The Pharmacokinetics of Enrofloxacin in Adult African Clawed Frogs (Xenopus laevis)

Abstract

Pharmacokinetics of enrofloxacin, a fluoroquinolone antibiotic, was determined in adult female Xenopus laevis after single-dose administration (10 mg/kg) by intramuscular or subcutaneous injection. Frogs were evaluated at various time points until 8 h after injection. Plasma was analyzed for antibiotic concentration levels by HPLC. We computed pharmacokinetic parameters by using noncompartmental analysis of the pooled concentrations (naive pooled samples). After intramuscular administration of enrofloxacin, the half-life was 5.32 h, concentration maximum was 10.85 µg/mL, distribution volume was 841.96 mL/kg, and area under the time–concentration curve was 57.59 µg×h/mL; after subcutaneous administration these parameters were 4.08 h, 9.76 µg/mL, 915.85 mL/kg, and 47.42 µg×h/mL, respectively. According to plasma pharmacokinetics, Xenopus seem to metabolize enrofloxacin in a manner similar to mammals: low levels of the enrofloxacin metabolite, ciprofloxacin, were detected in the frogs’ habitat water and plasma. At necropsy, there were no gross or histologic signs of toxicity after single-dose administration; toxicity was not evaluated for repeated dosing. The plasma concentrations reached levels considered effective against common aquatic pathogens and suggest that a single, once-daily dose would be a reasonable regimen to consider when treating sick frogs. The treatment of sick frogs should be based on specific microbiologic identification of the pathogen and on antibiotic susceptibility testing.

Xenopus laevis, the African clawed frog, is a fully aquatic amphibian species commonly used in biomedical research. Within the last year, Xenopus have been used in several hundred published studies including, but not limited to, research in reproduction, embryology, developmental biology, neurology, endocrinology, and genetics.^{1,2,10,15,18,19,26} Female Xenopus frogs consistently produce thousands of large, easily manipulated eggs, which provide researchers with a continuous, ready supply of biological material. X. laevis frogs are susceptible to infection and systemic disease caused by several opportunistic bacterial pathogens, including Mycobacterium chelonae, Mycobacterium liflandii, Flavobacterium meningosepticum (reclassified under the species Chryseobacterium), and Aeromonas and Pseudomonas species.^5,11,12,24

Effective treatment of infections in Xenopus is hampered by a lack of pharmacokinetic data on which to base antibiotic therapy. Current antibiotic treatment regimens are based primarily on extrapolations from pharmacokinetic studies in terrestrial reptiles, semiaquatic frogs, and mammalian species. However, the relevance of these studies to a fully aquatic species like Xenopus is unclear. In aquatic species, the animal's age, sex, water temperature, environmental temperature, and metabolic state can affect the pharmacokinetics of most drugs. In addition, active metabolites of various drugs can be excreted into the water and reabsorbed, further complicating dose predictions and plasma pharmacokinetics.

Enrofloxacin is a bactericidal fluoroquinolone antimicrobial with broad-spectrum activity against gram-negative and gram-positive bacteria, including the aquatic pathogens mentioned previously. Fluoroquinolones are concentration-dependent and produce their bactericidal properties by inhibiting the enzyme DNA gyrase (a type II topoisomerase), thereby preventing supercoiling and synthesis of bacterial DNA.²³ Enrofloxacin is metabolized in most mammals, at least partially, to the active metabolite ciprofloxacin.²³ In the current report, we examined the metabolism and pharmacokinetics of enrofloxacin after single-dose administration through intramuscular and subcutaneous injection of a fully aquatic laboratory animal, Xenopus laevis. Our objective was to determine plasma pharmacokinetics for enrofloxacin and to determine whether Xenopus do indeed metabolize the drug to produce its metabolically active metabolite, ciprofloxacin.

Materials and Methods

Frog colony.

This study was conducted under a protocol approved by the Stanford University Institutional Animal Care and Use Committee. All Xenopus were purchased from a single commercial vendor (Nasco, Madison, WI). These animals were used previously on an approved protocol involving superovulation (injections with pregnant mare serum gonadotropin as well as human chorionic gonadotropin to induce egg laying) for oocyte harvesting. The frogs had not been experimentally manipulated for approximately 1 y prior to this study.

