Temperature-dependent non-linear pharmacokinetics of florfenicol in Nile tilapia (Oreochromis niloticus) and its implementation in optimal dosing regimen determination

Journal Pre-proof Temperature-dependent non-linear pharmacokinetics of florfenicol in Nile tilapia (Oreochromis niloticus) and its implementation in optimal dosing regimen determination

Tirawat Rairat, Chia-Yu Hsieh, Wipavee Thongpiam, Niti Chuchird, Chi-Chung Chou PII:

S0044-8486(19)32892-3

DOI:

https://doi.org/10.1016/j.aquaculture.2019.734794

Reference:

AQUA 734794

To appear in:

aquaculture

Received date:

30 October 2019

Revised date:

28 November 2019

Accepted date:

28 November 2019

Please cite this article as: T. Rairat, C.-Y. Hsieh, W. Thongpiam, et al., Temperaturedependent non-linear pharmacokinetics of florfenicol in Nile tilapia (Oreochromis niloticus) and its implementation in optimal dosing regimen determination, aquaculture (2019), https://doi.org/10.1016/j.aquaculture.2019.734794

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© 2019 Published by Elsevier.

Journal Pre-proof

Temperature-dependent non-linear pharmacokinetics of florfenicol in Nile tilapia (Oreochromis niloticus) and its implementation in optimal dosing regimen determination Tirawat Rairat1 , Chia-Yu Hsieh1 , Wipavee Thongpiam1 , Niti Chuchird3 , Chi-Chung Chou1,2,* [email protected] Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing

of

1

Department and Graduate Institute of Pharmacology, National Defense Medical Center, Taipei,

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2

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University, Taichung, Taiwan

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Taiwan

Department of Fishery Biology, Faculty of Fisheries, Kasetsart University, Thailand

*

Corresponding author at: Department of Veterinary Medicine, College of Veterinary Medicine,

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3

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Abstract

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National Chung Hsing University, Taichung 402, Taiwan.

While the majority of drugs used clinically at therapeutic range usually follow dose-independent or linear pharmacokinetics (PK); as the administered dose is increased the PK behavior may become dose-dependent or non-linear without recognition and may lead to adverse effects. The current study aimed to investigate the extent of non-linear PK of florfenicol (FF) in Nile tilapia, and for the first time, to investigate the effect of temperature on non-linear PK behavior. For each temperature level (24, 28, and 32°C), the fish were orally administered with FF at a single dose of 10, 15, 30 and 45 mg/kg. The serum concentrations of FF were analyzed by HPLC-UV and the PK parameters were determined by 2-compartmental model. Regardless of the rearing

1

Journal Pre-proof temperature level, increasing the FF doses from 10 to 45 mg/kg resulted in about 1.3-1.5 fold increases in the dose-normalized area under the serum concentration-time curve (AUC/dose), longer elimination half-life (t1/2β), and slower clearance (CL/F). The dose-dependency of the above parameters but not absorption half-life (t1/2Ka) and volume of distribution (Vd) suggested that the FF non-linearity was most likely due to saturation of metabolism. For the first time, the effect of temperature on non-linearity was revealed and discovered that the non-linearity

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happened at lower dose in the warmer temperature (≥ 30 mg/kg at 28 and 32°C) than in the

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cooler temperature (≥ 45 mg/kg at 24°C), which correlated well with the lower Michaelis-

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Menten constant (K m ) at the warmer temperature. In the presence of non-linear PK, a modified

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Michaelis-Menten equation could be successfully applied to determine an optimal dosing regimen. The accuracy of the calculated dosage was verified by the oral multiple-dose study.

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Medication of Nile tilapia with a high dose of FF should be done with great caution especially at

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the warmer water as higher dosage may be required and that non-linearity may happen at lower

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dose such that it would cause a higher risks of drug toxicity and tissue residue violation.

1. Introduction

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Keywords: Amphenicols, Fish, Dose-dependent pharmacokinetics, Temperature effect

It is well known that bacterial diseases are among the major threats to intensive aquaculture development. Wise use of an antimicrobial drug can reduce fish mortality. However, inappropriate use of drug can bring harms to the industry. Specifically, problems with drug resistance, tissue residue violation, environmental pollution, treatment failure, and adverse effect to human consumers are all potentially related to inappropriate drug use (Liu et al., 2017; Okocha et al., 2018). Thus, selecting an appropriate drug for the disease and applying the

2

Journal Pre-proof optimal dosing regimen are the keys to successful treatment and minimizing the negative impacts in aquaculture. As water temperature has a strong influence on fish’s physiology and pharmacokinetics (PK) (Hayton, 1999; Killen et al., 2010), the selection of optimal dosing regimen (dosage) should take the temperature into account. Our previous study revealed that Nile tilapia (Oreochromis

of

niloticus) reared at 32°C eliminated florfenicol (FF) more rapid compared to those cultured at 24 and 28°C (Rairat et al., 2019a). Many other studies also reported faster elimination of FF at

ro

higher water temperature, including common carp (Cyprinus carpio) (Oshima et al., 2004),

-p

channel catfish (Ictalurus punctatus) (Liu et al., 2009), Japanese eel (Anguilla japonica) (Lin et

re

al., 2015), crucian carp (Carassius auratus gibelio) (Yang et al., 2018, 2019), and spotted halibut

lP

(Verasper variegates) (Chang et al., 2019). Intuitively, faster drug elimination at warmer water temperature can be translated to a higher dose requirement. The importance of applying optimal

na

dosing regimen in fish culture has been addressed by Rairat et al. (2019b) and the method for

ur

determining the optimal dosage of FF in Nile tilapia at a specific temperature and minimum inhibitory concentration (MIC) level by pharmacokinetic-pharmacodynamic (PK-PD) approach

Jo

was given. We proposed that applying optimal dosing regimen determined by PK-PD principle should be preferred over using a universally recommended dose such as 10-15 mg/kg/d (US. FDA, 2019) unconditionally. It is worth mentioning that FF is one of a few aquaculture drugs approved by US. FDA for treating bacterial diseases in food fish (US. FDA, 2019). The calculated optimal dosages increase with increasing water temperature and target MIC levels, ranging from 2.23 (MIC 1 µg/ml at 24°C) to 34.88 mg/kg/day (MIC 4 µg/ml at 32°C) (Rairat et al., 2019b). The proposed PK-PD method for optimal dosage determination was proven appropriate when the tilapia was reared at 24°C; the trough concentration at steady state (C min(ss))

