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Comparative pharmacokinetics of enrofloxacin and ciprofloxacin in lactating dairy cows and beef steers following intravenous administration of enrofloxacin
Research in Veterinary Science 89 (2010) 230–235
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Comparative pharmacokinetics of enrofloxacin and ciprofloxacin in lactating dairy cows and beef steers following intravenous administration of enrofloxacin O.R. Idowu *, J.O. Peggins, R. Cullison, J. von Bredow US Food and Drug Administration, Center for Veterinary Medicine, 8401 Muirkirk Road, Laurel, MD 20708, USA
a r t i c l e
i n f o
Article history: Accepted 31 December 2009
Keywords: Enrofloxacin Ciprofloxacin Pharmacokinetics Lactating cows Steers
a b s t r a c t The comparative pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin were investigated in lactating cows and beef steers. The plasma elimination half-life of either enrofloxacin or ciprofloxacin was shorter in cows than in steers. The overall production of ciprofloxacin was slightly higher in steers than in cows (metabolite ratio: 64% and 59%, respectively). There was no significant difference in plasma protein binding of enrofloxacin between cows (percent bound: 59.4%) and steers (percent bound: 60.8%). Ciprofloxacin was more extensively bound to plasma proteins in steers (percent bound: 49.6%) than in cows (percent bound: 33.8%). The steady state volume of distribution of enrofloxacin is comparable in cows (1.55 L/kg) and steers (1.59 L/kg). Within either bovine class, plasma elimination half-life of enrofloxacin and ciprofloxacin are comparable, while plasma protein binding was higher for enrofloxacin than for ciprofloxacin. Ciprofloxacin was more concentrated in milk than enrofloxacin. Published by Elsevier Ltd.
1. Introduction Fluoroquinolones (FQs) are antibacterial agents related to nalidixic acid. FQs are used in both human and veterinary medicine to treat a variety of infections (Campoli-Richards et al., 1988; Neu, 1988; Abadia et al., 1995; Brown, 1996). Enrofloxacin was developed exclusively for use in animals. Like other FQs, enrofloxacin exhibits a broad spectrum of antibacterial activity, against both Gram-positive and Gram-negative bacteria, in diseased animals. In the United States, enrofloxacin is approved for use in beef cattle and calves (excluding veal calves), chickens and turkeys not laying eggs for human consumption, and in cats and dogs. Ciprofloxacin is a major, active metabolite of enrofloxacin in different species and is formed by the de-ethylation of enrofloxacin. Ciprofloxacin was the first of the two compounds to be developed and is only approved for use in humans (Tyczkowska et al., 1989; Flammer et al., 1991). The present study was planned to characterize the pharmacokinetics of enrofloxacin and its major metabolite, ciprofloxacin, in lactating dairy cattle and beef steers after intravenous administration of enrofloxacin. The pharmacokinetics of enrofloxacin in lactating Ayshire cows have been reported (Kaartinen et al., 1995). However, the study may not have yielded a true picture of the pharmacokinetics of enrofloxacin in lactating cows because the data presented were based on a microbiological assay of enrofloxacin in serum which measured both enrofloxacin and its active * Corresponding author. Tel.: +1 91 2402768215; fax: +1 91 2402768118. E-mail address: [email protected] (O.R. Idowu). 0034-5288/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.rvsc.2009.12.019
metabolites, including ciprofloxacin which is known to be more active than enrofloxacin. The microbiological assay based approach also did not allow the separate characterization of the pharmacokinetics of ciprofloxacin formed from enrofloxacin. A short communication on the pharmacokinetics of enrofloxacin and ciprofloxacin in non-lactating Jersey cows was published during the present study (Varma et al., 2003). The analytical method used during this study could only measure the plasma concentrations of ciprofloxacin and enrofloxacin for 12 h following drug administration. There are no prior investigations of the pharmacokinetics of enrofloxacin or ciprofloxacin in beef steers. The purpose of the present study was to compare the pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin in lactating cows and beef steers in order to establish if there are differences in the disposition of the two compounds between these two bovine production classes. 2. Materials and methods 2.1. Animals Six lactating Holstein dairy cows (584–727 kg) were obtained from the USDA herd. Six Angus steers (159–213 kg) were obtained from a commercial sale barn. Animals assigned to the study were identified with a number 1–6. This study was approved by the FDA Center for Veterinary Medicine Animal Care and Use Committee and was in compliance with their guidelines on care and use of animals.