All of the Xenopus used in this project were physically healthy, sexually mature, adult female frogs (age, 4 to 5 y; snout to vent length, 10 to 12 cm; body weight, 112 to 175 g). They were fed a commercial diet (1 g per frog 3 times weekly; Frog Brittle, Nasco) given 2 h before a water change. Frogs were housed in an opaque, 300-L, self-flushing pond-style holding tank at a stocking density of approximately 1 frog per liter. Water temperature was maintained between 16 and 21 °C. Water quality parameters were tested regularly and maintained within the ranges considered to be safe for aquatic amphibians: pH, 7.0 to 8.5; average hardness, 15 to 30 dGH; total chlorine, less than 0.01 mg/L; chloramines, less than 0.01 mg/L; ammonia, less than 0.25 mg/L; nitrite, less than 0.20 mg/L; nitrate, 0.00 to 50.0 mg/L; copper, less than 0.02 g/L; water fecal coliform counts, fewer than 2000 per 100 mL; conductivity, 300 to 100 μOHM; and dissolved oxygen, 8.00 to 9.00 mg/L. The room was on a 12:12-h light cycle. Ambient temperature in the room was maintained at 23 to 25 °C.

Study design.

The number of frogs per group, sampling times, and routes of administration (Table 1) were based on data collected from small pilot studies conducted by our lab in preparation for this study. Frogs were placed randomly into 1 of 2 treatment groups according to the route of enrofloxacin (Baytril, Bayer HealthCare, Monheim, Germany) administration. After receiving an injection, each frog was housed individually in a polycarbonate small-rodent cage (dimensions, 25 × 12.5 × 7.5 cm) filled with 1 L clean water from the frog's holding tank. Water temperature was maintained between 21 and 24 °C.

Table 1.

Study design

Route of administration	No. of frogs evaluated at each time point (after dosing)										Total no. of frogs evaluated
	0 min	10 min	20 min	30 min	45 min	1 h	2 h	4 h	6 h	8 h
Intramuscular	n = 4	n = 4	n = 4	n = 4	n = 3	n = 4ab	n = 4	n = 4ab	n = 4	n = 4ab	39
Subcutaneous	n = 4	n = 4	n = 4	n = 3	n = 4	n = 4ab	n = 4	n = 4ab	n = 3	n = 4ab	38

^aWater sampled from one tank randomly

^bTissues collected from one frog randomly

Blood collection.

Approximately 5 min prior to scheduled blood collection time points, frogs were submerged in tricaine methanesulfonate (MS222) solution at a concentration of 5 g/L and buffered with 10 g sodium bicarbonate until both righting and toe-pinch reflexes were absent. The deeply anesthetized frogs were removed from the tricaine methanesulfonate solution and placed on a moist absorbent pad in dorsal recumbency. A paramedian abdominal incision was made to prevent blood loss from lacerating the ventral abdominal vein. The incision was extended anteriorly through the coelomic cavity. Chest cartilage was removed to better expose the heart. The base of the exposed heart was stabilized with a pair of forceps; blood was collected from the ventricle with a 22-gauge needle and a 3-mL heparinized syringe. Blood was placed into labeled 1.5-mL microcentrifuge tubes then spun at 14,000 × g for 15 min to allow maximal plasma collection. The plasma was decanted and placed into designated 1.5-mL microcentrifuge tubes. The plasma samples were placed in a −80 °C freezer until pharmacokinetic analysis.

Necropsy and tissue collection.

After blood collection, half of the frogs from each subgroup were chosen randomly for necropsy and tissue collection. The organs were examined for gross lesions, and the following tissues were taken for histologic examination: brain, heart, spleen, liver, kidney, lung, and quadriceps muscle at the injection site. Some tissues were frozen at −80 °C for future analysis. Remaining tissues were fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Tissues were evaluated by a veterinary pathologist for any gross or histologic changes resulting from enrofloxacin treatment.

Pharmacokinetic analysis.

HPLC was used to determine the plasma concentrations of enrofloxacin and the metabolite ciprofloxacin. ^3,4 Plasma drug concentrations were plotted on linear and semilogarithmic graphs for analysis and visual assessment of the best model for pharmacokinetic analysis. Because this study involved sparse sampling from individual animals, with multiple samples collected from the study population, a nonlinear mixed-effects modeling analysis was attempted (population pharmacokinetics) by using the program WinNonMix (Pharsight Corporation, Mountain View, CA). This analysis is used when it is not possible to estimate nonlinear regression parameters for each individual. However, we were not able to get a fit that provided meaningful population estimates, and a noncompartmental analysis using naïve averaged data from the time points was performed. Therefore, pharmacokinetic analysis was performed by using a commercial pharmacokinetic program (WinNonLin version 5.2, Pharsight Corporation). For the noncompartmental analysis, the area under the plasma concentration versus time curve (AUC) from time 0 to the last measured concentration was calculated using the log-linear trapezoidal method. The AUC from time 0 to infinity was calculated by adding the terminal portion of the curve, estimated from the relationship Cn/λ_Z, to the AUC_0-cn, where λ_Z is the terminal slope of the curve and Cn is the last measured concentration point. Values for the maximal plasma concentration after dosing (C_max) and time to maximum plasma concentration were taken directly from the data. Half-lives were calculated from the terminal slope: t_1/2 = ln 2.0 / (terminal rate constant), where ln 2.0 is the natural logarithm of 2.0. Secondary parameters were calculated according to previously described methods.¹⁴ Plasma values that were dependent on dose were listed as ‘per fraction absorbed.’ The limit of quantification was 0.05 µg/mL, which was the lowest level that gave a linear response on our calibration curve.