3

Journal Pre-proof of FF after 5-day oral administration at 4.5 mg/kg/d was similar to the predetermined MIC value (Rairat et al., 2019b). However, at 32°C, the calculated optimal dosing regimen (17.5 mg/kg/d) yielded higher-than-expected Cmin(ss). So, it was postulated that this unexpected outcome was probably related to the non-linear PK behavior of FF at higher dose.3 Non-linear PK (also known as dose-dependent and concentration-dependent PK) refers to

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the drug kinetics that is dependent on the drug amount or concentration in the body as opposed to the first-order kinetics (linear PK) in which the PK parameters such as the steady-state serum

ro

concentrations (C ss), half-life (t1/2 ), rate constant (K or β), total body clearance (CL), and volume

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of distribution (Vd) are constant irrespective of the drug amount or concentration (Hedaya, 2012;

re

Persky, 2013). In linear kinetics, if the administered dose was doubled, the drug concentration

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within the body and hence the area under the serum concentration-time curves (AUC) will also be doubled. In other words, the dose-normalized AUC (AUC/dose) is constant. Although the

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majority of drugs used clinically in human and veterinary medicine at their therapeutic dose (and hence

the

ur

concentrations usually follow linear PK, when the administered

corresponding concentration) was increased to a certain degree, one or more of the absorption,

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distribution, metabolism, and excretion (ADME) processes will gradually become saturated and the drug PK will no longer follow first-order kinetics (Ludden, 1991; Kwon, 2002; Rosenbaum, 2011; Persky, 2013). For the drug that follows non-linear PK due to saturable metabolism (also known as capacity-limited metabolism) which is the main cause of non-linearity in most drugs used clinically, the rate of drug metabolism can be described by Michaelis-Menten kinetics, i.e. rate = Vmax ·C/(K m+C). However, at very low drug concentration (C << K m) the rate of drug metabolism is proportional to the drug concentration, i.e. rate ≈ (Vmax /Km )·C and the process follows first-

4

Journal Pre-proof order kinetics. In contrast, at very high drug concentration (K m << C) the metabolizing enzyme is saturated such that the rate of drug metabolism is constant (independent of drug concentration), i.e. rate ≈ Vmax and the process becomes zero-order kinetics. (Rosenbaum, 2011; Hedaya, 2012). Non-linear PK refers to either Michaelis-Menten kinetics or zero-order kinetics. To adequately determine the occurrence of non-linear PK, the PK profiles at a minimum

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of 3 different dose levels are required; the dose-dependent changes in certain PK parameters such as t1/2 , CL, Vd, or AUC/dose are indicatives of the presence of non-linearity (Kwon, 2002).

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However, the occurrence of non-linear PK for aquaculture drug has yet to be comprehensively

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investigated in any fish species, even though some clues of dose-dependent PK were revealed.

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For example, the relationship between the administered dose and the peak concentration (C max )

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of FF in turbot appeared non-linear when the dose was higher than 50 mg/kg, possibly due to low solubility and/or dissolution rate of FF (De Ocenda et al., 2017). Saturation of plasma protein

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binding was reported for flumequine in the channel catfish (Ictalurus punctatus) (Plakas et al.,

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2000) and possibly in the European eel (Anguilla anguilla) as well (Boon et al., 1991). Dosedependency of some PK parameters and the AUC/dose are also observed in other antimicrobial

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drugs including oxytetracycline in Arctic charr (Salvelinus alpinus) (Haug and Hals, 2000), Japanese eel (Anguilla japonica) (Kim et al., 2008), and olive flounder (Paralichthys olivaceus) (Jung et al., 2008, 2009a); oxolinic acid in olive flounder (Jung et al., 2009b); and enrofloxacin in

rainbow

trout

(Oncorhynchus

mykiss)

(Bowser

et

al.,

1992)

and

grass

carp

no

detail

(Ctenopharyngodon idella) (Xu et al., 2013). Unfortunately,

other

than

incidental

observation

of dose-dependency,

information was provided in these papers, and no one studied the effect of water temperature on the presence of non-linear PK and subsequent dosage adjustment when the non-linearity

5

Journal Pre-proof appeared. Therefore, the primary objectives of the present study were to investigate the extent of non-linear PK of FF in Nile tilapia after single oral administration over a range of 4 different dose levels and to evaluate the effect of water temperature on the non-linearity. Possible mechanism of the observed non-linearity and its implication on dosing regimen adjustment were

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also investigated.

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2. Materials and Methods

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2.1 Chemicals

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Florfenicol analytical standard was purchased from Sigma-Aldrich (St. Louis, MO,

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USA). Acetonitrile (HPLC grade) and N,N-dimethylformamide were from Avantor Performance Materials (Center Valley, PA, USA). Propylene glycol was from AppliChem GmbH (Darmstadt,

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Germany). Sodium dihydrogen phosphate anhydrous (NaH2 PO4 ) and disodium hydrogen

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phosphate anhydrous (Na2 HPO 4 ) were from Panreac Química SLU (Barcelona, Spain).

2.2 Experimental fish

A total of 63 clinically healthy Nile tilapia (Oreochromis niloticus), weighing between 500-700 g obtained from a commercial fish farm in Chiayi County, Taiwan were reared in an outdoor concrete pond at College of Veterinary Medicine, National Chung Hsing University, Taiwan. One week before drug administration, each individual fish were acclimatized in a 70 Ltank at respective temperature of 24, 28, and 32 °C. Dissolved oxygen, pH, and total ammonia nitrogen were ≥ 5.0 ppm, 7.5-8.0, and < 1.0 ppm respectively. The animal study was approved

6

Journal Pre-proof by the Institutional Animal Care and Use Committee of National Chung Hsing University (IACUC approval No.: 107-147).

2.3 Drug administration and blood sample collection To investigate the effect of water temperature on non-linear PK of FF, the fish were

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administered with FF solution by oral gavage as described previously (Rairat et al., 2019a) at a

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dose of 10, 30, and 45 mg/kg at each water temperature level (n=7 for each group). The blood

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samples (approximately 0.40-0.45 mL) were collected from a caudal vessel at 0.25, 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 60, and 72 h post-administration without using neither anesthesia nor

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anticoagulant. The blood were allowed to clot at the room temperature and centrifuged at 3500

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rpm (2191×g; KN-70, Kubota, Japan) for 10 min; the supernatants (serum) were collected and

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kept at -20 °C until analysis (within a week). Note that the single oral dose experiments of 10, 30, and 45 mg/kg were conducted in the current study, whereas the PK data of the 15 mg/kg dose

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ur

were obtained from our previous publication (Rairat et al., 2019a).