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All animals were catheterized in the jugular vein the morning of dosing. Enrofloxacin (BaytrilÒ, 100 mg/ml, Bayer Corporation, Agriculture Division, Shawnee Mission, Kansas, USA) was administered (5 mg/kg body weight) as an I.V. bolus via the indwelling jugular catheter. Following dosing, blood samples were collected periodically via a needle, and heparin-treated syringe. Blood samples were collected at 0.17, 0.33, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 11, 13, 15, 17, 23, 29 and 35 h after administration of enrofloxacin. Plasma was separated by centrifuging the blood sample at 3500 rpm (2460g) for 15 min at 4 °C. Plasma samples were stored at 80 °C until analyzed. Each dairy cow was also milked periodically, starting at 2 h post-dosing and continuing until 131 h post-dosing. The milk was weighed and the weights recorded on data sheets. Aliquots of milk were taken and stored at 80 °C until analyzed. 2.3. HPLC analysis Concentrations of enrofloxacin and ciprofloxacin were measured in plasma and milk using reverse-phase high performance liquid chromatography (HPLC) with either fluorometric or mass spectrometric detection. Details of the method validation studies are described elsewhere (Idowu and Peggins, 2004). Briefly, a 200 lL aliquot of internal standard solution of sarafloxacin (concentration 1 ng/ml) was placed in a 15 mL, glass centrifuge tube using an adjustable pipette, followed by 1 mL of plasma (or milk) sample containing enrofloxacin and ciprofloxacin. For plasma sample, 100 lL of concentrated o-phosphoric acid was added (50 lL for milk sample). After shaking the mixture briefly (about 5 s) on a vortex-mixer, 2 mL of acetonitrile was added. The centrifuge tube was capped, and shaken at high speed on a vortex mixer for about 10 s. After centrifuging the mixture for 5 min at 4000 rpm and 4 °C, the supernatant was decanted into another 15 mL glass centrifuge tube and 3 mL of dichloromethane was added. The mixture was shaken on a vortex-mixer for about 10 s at high speed and then centrifuged for 5 min at 4000 rpm (2840g) and 4 °C. For chromatography, the upper, aqueous layer was transferred into an autosampler vial, using a Pasteur pipet. The aqueous plasma (or milk) extracts were analyzed for ciprofloxacin and enrofloxacin using the following HPLC conditions: Chromatographic system: Hewlett–Packard Series 1100. Column: PLRP-S 100A 5 l 150 4.6 mm (Phenomenex, Inc., Torrance, CA). Fluorescence detector: Hewlett–Packard 1046A; kexc: 280 nm; kemi: 460 nm. Mobile phase: isocratic: 20% aqueous formic acid:methanol:acetonitrile (75:13:12, v/v/v); flow rate 1 mL/min. Injection volume: 150 lL. The aqueous plasma (or milk) extracts were analyzed for enrofloxacin using the following HPLC– MS conditions: LC/MS SYSTEM: API2000 LC–MS/MS. Column – C8, 2.0 30 mm; 3.5 l particle size. Mobile phase: gradient of 0.1% formic acid (mobile phase A) and a 50:50 (v/v) mixture of acetonitrile and methanol (mobile phase B). The following mobile phase gradient was used: 0–0.5 min: 80% mobile phase A: 20% mobile phase B; 0.5–3.0 min: 30% mobile phase A: 70% mobile phase B. Total run time was about 6 min, including the time for re-equilibration of the column. Mass spectrometer: APCI source; MS/MS (Multiple Reaction Monitoring) mode. Ciprofloxacin levels in plasma were determined by HPLC with fluorescence detection, while enrofloxacin levels in plasma were determined by HPLC with mass spectrometric detection. Milk levels of both drugs were determined by HPLC with mass spectrometric detection.