Water analysis.

Individual water samples were collected for HPLC analysis to assess whether significant amounts of antibiotic enrofloxacin or the active metabolite ciprofloxacin were excreted from treated frogs into the surrounding water. Three water samples (approximately 50 mL each) were taken from randomly selected individual holding tanks.

Results

Subcutaneous administration.

The plasma profile after subcutaneous enrofloxacin administration to adult female Xenopus (Figure 1) best fit a noncompartmental model. Regarding pharmacokinetic results (Table 2), the terminal half-life, volume of distribution, and clearance were 4.08 h, 915.86 mL/kg, and 155.52 mL/min/kg, respectively. The 30-min and 6-h treatment groups each contained only 3 samples due to insufficient blood collection from 2 of the frogs (Table 1). Ciprofloxacin, the active metabolite of enrofloxacin, was detected at low levels in the plasma after subcutaneous injection (Figure 2).

Figure 1.

Concentration (µg/mL; mean ± 1 SD) of enrofloxacin in the plasma of Xenopus laevis after single-dose (10 mg/kg) subcutaneous injection of enrofloxacin (22.7 mg/mL) as analyzed by HPLC.

Table 2.

Pharmacokinetic parameters for enrofloxacin in plasma after subcutaneous and intramuscular administration of enrofloxacin to frogs (Xenopus laevis)

	Intramuscular dose (10 mg/kg)		Subcutaneous dose (10 mg/kg)
	Ciprofloxacin	Enrofloxacin	Ciprofloxacin	Enrofloxacin
Elimination rate (1/h)	0.09	0.13	0.12	0.17
Half-life (h)	7.36	5.32	5.70	4.08
Time to reach peak plasma concentration (h)	2	1	2	0.75
Peak plasma concentration (µg/mL)	0.24	10.85	0.27	9.79
AUC (h×µg/mL)	1.52	57.59	1.62	47.42
Distribution volume per fraction absorbed (mL/kg)	34144.6	841.96	30026.90	915.86
Clearance per fraction absorbed (mL/h/kg)	3216.53	109.61	3650.61	155.52
Mean residence time (h)	11.54	8.01	8.82	6.12

Figure 2.

Concentration (µg/mL; mean ± 1 SD) of ciprofloxacin in the plasma of Xenopus laevis after single-dose subcutaneous injection (10 mg/kg) of enrofloxacin (22.7 mg/mL) as analyzed by HPLC.

Intramuscular administration.

The plasma profile for intramuscular administration of enrofloxacin is shown in Figure 3. Regarding pharmacokinetic results from the noncompartmental analysis (Table 2), the terminal half-life, volume of distribution, and clearance were 5.32 h, 841.96 mL/kg, and 109.61 mL/min/kg, respectively. Ciprofloxacin was detected at low levels in the plasma (Figure 4).

Figure 3.

Concentration (µg/mL; mean ± 1 SD) of enrofloxacin in the plasma of Xenopus laevis after single-dose (10 mg/kg) intramuscular injection of enrofloxacin (22.7 mg/mL) as analyzed by HPLC.

Figure 4.

Concentration (µg/mL; mean ± 1 SD) of ciprofloxacin in the plasma of Xenopus laevis after single-dose intramuscular injection (10 mg/kg) of enrofloxacin (22.7 mg/mL) as analyzed by HPLC.

Water analysis.

HPLC results from water analysis are reported in Figures 5 and 6 and Table 3. The antibiotic concentrations in the water at 1, 4, and 8 h after frogs were treated with enrofloxacin either subcutaneously or intramuscularly are shown in Table 3.

Figure 5.

Enrofloxacin concentration (µg/mL; by HPLC) in water collected from holding tanks at 1, 4, and 8 h after antibiotic treatment of frogs.

Figure 6.

Ciprofloxacin concentration (µg/mL; by HPLC) of water collected from holding tanks 1, 4, and 8 h after antibiotic treatment of frogs.

Table 3.