2.4 Sample preparation, determination of FF concentration, and PK analysis The processes of sample preparation and determination of FF concentration and PK parameters were the same as described previously (Rairat et al., 2019a). Briefly, the 0.2 mL serum sample was extracted twice using 0.4 mL ethyl acetate as an extracting solvent. The supernatant was then combined and evaporated until dry. The residues were reconstituted with the mobile phase (acetonitrile and 10 mM NaH2 PO4 -Na2 HPO 4 , pH 5 at a ratio of 30:70 v/v), filtered through 0.2 μm-nylon membrane and injected (50 µL) into the HPLC system (Waters 7

Journal Pre-proof 1525, Waters, Milford, MA, USA) with a 5 μm-particle C-18 column, 150×4.6 mm (Apollo, Hichrom, UK). The flow rate and detection wavelength were 1 mL/min and 224 nm, respectively. The relevant PK parameters, namely area under the serum concentration-time curve (AUC),

absorption

half-life

(t1/2Ka),

elimination

half-life

(t1/2β),

clearance

relative

to

bioavailability (CL/F), and volume of distribution at steady-state relative to bioavailability (Vss/F) were determined by 2-compartmental model with a weighting scheme of 1/C using

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PKSolver 2.0 software (China Pharmaceutical University, Nanjing, China) (Zhang et al., 2010).

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2.7 Estimation of Michaelis-Menten kinetic parameters at 24 and 32°C

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The 2 Michaelis-Menten kinetic parameters, Michaelis-Menten constant (K m) and

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maximal velocity (Vmax ), were estimated by mathematical method (Hedaya, 2012; Persky, 2013).

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This approach requires the determination of average steady-state serum concentrations (C av(ss))

( )

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using Equation (1).

ur

after 2 different dosing rates (Dose/τ). The relationship between these 2 variables was established

( )

( )

where Dose is the amount of administered drug (mg/kg); τ is the dosing interval (h); Dose/τ is the dosing rate (mg/kg/d); Vmax is the maximal rate of the enzymatic reaction (mg/kg/d); K m is Michaelis-Menten constant (the drug concentration at which the reaction velocity is 50% of the Vmax ); Cav(ss) is the average steady-state serum concentration (μg/ml); and F is the bioavailability. To determine K m and Vmax , a pair of Dose/τ and their corresponding C av(ss) are required. At a specific temperature level, the C av(ss) of the 10 mg/kg (which followed linear PK as would be

8

Journal Pre-proof demonstrated in the Result section) was calculated as C av(ss) = AUC0-∞/τ. The AUC0-∞ is AUC from time zero to infinity after single-dose administration, determined by PKSolver 2.0 software. Another Cav(ss) values was calculated as C av(ss) = AUCss/τ. The AUCss is AUC at steady-state after multiple-dose administration, estimated by the trapezoidal method using the steady-state concentration data from our recent study (Rairat et al., 2019b). To minimize the inaccuracy associated with the trapezoidal rule, the linear up/log down approach was applied; namely, the

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linear trapezoidal method was used during the ascending phase (before C max ) and the log

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trapezoidal method was applied during the descending phase (after C max ) (Fan and de Lannoy,

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2014).

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When information of C av(ss) at the 2 different Dose/τ was available, 2 different equations

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in the same form as Equation (1) can be constructed. These 2 equations which contain 2 different unknowns (Vmax and K m) can be solved simultaneously to determine Vmax and K m. It should be

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remembered that as the drug was given only by oral route in the current study, the absolute

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bioavailability data was unavailable. Therefore, the maximal velocity would be presented as Vmax

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relative to bioavailability (Vmax /F).

2.8 Determining the optimal dosing regimen in the presence of non-linear PK and verification of the determined dosing regimen Determination of optimal dosage in the presence of non-linearity was fundamentally different from those of linear PK. To calculate the optimal dosing regimen for a predetermined MIC value, Equation (2) was proposed.

9

Journal Pre-proof (

( )

)

(

( )

( )

)

Equation (2) was a modified version of Equation (1) by replacing Cav(ss) with Cmin(ss) such that the corresponding Vmax /F and K m became Vmax (Cmin)/F and K m (Cmin), respectively. The Vmax and K m

(Cmin)

(Cmin) /F

were first calculated by the mathematical method using a pair of known Dose/τ and

their corresponding C min(ss) analogous to the case of Vmax /F and K m and their corresponding C av(ss)

of

in Equation (1). Then, these 2 parameters were used to calculate the optimal dosage (Dose/τ) at a

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given MIC by replacing the C min(ss) in Equation (2) with the target MIC value.

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To verify the appropriateness of this approach, 5 tilapia reared at 32°C in a similar

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environment as the PK study were orally administered with FF at the determined dosage (13

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mg/kg/d for the MIC of 2 µg/mL, see the Result below) once daily for 5 consecutive days and the average C min(ss) after the 5th dose was compared to the predetermined MIC level (Rairat et al.,

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2019b). The agreement between the observed C min(ss) and the target MIC should indicate if

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Equation (2) could be applied for the determination of the optimal dosing regimens in the

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presence of non-linear PK.

2.9 Statistical analysis The differences of PK parameters among 3 water temperatures and 4 doses were analyzed by two-way ANOVA, followed by Bonferroni post-hoc test. Statistical analysis was performed using IBM SPSS Statistics version 22 software (IBM Corporation, Armonk, NY, USA). In all cases, the p‐ value < 0.05 was considered statistically significant.

10

Journal Pre-proof 3. Results The serum concentrations of FF versus time after a single oral administration at 4 different doses for 3 water temperatures (24, 28, and 32°C) were shown in Fig. 1-3, respectively. Their corresponding PK parameters, namely AUC/dose, t1/2β, and CL/F were presented in Table 1. In general, increasing the administered doses resulted in disproportional increase in the AUC

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regardless of water temperature level. At 24°C, the AUC/dose were similar for the 10, 15, and 30 mg/kg groups, (20.72-22.82) (Table 1), but significantly lower than those of 45 mg/kg (27.73).