Centricon-10 (Millipore) centrifugal filter (cut off 10,000 Da) device and centrifuged at 5000g for 9 h in Sorval RC-5 refrigerated centrifuge at 10 °C. A 500 lL aliquot of the plasma ultra-filtrate was placed in a test tube containing 200 lL of the sarafloxacin internal standard solution followed by 50 lL of concentrated ophosphoric acid. The mixture was mixed and then transferred to an autosampler vial, for analysis using the HPLC method described above. A set of plasma samples from one of the cows and a set of samples from one of the steers were used for the plasma protein binding studies. 2.5. Data analysis The plasma concentration–time data were analyzed using WinNonlin (Pharsight Corp., Cary, NC, USA), a recursive nonlinear pharmacokinetic curve fitting program. The most appropriate model was selected based on the Akaike information criteria (Yamaoka et al., 1978). The data for each animal were fit using a simple sum of 2 or 3 exponentials model. The dose event was modeled as a first order, bolus input. Statistical analysis of pharmacokinetic parameter estimates was performed using a two-tailed nonparametric t-test. Differences between means were considered significant where p 6 0.05.
3. Results The plasma concentration vs time curves for enrofloxacin and ciprofloxacin are shown in Fig. 1. Pharmacokinetic parameters, calculated from the plasma concentration–time data for enrofloxacin in lactating dairy cows, are shown in Table 1. These are terminal half-life (t1/2); mean residence time (MRT); volume of distribution (Vd); steady state volume of distribution (Vss); total body clearance (Cl); and area under the curve (zero to infinity) (AUC0–1). Similarly, pharmacokinetic parameters calculated from the plasma concentration–time data for enrofloxacin in beef steers are shown in Table 2. Pharmacokinetic parameters calculated from the plasma concentration–time data for ciprofloxacin in lactating dairy cows are shown in Table 3, while the pharmacokinetic parameters for ciprofloxacin in beef steers are shown in Table 4. A comparison of the pharmacokinetic parameters for enrofloxacin between the two groups of animals is shown in Table 5, while a comparison of the pharmacokinetic parameters for ciprofloxacin between the two groups of animals is shown in Table 6.
10000
Conc (ng/mL)
2.2. Animal dosing and sample collection
Cipro in Cows Cipro in Steers Enro in Cows Enro in Steers
1000
100
10
1 0
2.4. Protein binding Protein binding was measured by ultra-filtration. A set of plasma samples from a cow (or a steer) was transferred to
5
10
15
20
25
30
35
40
Time (h) Fig. 1. Plasma enrofloxacin and ciprofloxacin levels in lactating dairy cows and steers after after I.V. administration of enrofloxacin to dairy cows.
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Table 1 Pharmacokinetic data for enrofloxacin in lactating dairy cows following intravenous administration of enrofloxacin.
Cow 1 Cow 2 Cow 3 Cow 4 Cow 5 Cow 6 Mean ± S.E.
T1/2 (h)
MRT (h)
Vss (L/kg)
Vd (L/kg)
Cl (L/h/kg)
AUC0–1 (lgh/mL)
4.63 4.31 2.05 4.07 4.6 2.48 3.69 ± 0.41
1.23 1.05 0.66 1.21 0.96 1.18 1.05 ± 0.08
2.37 1.69 0.63 1.66 1.29 1.73 1.56 ± 0.21
1.01 0.86 0.13 1.22 0.60 0.94 0.79 ± 0.16
1.93 1.61 0.96 1.38 1.35 1.46 1.45 ± 0.12
2.59 3.11 5.23 3.63 3.71 3.43 3.62 ± 0.36
Table 2 Pharmacokinetic data for enrofloxacin in beef steers following intravenous administration of enrofloxacin.
Steer 1 Steer 2 Steer 3 Steer 4 Steer 5 Steer 6 Mean ± S.E.
T1/2 (h)
MRT (h)
Vss (L/kg)
Vd (L/kg)
Cl (L/h/kg)
AUC0–1 (lgh/mL)
5.64 7.79 3.87 4.92 3.53 5.16 5.15 ± 0.57
2.54 2.40 2.04 2.33 2.23 2.09 2.27 ± 0.07
1.81 1.94 1.41 1.48 1.47 1.45 1.59 ± 0.08
0.90 0.96 0.72 0.86 1.00 1.19 0.94 ± 0.04
0.71 0.81 0.69 0.64 0.66 0.70 0.70 ± 0.02
7.00 6.18 7.22 7.88 7.61 7.19 7.18 ± 0.24
Table 3 Pharmacokinetic data for ciprofloxacin in lactating dairy cows following intravenous administration of enrofloxacin.
Cow 1 Cow 2 Cow 3 Cow 4 Cow 5 Cow 6 Mean ± S.E.
