR administration at 13°C. The bioavailabilities (F) of DO after OR and IM administrations were 32.29% and 57.02%, respectively. F greater than 30% is acceptable for antibacterial drugs in fish (Bowser & Babish, 1991). These absorption parameters were higher than Yang et al. (2014) for DO in tilapia following OR administration at 24 ± 0.5°C (F 23.41%, AUC∞ 113.45 h µg/mL, C max: 2.27 µg/mL, T max: 2 h). In particular, the fact that the OR and IM bioavailability rates obtained in this study are higher than 30% indicates that DO can be used by IM and OR routes.

In rainbow trout, drug or food absorption takes place mainly in the gut (Khojasteh, 2012), and the stomach begins to empty 10 h after food intake in rainbow trout (Olsson et al., 1999). Gastric emptying also varies with temperature and fish weight (Huebner & Langton, 1982; Lee et al., 2000); moreover, about 90% of the trout's musculature consists of white muscles with a low level of vascularization (White et al., 1988). The involvement of these mechanisms for DO in gastric absorption may explain why DO is absorbed more slowly with OR administration compared with IM routes of administration in rainbow trout. These results can be explained as the reason for the delay of DO elimination is that the absorption of orally administered DO is slower than the IM routes due to environmental and physical conditions such as water temperature, body weight and gastric emptying rate.

Anaesthetic drugs are used in PK studies in fish to ensure proper experimental procedure and animal welfare (Sneddon, 2012). However, in the literature review, no interaction studies were found between the relevant MS‐222 and DO. Therefore, we completed our PK study assuming that this undesirable effect was negligible and therefore the MS‐222 did not interact with DO. However, based on this incomplete aspect of our study, we believe that more comprehensive studies should be conducted to reveal whether there is an interaction between DO and MS‐222 in rainbow trout.

Most tetracyclines are classified as time‐dependent drugs, and the commonly used parameter for PK/PD integration is the time above the MIC, as a percentage (% T > MIC) after different dosage treatments (Xu et al., 2019b, 2021). However, for tetracycline, AUC0–24/MIC is the best surrogate index that correlates with a successful therapeutic effect, as different % T > MIC values cannot be obtained at static drug concentrations (De Lucas et al., 2021; Dorey et al., 2017; Hesje et al., 2007; Zhang et al., 2021). In previous studies, the MIC90 value was used instead of the MIC to ensure optimal treatment with tetracycline in fish (Vasuntrarak et al., 2022; Yang et al., 2014). For this reason, we used AUClast/MIC90 for PK/PD integration of DO against A. hydrophila and A. sobria in this study.

The MIC values of DO were 0.25–4 µg/mL for A. hydrophila and 0.5–4 µg/mL for A. sobria in this study. Previous studies performed in animal species demonstrate that a favourable therapeutic target is AUClast/MIC90 ratio 100–125 for DO (Cunha et al., 2000; Prats et al., 2005). Based on this value, it is seen that DO administered to rainbow trout at a dose of 20 mg/kg IM reached the AUClast/MIC90 target for A. sobria and A. hydrophila with MIC90 ≤4 µg/mL. The authors have suggested that when using the PK/PD indices (AUClast/MIC90) to optimize dosages, a scaling factor should be determined by the corresponding to the last measured plasma concentration (Toutain et al., 2007, 2021). Scaling factors (for 192 h) of DO administered at a dose of 20 mg/kg by the IM and OR routes of administration were found to be 0.54 and 0.30, respectively, for A. hydrophila and A. sobria. As the ideal scaling factor of DO for A. hydrophila and A. sobria has not been determined before, these scaling factors could not be evaluated in this study.

5. CONCLUSION

In summary, in this study, DO administered IV, IM and OR at a dose of 20 mg/kg did not cause any adverse reactions in fish and showed favourable PK properties, such as a high apparent volume of distribution, acceptable bioavailability and long half‐life. Calculations based on PK–PD integration obtained in this study showed that DO (20 mg/kg) administered by IM routes in rainbow trout would be effective against susceptible pathogens such as A. sobria and A. hydrophila with MIC90 ≤4 µg/mL. As the appropriate AUC/MIC and scaling factor of DO for fish are not known, the results obtained by PK/PD analysis could not be evaluated. Therefore, further studies are needed to scaling factor of DO in fish and clinical efficacy against specific pathogens.

AUTHOR CONTRIBUTIONS

Conceptualization; investigation; methodology; project administration; resources; supervision; writing – original draft; writing – review and editing: Feray ALTAN. Methodology; project administration; writing – review and editing: Orhan Corum. Investigation; methodology: Duygu Durna Corum, Ertugrul Terzi, Soner Bilen and Adem Yavuz Sonmez. Writing – review and editing: Kamil Uney and Muammer Elmas.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

ETHICS STATEMENT

The experiment was approved (2019/01) by the Kastamonu University Animal Experiments Local Ethics Committee, Turkiye and carried out in accordance with the European Directive (2010/63/EU).

PEER REVIEW

The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer‐review/10.1002/vms3.1419

ACKNOWLEDGEMENTS

This study was supported by the Coordination of Scientific Research Projects, University of Dicle, Turkiye (Project No. VETERINER.19.009).

Altan, F. , Corum, O. , Durna Corum, D. , Uney, K. , Terzi, E. , Bilen, S. , Sonmez, A. Y. , & Elmas, M. (2024). Pharmacokinetic behaviour and pharmacokinetic–pharmacodynamic integration of doxycycline in rainbow trout (Oncorhynchus mykiss) after intravascular, intramuscular and oral administrations. Veterinary Medicine and Science, 10, e1419. 10.1002/vms3.1419

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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