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At warmer temperature, significant differences in the AUC/dose were seen for doses higher than

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15 mg/kg. Specifically, at 28 and 32°C the AUC/dose of 10 and 15 mg/kg were significantly

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smaller than those of 30 mg/kg. Consequently, the non-linear PK of FF in Nile tilapia was

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evident when the dose was ≥ 45 mg/kg at 24°C and ≥ 30 mg/kg at 28 and 32°C. While there was no significant difference in t1/2Ka and Vss/F among 4 different doses

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(data not shown), the statistical differences were observed in t1/2β and CL/F (Table 1). The extent

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of PK parameters change was more noticeable at the higher temperature. At 24°C the AUC/dose,

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t1/2β, and CL/F for the 45 mg/kg group were 1.32, 1.30, and 1.36-times different from those of the 10 mg/kg, whereas at the 32°C the difference in these 3 parameters were 1.50, 1.42, 1.47-times, respectively. Regardless of the dose administered, increasing the water temperature lead to significantly decreased in AUC/dose and t1/2β and significantly increased in CL/F (Table 1). Given that the fish eliminated FF by hepatic metabolism to a significant degree (see the Discussion below), the 2 Michaelis-Menten kinetic parameters (K m and Vmax /F) were estimated to evaluate the temperature effect on non-linear behavior. At 24°C, the C av(ss) of the 4.5 and 10 mg/kg were 3.86 and 8.66 µg/mL, respectively and the corresponding K m and Vmax /F were

11

Journal Pre-proof calculated as 496.90 µg/mL and 583.78 mg/kg/d, respectively. At 32°C, the C av(ss) of 10 (4.47 µg/mL) and 17.5 mg/kg (11.91 µg/mL) resulted in the K m and Vmax /F values of 9.77 µg/mL and 31.85 mg/kg/d, respectively (Table 2). As our result revealed that at 32°C the characteristic PK of FF after administration of ≥ 30 mg/kg/d would follow non-linear PK, the optimal dosing regimen was determined by Equation (2). Using the data from our previous publication which indicated that the Cmin(ss) after

(Cmin) /F

and K m

(Cmin)

were calculated as 26.34 mg/kg/d and 2.02 µg/mL,

( )

( )

( )

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re

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respectively. Therefore, Equation (3) was obtained.

ro

2019b), the Vmax

of

oral administration of 8.72 and 17.5 mg/kg/d were 1 and 4 µg/mL, respectively (Rairat et al.,

For the target MIC of 2 µg/mL, the optimal dosing regimen was determined as 13 mg/kg/d based

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on Equation (3). Experimental validation revealed that multiple oral administration of 13

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MIC value of 2 µg/mL.

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mg/kg/d for 5 days at 32°C yielded an average C min(ss) of 1.96 µg/mL, very close to the target

4. Discussion To reveal the occurrence of non-linear PK, the dose proportionality study is usually conducted using a minimum of 3 different dose levels (Kwon, 2002). The parameter that is often used to check for linearity is AUC from time zero to infinity after single-dose administration (Rosenbaum, 2011; Hedaya, 2012). The AUC that is directly proportional to the dose is indicative of linear PK (within the range of the studied doses). In the current study, the AUC

12

Journal Pre-proof were increased disproportionally large with the increasing dose from 10 to 45 mg/kg in all cases. However, the increment of AUC was influenced by the temperature level. At 24°C, the AUC/dose of the 45 mg/kg were significantly higher than those ≤ 30 mg/kg suggesting that FF exhibit non-linear PK when the dose is at least 45 mg/kg. In contrast, at 28 and 32°C the upper linear range of FF was only up to less than 30 mg/kg. To the best of the authors’ knowledge this was the first report indicating that water temperature exerted a differential effect on the

of

occurrence of non-linear PK in fish.

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The present/absent of specific PK parameters changes and the direction of the changes

-p

are useful diagnostic features to determine the possible mechanism(s) behind the observed non-

re

linearity (Kwon, 2002). For example, a decrease in AUC/dose without a change in CL, t1/2β, and

lP

Vss with increasing dose levels usually indicate saturation of the absorption process, possibly as a result of poor aqueous solubility/slow dissolution, carrier-mediated absorption, saturable first-

na

pass effect (Kwon, 2002; Hedaya, 2012). A decrease in AUC/dose and increases in CL and Vss

ur

with increasing dose levels are usually indicative of saturation of plasma/serum protein binding, whereas an increase in AUC/dose and t1/2β, decrease in CL, and no change in Vss are

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characteristics of saturable drug metabolism. In our case, irrespective of the rearing temperature, increasing the administered doses from 10 to 45 mg/kg generally resulted in a larger AUC/dose, longer t1/2β, slower CL/F, but no change in t1/2Ka and Vss/F, suggesting that the underlying mechanism of the observed non-linear PK was most likely due to the saturation of FF metabolism. The dose-dependent PK in a way similar to our result (i.e., increasing AUC/dose and t1/2β, and decreasing CL/F at the higher dose) was also seen in the cases of oxytetracycline in Arctic charr over the dose range of 50 to 100 mg/kg at 6.3°C (Haug and Hals, 2000) and enrofloxacin in

13

Journal Pre-proof grass carp over the 10 to 30 mg/kg range at 22°C (Xu et al., 2013). However, the limited available data often prevents us from evaluating the possible mechanism behind these observations. It is well-known that FF was metabolized to florfenicol amine (FFA) as a major metabolite in several animal species including cattle (EMEA, 1996), chicken (EMEA, 1999a),

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pig (EMEA, 1999b), Atlantic salmon (Horsberg et al., 1996; EMEA, 2000), pacu (Piaractus mesopotamicus) (Marques et al., 2018), crucian carp (Carassius auratus gibelio) (Yang et al.,

ro

2018, 2019), Asian swamp eel (Monopterus albus) (Xie et al., 2013), yellow catfish (Tachysurus

-p

fulvidraco) (Yang et al., 2013), orange-spotted grouper (Epinephelus coioides) (Feng et al.,

re

2016), and Wuchang bream (Megalobrama amblycephala) (Huang et al., 2019). Following

lP

single oral administration, the ratio of AUC FFA/AUCFF in the fish liver ranges from 44% in Asian swamp eel (Xie et al., 2013), 76% in yellow catfish (Yang et al., 2013), 84% in orange-spotted

na

grouper (Feng et al., 2016), and 43-94% in Wuchang bream (Huang et al., 2019) indicating that a

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significant portion of FF can be metabolized to FFA in many fish species. Therefore, even though the information regarding FF metabolism in Nile tilapia is unavailable, it is reasonable to

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assume that FF is probably eliminated by metabolism to FFA to a significant degree. Although the identity of the drug-metabolizing enzyme responsible for the biotransformation of FF has yet to be revealed, circumstantial evidence indicated that CYP 3A may involve in FF metabolism in rabbits (Liu et al., 2012) and chickens (Wang et al., 2018). Treating the Pacific white shrimp with FF caused up-regulations of CYP 1A1 and CYP 3A gene expression (but not CYP 4) in the hepatopancreas in a dose-dependent manner, but the role of these enzymes in FF metabolism remained unknown (Ren et al., 2016) and warrants further elucidation.