MRT (h)
T1/2 (h)
Cmax (lg/mL)
AUC0–1 (lgh/mL)
2.66 2.66 3.06 3.59 3.09 2.64 2.95 ± 0.15
2.07 2.60 3.35 3.99 3.44 2.32 2.96 ± 0.30
1.41 1.11 0.83 1.03 1.34 1.38 1.19 ± 0.09
1.92 1.93 2.10 2.45 2.31 2.14 2.14 ± 0.08
Table 4 Pharmacokinetic data for ciprofloxacin in beef steers following intravenous administration of enrofloxacin.
Steer 1 Steer 2 Steer 3 Steer 4 Steer 5 Steer 6 Mean ± S.E.
T1/2 (h)
Cmax (lg/mL)
Tmax (h)
AUC0–1 (lgh/mL)
5.46 17.9 2.80 3.63 3.44 12.4 7.60 ± 2.5
0.75 0.66 0.74 0.64 0.46 0.54 0.63 ± 0.04
0.88 0.65 0.91 0.89 1.17 0.99 0.92 ± 0.08
6.58 6.11 3.76 3.94 2.91 4.28 4.60 ± 0.57
Table 5 Comparison of pharmacokinetic parameters for enrofloxacin in lactating dairy cows and beef steers.
Dairy cows Beef steers
T1/2 (h)
MRT (h)
Cl (L/h/kg)
Vss (L/kg)
Vd (L/kg)
3.69 ± 0.41 5.15 ± 0.57
1.05 ± 0.08 2.27 ± 0.07
1.45 ± 0.12 0.70 ± 0.02
1.56 ± 0.21 1.59 ± 0.08
0.79 ± 0.16 0.94 ± 0.04
Table 6 Comparison of pharmacokinetic parameters for ciprofloxacin in lactating dairy cows and beef steers.
Dairy cows Beef steers
Half-life, t1/2 (h)
Cmax (lg/mL)
AUCciprofloxacin/AUCenrofloxacin
2.96 ± 0.3 7.60 ± 2.5
1.19 ± 0.09 0.630 ± 0.04
0.59 0.64
Plots of the ratio of the concentration of free (unbound) enrofloxacin to the total enrofloxacin concentration in plasma from cows and steers are shown in Fig. 2. Similar plots of the ratio of
the concentration of free (unbound) ciprofloxacin to the total ciprofloxacin concentration in plasma from cows and steers are shown in Fig. 3. In order to compare the protein binding of enrofloxacin to that of ciprofloxacin, a plot of the ratio of free (unbound) to total concentration for the two compounds in cow plasma is presented in Fig. 4. The protein binding data, expressed in terms percentage of enrofloxacin or ciprofloxacin bound to plasma proteins from the two bovine classes are summarized in Table 7. The variation of the milk concentration of enrofloxacin and ciprofloxacin with time following I.V. administration of enrofloxacin to lactating cows is illustrated in Fig. 5.
4. Discussion The plasma concentration–time profile following a single I.V. dose of enrofloxacin in all 6 lactating cows and five of the steers was best fitted by a 3-compartment open model. This is comparable with plasma concentration–time profile following a single I.V. dose of enrofloxacin in pregnant mid-lactating dairy cows (Malbe et al., 1996). This is in contrast with literature reports in which a 2-compartment model was found to provide the best fit for I.V. enrofloxacin plasma concentration–time data in some species, including non-lactating cows (Varma et al., 2003; Bregante et al., 1999) non-lactating sheep (Bregante et al., 1999), and lactating sheep (Haritova et al., 2003). The plasma concentration–time profile in one of the steers used in the present study was best fitted by a 2-compartment open model. A comparison of the pharmacokinetic parameters for enrofloxacin in the two classes of animals, as summarized in Table 5, show that the clearance of enrofloxacin was significantly faster in cows compared to steers. This is further reflected in the significantly longer elimination half-life and Mean Residence Time (MRT) in steers. There was no significant difference between cows and steers in the steady state volume of distribution, or volume of distribution. This suggests that the differences in half-life and clearance between cows and steers were not due to differences in the body distribution of enrofloxacin in the two animal classes. The clearance of enrofloxacin in lactating dairy cows observed in the present study (1.45 L/h/kg) is comparable with that reported for mid-lactating pregnant cows (1.26 L/ h/kg/; Malbe et al., 1996) and non-lactating cows (1.14 L/h/kg; Varma et al., 2003).