Antibiotic concentrations (µg/mL) in habitat water at various times after treatment of frogs with enrofloxacin

	Intramuscular dosage		Subcutaneous dosage
Time (h)	Enrofloxacin	Ciprofloxacin	Enrofloxacin	Ciprofloxacin
1	0.014	0.007	0.055	0.000
4	0.075	0.016	0.090	0.019
8	0.057	0.023	0.170	0.068

Discussion

Pharmacokinetic studies in aquatic species present many challenges. Water quality, aestivation or hibernation state, age, sex, and environmental temperatures can affect the metabolism of aquatic frogs and thus affect the pharmacokinetics of systemically administered drugs. For example, high environmental temperature is known to increase the systemic clearance of enrofloxacin in nonmammals.²³ In addition, the aquatic environment can become contaminated with the drug or its active metabolites because of normal drug metabolism and excretion. Once the environment is contaminated, reabsorption can occur, especially in aquatic amphibians whose skin is highly permeable to certain compounds and who may ingest water during feeding. Our HPLC water analysis (Figures 5 and 6) suggests that the enrofloxacin concentration in the aquatic habitat increases over time—for example, the drug accumulates in the water. If the frogs reabsorb excreted drug, there is potential for drug toxicity.

Administration of a single 10-mg/kg intramuscular or subcutaneous dose of enrofloxacin was not associated with clinical signs of illness in our frogs. Necropsy revealed no negative affects (liver or kidney toxicity, skin irritation) in organs or tissues based on gross and histologic examination. However, toxicity was not evaluated for repeated dosing over time.

The high peak and rapid increase in the plasma concentrations of enrofloxacin in Xenopus suggests good absorption when the drug is given intramuscularly or subcutaneously. The subcutaneous:intramuscular AUC ratio was 0.82. This high ratio suggests that there is no significant difference between the 2 routes of administration when the variability associated with plasma concentration data is taken into consideration. Bioavailability (extent of systemic absorption) can only be measured by comparing the intramuscular or subcutaneous dose AUC with the AUC from an intravenous dose. Because it is not possible to administer an intravenous dose in this species, the bioavailability after intramuscular and subcutaneous injections could not be measured in this study.

The plasma levels achieved in this study indicate efficacy against several common aquatic pathogens, including Aeromonas salmonicida subsp. salmonicida, Aeromonas hydrophila, and Escherichia coli ^13,21 (Table 4). Ideally the ratio between the AUC_0-∞ and minimal inhibitory concentration (MIC) of quinolones should be at least 100 to 125 for Pseudomonas aeruginosa and gram-negative bacteria, but an AUC_0-∞:MIC ratio of 30 or greater seems sufficient in most gram-positive bacteria.^9,20,22,27 The C_max:MIC ratio should be at least 8 to 10 for effective bactericidal activity.²¹ Both the AUC_0-∞:MIC and C_max: MIC ratios for enrofloxacin were well above the recommended therapeutic levels for the bacteria listed. Although the enrofloxacin plasma concentration varied slightly among individual frogs within a given subgroup, we believe the pharmacokinetic analysis (Table 2) in this study provides a reliable estimate of our sample population. In light of the values for t_1/2 and C_max, a single dose of 10 mg/kg enrofloxacin given either subcutaneously or intramuscularly likely will provide an effective dose against pathogens known to be susceptible to such concentrations. The half-life also gives a basis for the appropriate dosing frequency for this antibiotic. The half-lives of enrofloxacin for dogs (4.07 h), cats (6.7 h), horses (6.7 h), salmon (34.2 h), carp (17.9 h), and catfish (17.44 h) suggest that Xenopus metabolize enrofloxacin more similarly to mammals than to fish.^6,14,16,25

Table 4.

Pharmacokinetic and pharmacodynamic relationships of enrofloxacin and select aquatic bacteria.

	MIC (µg/mL)	Reference	AUC0-∞:MIC	Cmax: MIC
Escherichia coli	0.004–0.015	23	>3000	>600
Aeromonas salmonicida subsp. salmonicida	0.008–0.03	23	>1000	>300
Aeromonas hydrophila	0.25†	13	>180	> 30

Enrofloxacin is well known to be converted to the metabolically active metabolite ciprofloxacin in many mammalian species, but the extent of this conversion is somewhat variable in other species.^7,8,17 In the present study, we found detectable levels of ciprofloxacin in all samples of treated animals (Tables 2 and 3), but the levels were fairly low and will require further investigation.

In summary, enrofloxacin was absorbed rapidly by laboratory Xenopus after either subcutaneous or intramuscular administration at a dose of 10 mg/kg, with no signs of toxicity associated with this dose or routes of administration. Small amounts of enrofloxacin's active metabolite, ciprofloxacin, were found in the plasma and excreted in the water. The peak plasma concentrations, half-life, and AUC produced after a single dose of enrofloxacin were sufficient to reach therapeutic levels considered effective against common aquatic pathogens and suggest that a single, once-daily dose would be a starting point to consider when treating sick frogs. To minimize drug reabsorption after excretion, the habitat water should be changed daily during treatment. Treatment of sick frogs ideally is based on microbiologic identification of the specific pathogen and susceptibility testing.