14

Journal Pre-proof For the drug that exhibits dose-dependent PK due to saturable metabolism, the K m and Vmax /F can be determined to characterize the rate of enzymatic reaction at a specific drug concentration. Unfortunately, these 2 Michaelis-Menten kinetic parameters could not be accurately determined in the current study since our original experimental design prevented us from accurately calculate the C av(ss). In our previous 5-day multiple oral study (Rairat et al., 2019b), the occurrence of non-linear PK was unforeseen before conducting the experiment, such

of

that only Cmin and Cmax of each day were designed to be measured. As a result, the complete

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serum concentration-time profiles at steady-state necessary for precisely determination of the

-p

AUCss (which is in turn essential for the calculation of the C av(ss)) was unavailable. Nevertheless, the AUCss could be estimated based on the 3 time points (i.e., at Cmin after the 4th dose, Cmax after

re

the 5th dose, and Cmin after the 5th dose). Despite this limitation, the effect of water temperature

lP

on the K m and Vmax /F was clearly demonstrated in the current study. Both K m and Vmax /F were

na

decreased with increasing water temperature. The lower Km indicated that the enzyme was saturated at lower drug concentration which is consistent with our finding that non-linear PK of

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FF appeared the lower dose at the warmer temperature (≥ 30 mg/kg at 28 and 32°C) compared to

Jo

the cooler temperature (≥ 45 mg/kg at 24°C). It is worth mentioning that increasing temperature generally causing an increase in K m (reduced ligand binding ability) for most enzyme systems (Somero, 1995; Schulte, 2015). Nevertheless, there is some exception to this general trend. For instance, while the K m of aryl hydrocarbon hydroxylase from rainbow trout liver microsomes was higher at 18°C than 10°C (Carpenter et al., 1990), the K m of benzo(a)pyrene hydroxylase from bluegill liver microsomes was reduced when the acclimation temperature was increased from 10 to 30°C (Karr et al., 1985). These studies indicate that the effect of temperature on K m may be multifactorial. In fact,

15

Journal Pre-proof it was shown that the K m of lactate dehydrogenase from brook trout and lake trout muscles (Hochachka and Somero, 1968) and from embryos of several fish species (Klyachko and Ozernyuk, 1994) was increased when the temperature was either decreased or increased from the optimal temperature. The change in K m as a function of temperature could be attributed to protein conformational change (Somero,

1995; Schulte,

2015) or

differential isozymes

production at different temperature levels (Hochachka and Somero, 1968; Karr et al., 1985;

of

Carpenter et al., 1990). In contrast to the K m value, the Vmax was either not affected or less

ro

affected by changing temperature (Hochachka and Somero, 1968; Karr et al., 1985; Carpenter et

-p

al., 1990); and our result was in agreement with this observation.

re

The current study clearly demonstrated that in addition to the water temperature the

lP

administered dose also played a significant role in determining the PK profiles for the drug that exhibits non-linear PK. For the dose level that follow linear PK, dose-independent PK

na

parameters allow us to anticipate the serum concentration (and hence the drug effect, duration of

ur

action, depletion rate, etc.) after multiple dosing or when the administered dose and/or dosing regimen are changed. For instance, the Cav(ss) can be predicted using the serum concentration

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attained after administration of a single dose. In addition, it can be assumed that doubling the dose usually increase the duration of action by one t1/2 (Fan and de Lannoy, 2014). However, when non-linearity present, the serum concentration and PK behavior could not be accurately predicted by classical PK equations that assuming first-order kinetics. In our case, if a fish farmer treat Nile tilapia reared at 32°C with FF at double (30 mg/kg) or triple the recommended dose (45 mg/kg), the drug would remain in the fish body for a longer time and deplete with a slower rate than those expected from linear PK principle, making the fish more prone to suffer drug toxicity and tissue residue violation.

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Journal Pre-proof Comparing to our previous result (Rairat et al., 2019b), now it is clear that the determined optimal dosage at 24°C (4.5 mg/kg/d) produced the C min(ss) similar to the desired MIC because it was in the linear dose range (≤ 30 mg/kg). In contrast, the determined optimal dosage at 32°C (17.5 mg/kg/d) overestimated the expected C min(ss) because it was beyond the linear range (> 15 mg/kg). Regarding the determined optimal dosage at 28°C (11.0 mg/kg/d), despite the fact that this dose level was proven within the linear range (≤ 15 mg/kg), yet it slightly overestimated the

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expected Cmin(ss), possibly due to the drug accumulation after multiple dosing such that the linear

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range was narrower than those of single oral administration. Nevertheless, this slight mismatch

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seems of little significance clinically as the observed C min(ss) was not far greater than the desired

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MIC (i.e., 3.4 vs 2 µg/mL) and apparently the drug efficacy would not be compromised.

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For the drugs that follow first-order kinetics for the whole therapeutic range, the optimal dosing regimen can be determined by the regular PK-PD approach (Rairat et al., 2019b).

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Unfortunately, the determination of optimal dosing regimen in the presence of non-linearity is

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more complicated. Provided that the K m and Vmax /F at a given temperature were known,

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Equation (1) can be applied to calculate the optimal dosage to obtain the desired C av(ss). However, the Cav(ss) has only limited application for antimicrobial agents whereas C min(ss) is more meaningful for a time-dependent drug since its PK-PD index is T>MIC (the duration of drug concentration that over the MIC). To the best of the authors’ knowledge, no similar equation exists to determine the dose that produces the desired C min(ss) for the drug that exhibits dosedependency. Here, we proposed Equation (2) for establishing the optimal dosing regimens of FF in the presence of non-linearity when the target concentration was based on Cmin(ss) instead of the regular Cav(ss). Our result revealed that following administration of 13 mg/kg/d FF for 5 days the average Cmin(ss) was 1.96 µg/mL, very similar to the target MIC of 2 µg/mL, suggesting that

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Journal Pre-proof Equation (2) was applicable. By comparison, 5-day administration of FF at the dose rate of 17.5 mg/kg/d which was supposed to be the optimal dosage for the bacterial MIC of 2 µg/mL determined by PK-PD approach in our previous study assuming first-order kinetics (Rairat et al., 2019b) resulted in the C min(ss) of 4.0 µg/mL, a 2-fold overestimation of the target MIC. These findings clearly demonstrated that using the equation for linear PK to calculate the optimal dosing regimen when the assumption of first-order kinetics was violated would render the

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determined dosage inaccurate. Taken together the result of our present study and previous

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publication (Rairat et al., 2019b), it could be suggested that the optimal dosing regimens of FF in

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Nile tilapia cultured at 32°C for the bacterial MIC of 1, 2, and 4 µg/mL were 8.7, 13.0, and 17.5

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mg/kg, respectively.

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5. Conclusions

The study revealed the dose-dependent PK of FF in Nile tilapia reared at 3 different temperature

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levels. Irrespective of the rearing temperature, increasing the administered doses from 10 to 45

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mg/kg generally resulted in a larger AUC/dose, longer t1/2β, and slower CL/F, suggesting that the saturation of FF metabolism was most likely responsible for the observed non-linearity. At 28 and 32°C, the FF exhibited non-linear PK at a lower dose (≥ 30 mg/kg) compared to the 24°C (≥ 45 mg/kg) and these observations were correlated with the lower K m at the higher temperature. The formula for determination of optimal dosing regimen in the presence of non-linear PK was proposed based on the modification of the Michaelis-Menten equation and demonstrated in animal study with a satisfactory result.