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0.5 0.45 0.4
Free/Total ratio
0.35 0.3 Cow
0.25
Steer 0.2 0.15 0.1 0.05 0 0.17 0.33 0.5
0.75
1
1.5
2
2.5
3 4 Time (h)
5
6
7
8
9
11
Fig. 2. Ratio of free to total plasma levels of enrofloxacin in lactating dairy cows and beef steers.
Table 7 Comparison of protein binding data for enrofloxacin and ciprofloxacin in lactating dairy cows and beef steers.
0.90
0.70
Percent plasma protein binding (mean ± S.E.)
0.60 Steer plasma Cow plasma
0.50
Enrofloxacin
Ciprofloxacin
60.8 ± 1.05 59.4 ± 1.48
49.6 ± 2.58 33.75 ± 1.73
0.40 Cow Steer
0.30 0.20
A comparison of the pharmacokinetic parameters for ciprofloxacin in the two classes of animals, as summarized in Table 6, show that the elimination half-life of ciprofloxacin was significantly longer in steers compared to cows. The mean plasma half-life of ciprofloxacin in steers is apparently skewed by the relatively high half-life values for two of the steers. However, even after excluding the data from these two steers from the calculations, the mean half-life of ciprofloxacin in the remaining 4 steers (3.83 ± 0.57 h)
0.10 0.00 1.00
2.00
3.00
5.00
7.00
9.00
Time (h) Fig. 3. Ratio of free to total plasma levels of ciprofloxacin in lactating dairy cows and beef steers.
Cow Enro Free/Total ratio
0.9
Cow Cipro Free/Total ratio 0.8 0.7
0.5 0.4 0.3 0.2 0.1
11
9
8
7
6
2. 5
2
1
1. 5
5
75
0.
0.
03
.3
0 0. 17
Free/Total ratio
0.6
5
0.50
4
0.17
3
Free/Total ratio
0.80
Time (h) Fig. 4. Ratio of free to total plasma levels of enrofloxacin and ciprofloxacin in dairy cows.
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10000
Milk Enrofloxacin
1000
Conc (ng/mL)
Mlk Ciprofloxacin
100
10
1
0
20
40
60
80
100
120
140
Time (h) Fig. 5. Enrofloxacin and ciprofloxacin levels in milk after I.V. administration of enrofloxacin to dairy cows.
is still significantly longer than the mean half-life of ciprofloxacin lactating dairy cows. To compare the production levels of ciprofloxacin in the two animal classes the ratio of AUC (ciprofloxacin) to AUC (enrofloxacin) were calculated for the two classes. As shown in Table 6, the metabolic conversion of enrofloxacin to ciprofloxacin was 59% in dairy cows and 64% in beef steers. The greater overall production level of ciprofloxacin in steers may partly account for the longer persistence (longer half-life) of ciprofloxacin in steer plasma compared to lactating cows. The metabolic conversion of enrofloxacin to ciprofloxacin was 29% in mid-lactating pregnant cows (Malbe et al., 1996). The average metabolic conversion of enrofloxacin to ciprofloxacin in lactating Ayrshire cows was reported to be 35% by (Kaartinen et al., 1995). Compared to other animals, the metabolic conversion of enrofloxacin to ciprofloxacin, after I.V. administration of enrofloxacin, was 35% in sheep (Mengozzi et al., 1996), and 23% in female goats (Rao et al., 2002). As shown in Fig. 1, the maximum concentration of ciprofloxacin in lactating cows was attained very rapidly and Tmax (time to reach maximum concentration) had been attained before the first plasma sample was taken at 10 min post-dose. Ciprofloxacin has been reported to appear in plasma, at a relatively high concentration, within 2 min following I.V. administration of enrofloxacin to non-lactating Jersey cows, even though peak plasma concentration was attained 30 min post-dose (Varma et al., 2003). Data from the present study suggest a much more rapid metabolism of enrofloxacin to ciprofloxacin by the lactating dairy cow compared to the non-lactating cow. In contrast, the production of ciprofloxacin was relatively slower in the beef steers. This difference in the rate of production of ciprofloxacin between the two animal classes is also reflected in the significantly higher maximum plasma concentration (Cmax) of ciprofloxacin in dairy cows, as shown in Table 6. It should be noted, however, that Tmax for ciprofloxacin was attained in about an hour in the beef steers, and this is still later than the Tmax of 30 min reported for non-lactating cows (Varma et al., 2003). Thus it seems, in general, beef steers metabolize enrofloxacin more slowly than cows, even though the overall production of ciprofloxacin from enrofloxacin is slightly higher in steers than in lactating cows. The plasma protein binding data, illustrated in Figs. 2–4 (and summarized in Table 7) show that the extent of plasma protein binding of enrofloxacin was similar in both classes of animals (Fig. 2). This is confirmed with the data in Table 7 which shows there is no significant difference in the percentage of enrofloxacin
bound to plasma protein in lactating cows compared to steers. In contrast, the percentage of ciprofloxacin bound to plasma protein in steers is significantly higher than the percentage of ciprofloxacin bound to plasma protein in lactating cows (Fig. 3 and Table 7). The more extensive plasma protein binding of ciprofloxacin in steers may explain, in part, the longer elimination half-life of ciprofloxacin in steers compared to lactating cows. Within either bovine class, enrofloxacin was more extensively bound to plasma protein than ciprofloxacin (Fig. 4 and Table 7). As shown in Fig. 4, the free fraction of ciprofloxacin was over one and a half times greater than that of enrofloxacin. The in vivo percentage of bound enrofloxacin (59.4%) and bound ciprofloxacin (33.8%) reported in the present study is in close agreement with the respective values of 56% and 31% reported earlier, based on the in vitro incubation of enrofloxacin and ciprofloxacin with bovine plasma (Villa et al., 1997). The disposition of enrofloxacin and ciprofloxacin in milk following a single oral dose of enrofloxacin in lactating dairy cows is illustrated in Fig. 5, showing the rapid disappearance of enrofloxacin from milk and the higher concentration of ciprofloxacin in milk, compared to enrofloxacin. In the present study, enrofloxacin could not be detected in milk from any of the cows beyond 17 h postdose. In contrast, ciprofloxacin was detectable in milk from two of the cows 131 h post-dose. Only about 0.04% of the enrofloxacin dose appeared in milk. In contrast, 0.29–1.7% (0.74 ± 1.2%) of the enrofloxacin dose appeared in milk as ciprofloxacin. After intravenous injection of enrofloxacin in mid-lactating pregnant cows 0.04% of the total enrofloxacin dose was recovered in milk and another 0.8% was recovered as ciprofloxacin (Malbe et al., 1996). The greater concentration of ciprofloxacin in milk relative to enrofloxacin has also been observed in lactating rabbits (Aramayona et al., 1996). It has been suggested that the more rapid disappearance of enrofloxacin from milk relative to ciprofloxacin could be due to the slow metabolism of enrofloxacin to ciprofloxacin within the udder and the trapping of ciprofloxacin in milk due to its ionization in the relatively low pH environment of milk (Malbe et al., 1996). The protein binding data obtained in the present study also suggest that the greater concentration of ciprofloxacin in milk relative to enrofloxacin may reflect the lesser binding of ciprofloxacin to plasma proteins, and the corresponding higher concentration of transferable ciprofloxacin in plasma. Although, in absolute terms, more of ciprofloxacin was excreted into milk, the plasma levels of ciprofloxacin were higher than the plasma levels of enrofloxacin throughout most of the sampling period, as shown in Fig. 1. Therefore, clearance into milk does not appear to play a major role in the differences observed in the plasma kinetics of enrofloxacin and ciprofloxacin in cows. However, the greater plasma clearance of both enrofloxacin and ciprofloxacin in cows as compared to steers may, in part, be due to the excretion of the compounds into milk.