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Journal Pre-proof Acknowledgments The authors would like to thank Gao Zheng farm, Chiayi County, Taiwan, for kindly supplied us the tilapia. This research was partially supported by the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education,

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Taiwan.

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References

-p

Boon, J.H., Nouws, J.M.F., Van der Heijden, M.H.T., Booms, G.H.R., Degen, M., 1991.

re

Disposition of flumequine in plasma of European eel (Anguilla anguilla) after a single

lP

intramuscular injection. Aquaculture. 99, 213-223. Bowser, P.R., Wooster, G.A., Stleger, J., Babish, J.G., 1992. Pharmacokinetics of enrofloxacin in

na

fingerling rainbow trout (Oncorhynchus mykiss). J Vet Pharmacol Ther. 15, 62-71.

ur

Carpenter, H.M., Fredrickson, L.S., Williams, D.E., Buhler, D.R., Curtis, L.R., 1990. The effect

Jo

of thermal acclimation on the activity of arylhydrocarbon hydroxylase in rainbow trout (Oncorhynchus mykiss). Comp Biochem Physiol C. 97, 127-132. Chang, Z., Chen, Z., Gao, H., Zhai, Q., Li, J., 2019. Pharmacokinetic profiles of florfenicol in spotted halibut, Verasper variegatus, at two water temperatures. J Vet Pharmacol Ther. 42, 121-125. De Ocenda, V.R., Almeida‐ Prieto, S., Luzardo‐ Álvarez, A., Barja, J.L., Otero‐ Espinar, F.J., Blanco‐ Méndez,

J.,

2017.

Pharmacokinetic

19

model

of

florfenicol

in

turbot

Journal Pre-proof (Scophthalmus maximus): Establishment of optimal dosage and administration in medicated feed. J Fish Dis. 40, 411-424. EMEA, 1996. Florfenicol, Summary Report (1). Committee for Veterinary Medicinal Products. EMEA, 1999a. Florfenicol (Extension to Chicken), Summary Report (3), EMEA/MRL/589/99Final. Committee for Veterinary Medicinal Products.

ro

Committee for Veterinary Medicinal Products.

of

EMEA, 1999b. Florfenicol (Extension to Pig), Summary Report (4), EMEA/MRL/591/99-Final.

-p

EMEA, 2000. Florfenicol (Extension to Fish), Summary Report (5), EMEA/MRL/760/00-Final.

re

Committee for Veterinary Medicinal Products.

lP

Fan, J., de Lannoy, I.A.M., 2014. Pharmacokinetics. Biochem Pharmacol. 87, 93-120. Feng, J.B., Huang, D.R., Zhong, M., Liu, P., Dong, J.D., 2016. Pharmacokinetics of florfenicol behaviour

of its metabolite

na

and

florfenicol amine in

orange‐ spotted

grouper

ur

(Epinephelus coioides) after oral administration. J Fish Dis. 39, 833-843.

Jo

Haug, T., Hals, P.A., 2000. Pharmacokinetics of oxytetracyline in Arctic charr (Salvelinus alpinus L.) in freshwater at low temperature. Aquaculture. 186, 175-191. Hayton, W.L., 1999. Considerations in compartmental pharmacokinetic modeling in fish, in: Smith, D.J., Gingerich, W.H., Beconi-Barker, M.G. (Eds.), Xenobiotics in Fish, Springer Science+Business Media, New York, pp. 55-72. Hedaya, M.A., 2012. Basic Pharmacokinetics. 2nd ed. CRC Press, Taylor & Francis Group, Boca Raton, Florida.

20

Journal Pre-proof Hochachka, P.W., Somero, G.N., 1968. The adaptation of enzymes to temperature. Comp Biochem Physiol. 27, 659-668. Horsberg, T.E., Hoff, K.A., Nordmo, R., 1996. Pharmacokinetics of florfenicol and its metabolite florfenicol amine in Atlantic salmon. J Aquat Anim Health. 8, 292-301. Huang, Y., Chen, X., Wang, H., Zhao, H., Luo, Y., Wu, Z., 2019. Pharmacokinetics of amblycephala) at two

water

of

florfenicol in blunt‐ snout bream (Megalobrama

ro

temperatures with single‐ dose oral administration. J Vet Pharmacol Ther. 42, 564-571.

-p

Jung, S.H., Choi, D.L., Kim, J.W., Jo, M.R., Seo, J.S., Jee, B.Y., 2008. Pharmacokinetics of oxytetracycline in olive flounder (Paralichthys olivaceus) by dipping and oral

lP

re

administration. J Fish Pathol. 21, 107-117 [in Korean]. Jung, S.H., Choi, D.L., Kim, J.W., Jo, M.R., Seo, J.S., Ji, B.Y., 2009a. Pharmacokinetics of

na

oxytetracycline in olive flounder (Paralichthys olivaceus) by intramuscular injection. J

ur

Fish Pathol. 22, 91-95 [in Korean]. Jung, S.H., Choi, D.L., Kim, J.W., Jo, M.R., Jee, B.Y., Seo, J.S., 2009b. Pharmacokinetics of

Jo

oxolinic acid in cultured olive flounder Paralichthys olivaceus by oral administration, injection and dipping. J Fish Pathol. 22, 125-135 [in Korean]. Karr, S.W., Reinert, R.E., Wade, A.E., 1985. The effects of temperature on the cytochrome P450 system of thermally acclimated bluegill. Comp Biochem Physiol C. 80, 135-139. Killen, S.S., Atkinson, D., Glazier, D.S., 2010. The intraspecific scaling of metabolic rate with body mass in fishes depends on lifestyle and temperature. Ecol Lett. 13, 184-193.

21

Journal Pre-proof Kim, J.-D., Seo, J.S., Kim, J.-W., Lee, J.S., Jung, S.H., Jee, B.-Y., Kim, J.W., Kim, E.-O., 2008. Pharmacokinetics of oral administration of oxytetracycline in eel, Anguilla japonica. J Fish Pathol. 21, 119-127 [in Korean]. Klyachko, O.S., Ozernyuk, N.D., 1994. The effect of temperature on the kinetic properties of lactate dehydrogenase from embryos of various fish species. Comp Biochem Physiol.

of

107, 593-595.

ro

Kwon, Y., 2002. Handbook of Essential Pharmacokinetics, Pharmacodynamics and Drug

-p

Metabolism for Industrial Scientists. Kluwer Academic Publishers, New York. Lin, M., Chen, Z., Ji, R., Yang, X., Wang, J., 2015. Comparative pharmacokinetics of florfenicol

lP

re

in Japanese eels at different temperature. Anim Husb Feed Sci. 7, 126-130. Liu, Y.T., Ai, X.H., Yang, H., 2009. Pharmacokinetics of florfenicol in channel catfish (Ictalurus

na

punctatus) at different water temperatures. Acta Hydrobiol Sin. 33, 1-6 [in Chinese].

ur

Liu, N., Guo, M., Mo, F., Sun, Y.H., Yuan, Z., Cao, L.H., Jiang, S.X., 2012. Involvement of P‐ glycoprotein and cytochrome P450 3A in the metabolism of florfenicol of rabbits. J Vet

Jo

Pharmacol Ther. 35, 202-205. Liu, X., Steele, J.C., Meng, X.-Z., 2017. Usage, residue, and human health risk of antibiotics in Chinese aquaculture: A review. Environ Pollut. 223, 161-169. Ludden, T.M., 1991. Nonlinear pharmacokinetics: clinical Implications. Clin Pharmacokinet. 20, 429-446.

22

Journal Pre-proof Marques, T.V., Paschoal, J.A.R., Barone, R.S.C., Cyrino, J.E.P., Rath, S., 2018. Depletion study and estimation of withdrawal periods for florfenicol and florfenicol amine in pacu (Piaractus mesopotamicus). Aquacult Res. 49, 111-119. Okocha, R.C., Olatoye, I.O., Adedeji, O.B., 2018. Food safety impacts of antimicrobial use and their residues in aquaculture. Public Health Rev. 39, 1-22.

of

Oshima, Y., Takeda, T., Katayama, S., Inoue, Y., Inoue, S., Nakayama, K., Shimasaki, Y.,

ro

Honjo, T., 2004. Relationship between temperature and pharmacokinetic parameters of

-p

florfenicol in carp (Cyprinus carpio). Jpn J Environ Toxicol. 7, 61-68. Persky, A.M., 2013. Foundations in Pharmacokinetics. UNC Eshelman School of Pharmacy,

lP

re

University of North Carolina, Chapel Hill, North Carolina. Plakas, S.M., El Said, K.R., Musser, S.M., 2000. Pharmacokinetics, tissue distribution, and

ur

14.

na

metabolism of flumequine in channel catfish (Ictalurus punctatus). Aquaculture. 187, 1-

Rairat, T., Hsieh, C.Y., Thongpiam, W., Sung, C.H., Chou, C.C., 2019a. Temperature-dependent

Jo

pharmacokinetics of florfenicol in Nile tilapia (Oreochromis niloticus) following single oral and intravenous administration. Aquaculture. 503, 483-488. Rairat, T., Hsieh, C.Y., Thongpiam, W., Chou, C.C., 2019b. Pharmacokinetic-pharmacodynamic modelling for the determination of optimal dosing regimen of florfenicol in Nile tilapia (Oreochromis niloticus) at different water temperatures and antimicrobial susceptibility levels. J Fish Dis. 42, 1181-1190.

23

Journal Pre-proof Ren, X., Pan, L., Wang, L., 2016. Tissue distribution, elimination of florfenicol and its effect on metabolic enzymes and related genes expression in the white shrimp Litopenaeus vannamei following oral administration. Aquacult Res. 47, 1584-1595. Rosenbaum, S., 2011. Basic Pharmacokinetics and Pharmacodynamics: An Integrated Textbook and Computer Simulations. John Wiley & Sons, Hoboken, New Jersey.

of

Schulte, P.M., 2015. The effects of temperature on aerobic metabolism: Towards a mechanistic

ro

understanding of the responses of ectotherms to a changing environment. J Exp Biol.

-p

218, 1856-1866.

re

Somero, G.N., 1995. Proteins and temperature. Annu Rev Physiol. 57, 43-68.

Available

source

lP

U.S. FDA, 2019. Approved Aquaculture Drugs [online]. U.S. Food and Drug Administration. https://www.fda.gov/animal-veterinary/aquaculture/approved-

na

aquaculture-drugs. (accessed September, 29 2019).

ur

Wang, G.Y., Zheng, H.H., Zhang, K.Y., Yang, F., Kong, T., Zhou, B., Jiang, S.X., 2018. The roles of cytochrome P450 and P-glycoprotein in the pharmacokinetics of florfenicol in

Jo

chickens. Iran J Vet Res. 19, 9-14. Xie, L.L., Wu, Z.X., Chen, X.X., Li, Q., Yuan, J., Liu, H., Yang, Y., 2013. Pharmacokinetics of florfenicol and its metabolite, florfenicol amine, in rice field eel (Monopterus albus) after a single‐ dose intramuscular or oral administration. J Vet Pharmacol Ther. 36, 229-235.

24

Journal Pre-proof Xu, L., Wang, H., Yang, X., Lu, L., 2013. Integrated pharmacokinetics/pharmacodynamics parameters-based dosing guidelines of enrofloxacin in grass carp Ctenopharyngodon idella to minimize selection of drug resistance. BMC Vet Res. 9, 126. Yang, Q., Xie, L., Wu, Z., Chen, X., Yang, Y., Liu, J., Zhang, Q., 2013. Pharmacokinetics of florfenicol after oral administration in yellow catfish, Pelteobagrus fulvidraco. J World

of

Aquacult Soc. 44, 586-592.

ro

Yang, F., Yang, F., Kong, T., Wang, G., Bai, D., Liu, B., 2018. Pharmacokinetics of florfenicol and its metabolite florfenicol amine in crucian carp (Carassius auratus) at three

-p

temperatures after one single intramuscular injection. J Vet Pharmacol Ther. 41, 739-

re

745.

lP

Yang, F., Yang, F., Wang, G., Kong, T., Liu, B., 2019. Pharmacokinetics of florfenicol and its

na

metabolite florfenicol amine in crucian carp (Carassius auratus) at three temperatures after single oral administration. Aquaculture. 503, 446-451.

ur

Zhang, Y., Huo, M., Zhou, J., Xie, S., 2010. PKSolver: An add-in program for pharmacokinetic

Jo

and pharmacodynamic data analysis in Microsoft Excel. Comput Methods Programs Biomed. 99, 306-314.

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Journal Pre-proof Tables Table 1. Dose-normalized area under the serum concentration-time curve (AUC/dose), elimination half-life (t1/2β), and clearance (CL/F) (mean ±SD) following oral administration of 10, 15, 30, and 45 mg/kg FF at 3 temperature levels (n=7). Dose

24°C

28°C

32°C

AUC/dose a,A

10 mg/kg

20.79 ± 2.65

15 mg/kg

21.85 ± 2.94

30 mg/kg

22.82 ± 2.74

45 mg/kg

27.73 ± 3.05

10 mg/kg

12.72 ± 1.35

15 mg/kg

12.49 ± 1.44

30 mg/kg

14.58 ± 1.29

45 mg/kg

16.50 ± 1.90

10 mg/kg

0.049 ± 0.006

15 mg/kg

0.046 ± 0.005

30 mg/kg

0.044 ± 0.005

12.10 ± 1.34

a,A

a,A b,A

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9.03 ± 0.45 9.40 ± 0.89

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a,B

11.99 ± 1.30 CL/F (L/kg/h)

a,A

ab,A ab,A

0.036 ± 0.004

b,A

c,B

a,B

11.50 ± 2.39

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c,A

b, B

19.45 ± 1.36 t1/2β (h)

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ab,A

16.33 ± 2.88

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b,A

a,B

10.72 ± 0.88

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a,A

ab,B

b,B b,B

0.073 ± 0.004

ab,B

0.084 ± 0.010 0.063 ± 0.011

a,B

bc,B

0.052 ± 0.004

c,B

9.39 ± 1.51

13.72 ± 1.16

ab,C

a,C bc, B

16.04 ± 2.41 6.76 ± 0.42 7.90 ± 0.93

c,B

a,C

ab,B

7.69 ± 0.58 9.61 ± 1.51

a,C b,C

0.094 ± 0.007 0.109 ± 0.015 0.073 ± 0.006 0.064 ± 0.010

b,C a,C c,B c,C

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45 mg/kg

13.67 ± 0.79

For each PK parameter, means with different small superscripts in each column and means with different capital superscripts in each row are significantly different from each other (p<0.05).

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Journal Pre-proof Table 2. Estimation of the Michaelis-Menten constant (K m) and maximal velocity (Vmax /F) at 24 and 32°C by mathematical method using the Equation (1) 32°C

Dose 1/τ (mg/kg/d)1

10

10

Dose 2/τ (mg/kg/d)2

4.5

17.5

Cav(ss) 1 (μg/ml)3

8.66

4.47

Cav(ss) 2 (μg/ml)4

3.86

11.91

Km (μg/ml)

496.90

9.77

Vmax /F (mg/kg/d)

583.78

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24°C

31.85

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Parameters

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Note: The single oral dose used in the present study

2

The multiple oral dose from our previous study (Rairat et al., 2019b)

3

Calculated as C av(ss) = AUC0-∞/τ. The AUC0-∞ after single oral administration is determined by

Calculated as C av(ss) = AUCss/τ. The AUCss after multiple oral administration is estimated by the

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4

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PKSolver 2.0.

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1

trapezoidal method using the steady-state concentration data from our previous study (Rairat et al., 2019b). 5

Abbreviation: Dose/τ, dosing rate; C av(ss), average steady-state serum concentration

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Journal Pre-proof Figure legends Fig. 1. Linear (above) and semi-logarithmic plots (below) of serum concentration-time profile (mean ± SD) of 10, 15, 30, and 45 mg/kg florfenicol at 24°C (n=7).

Fig. 2. Linear (above) and semi-logarithmic plots (below) of serum concentration-time profile

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(mean ± SD) of 10, 15, 30, and 45 mg/kg florfenicol at 28°C (n=7). Note that the florfenicol

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concentrations of 5/7 and 4/7 fish of the 10 and 15 mg/kg group, respectively, declined below the

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limit of quantification (33 ng/mL) at 72 h post-administration.

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Fig. 3. Linear (above) and semi-logarithmic plots (below) of serum concentration-time profile (mean ± SD) of 10, 15, 30, and 45 mg/kg florfenicol at 32°C (n=7). Note that the florfenicol

na

concentrations of 4/7 fish of the 10 mg/kg group declined below the limit of quantification (33

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ng/mL) at 60 h post-administration and all fish except the 45 mg/kg group declined below the

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limit of quantification at 72 h post-administration.

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Journal Pre-proof Figure captions

70

10 15 30 45

50

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40 30

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FF concentration (µg/mL)

60

20

0 0

12

24

48

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100

36

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-p

10

na

10 15 30 45

72

mg/kg mg/kg mg/kg mg/kg

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10

60

1

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FF concentration (µg/mL)

mg/kg mg/kg mg/kg mg/kg

0.1

0.01 0

12

24

36

48

60

72

time (h)

Fig. 1. Linear (above) and semi-logarithmic plots (below) of serum concentration-time profile (mean ± SD) of 10, 15, 30, and 45 mg/kg florfenicol at 24°C (n=7).

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Journal Pre-proof

70

10 15 30 45

50 40 30 20

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10

0

12

24

-p

0 36

48

lP

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100

10 15 30 45

72

mg/kg mg/kg mg/kg mg/kg

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10

60

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1

0.1

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FF concentration (µg/mL)

mg/kg mg/kg mg/kg mg/kg

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FF concentration (µg/mL)

60

0.01 0

12

24

36

48

60

72

time (h)

Fig. 2. Linear (above) and semi-logarithmic plots (below) of serum concentration-time profile (mean ± SD) of 10, 15, 30, and 45 mg/kg florfenicol at 28°C (n=7). Note that the florfenicol concentrations of 5/7 and 4/7 fish of the 10 and 15 mg/kg group, respectively, declined below the limit of quantification (33 ng/mL) at 72 h post-administration.

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Journal Pre-proof

10 15 30 45

80

60

of

40

20

0

12

24

-p

0 36

48

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100

lP

10

10 15 30 45

72

mg/kg mg/kg mg/kg mg/kg

na

1

60

ur

0.1

0.01

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FF concentration (µg/mL)

mg/kg mg/kg mg/kg mg/kg

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FF concentration (µg/mL)

100

0.001 0

12

24

36

48

60

72

time (h)

Fig. 3. Linear (above) and semi-logarithmic plots (below) of serum concentration-time profile (mean ± SD) of 10, 15, 30, and 45 mg/kg florfenicol at 32°C (n=7). Note that the florfenicol concentrations of 4/7 fish of the 10 mg/kg group declined below the limit of quantification (33

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Journal Pre-proof ng/mL) at 60 h post-administration and all fish except the 45 mg/kg group declined below the

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limit of quantification at 72 h post-administration.

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Journal Pre-proof

Highlights

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 The occurrence of non-linear pharmacokinetics of florfenicol was demonstrated  Temperature exerted differential effects on non-linear pharmacokinetics  The observed non-linearity was likely due to saturation of florfenicol metabolism  Method for determining optimal dosage in the presence of non-linearity was proposed

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Journal Pre-proof Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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