5. Conclusions Pharmacokinetic parameters were determined for enrofloxacin and its metabolite, ciprofloxacin, following administration of enrofloxacin to 6 healthy lactating dairy cows and 6 healthy beef steers. The clearance of enrofloxacin was significantly faster in cows compared to steers. This is correlated with the significantly longer elimination half-life and Mean Residence Time in steers. No difference was detected between cows and steers in the steady state volume of distribution, indicating that whatever the reasons for the differences in half-life and clearance, there do not appear to be differences in distribution between the two classes. There was no difference in the extent of binding of enrofloxacin to plasma proteins in dairy cows and beef steers. Ciprofloxacin was more bound to plasma proteins in steer than in lactating cows. In either bovine
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class, plasma protein binding was higher for enrofloxacin than for ciprofloxacin. Enrofloxacin was apparently more rapidly metabolized to ciprofloxacin by the lactating dairy cow, although the extent of overall production of ciprofloxacin was higher in steers. Ciprofloxacin was more concentrated in milk than enrofloxacin. However, for most of the sampling period, the plasma levels of ciprofloxacin were higher than that of enrofloxacin. This suggests that for the dairy cow, clearance into milk does not appear to play a major role in the differences observed in the plasma kinetics of enrofloxacin and its metabolite, ciprofloxacin. In contrast, clearance into milk may play a role in the differences between steers and dairy cows in the pharmacokinetics of enrofloxacin and ciprofloxacin. Although only a very small proportion of the enrofloxacin dose appeared in milk as either unchanged enrofloxacin or as ciprofloxacin, the greater plasma clearance of both enrofloxacin and ciprofloxacin in cows as compared to steers may in part be due to the excretion of the compounds into milk. References Abadia, A.R., Aramayona, J.J., Muñoz, M.J., Pla-Delfina, J.M., Bregante, M.A., 1995. Ciprofloxacin pharmacokinetics in dogs following oral administration. Zentralblatt für Veterinärmedizin (A) 42, 505–511. Aramayona, J.J., Mora, J., Fraile, L.J., Garcia, M.A., Abadia, A.R., Bregante, M.A., 1996. Penetration of enrofloxacin and ciprofloxacin into breast milk, and pharmacokinetics of the drugs in lactating rabbits and neonatal offspring. American Journal of Veterinary Research 57, 547–553. Bregante, M.A., Saez, P., Aramayona, J.J., Fraile, L., Garcia, M.A., Solans, C., 1999. Comparative pharmacokinetics of enrofloxacin in mice, rats, rabbits, sheep and cows. American Journal of Veterinary Research 60, 1111–1116. Brown, S.A., 1996. Fluoroquinolones in animal health. Journal of Veterinary Pharmacology and Therapeutics 19, 1–14. Campoli-Richards, D.M., Monk, J.P., Price, A., Benfield, P., Todd, P.A., Ward, A., 1988. Ciprofloxacin. A review of its antibacterial activity, pharmacokinetic properties and therapeutic use. Drugs 35, 373–447.
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Flammer, K., Aucoin, D.P., Whitt, D.A., 1991. Intramuscular and oral disposition of enrofloxacin in African grey parrots following single and multiple doses. Journal of Veterinary Pharmacology and Therapeutics 14, 359–366. Haritova, R., Lashev, L., Pashov, D., 2003. Pharmacokinetics of enrofloxacin in lactating sheep. Research in Veterinary Science 74, 241–245. Idowu, O.R., Peggins, J.O., 2004. Simple, rapid determination of enrofloxacin and ciprofloxacin in bovine milk and plasma by high-performance liquid chromatography with fluorescence detection. Journal Pharmaceutical and Biomedical Analysis 35, 143–153. Kaartinen, L., Salonen, M., Alli, L., Pyorala, S., 1995. Pharmacokinetics of enrofloxacin after single intravenous, intramuscular and subcutaneous injections in lactating cows. Journal of Veterinary Pharmacology and Therapeutics 18, 357–362. Malbe, M., Salonen, M., Fang, W., Oopik, T., Jalakas, M., Klaassen, M., Sandholm, M., 1996. Disposition of enrofloxacin (BaytrilÒ) into the udder after intravenous and intra-arterial injections into dairy cows. Journal of Veterinary Medicine, Series A 43, 377–386. Mengozzi, G., Intorre, L., Bertini, S., Soldani, G., 1996. Pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin after intravenous and intramuscular administration in sheep. American Journal of Veterinary Research 57, 1040–1043. Neu, H.C., 1988. Quinolones: a new class of antimicrobial agents with wide potential uses. Medical Clinics of North America 72, 623–636. Rao, G.S., Ramesh, S., Ahmad, A.H., Tripathi, H.C., Sharma, L.D., Malik, J.K., 2002. Disposition kinetics of enrofloxacin and ciprofloxacin following intravenous administration of enrofloxacin in goats. Small Ruminant Research 44, 9–15. Tyczkowska, K., Hedeen, K.M., Aucoin, D.P., Aronson, A.L., 1989. High-performance liquid chromatographic method for the simultaneous determination of enrofloxacin and its primary metabolite ciprofloxacin in canine serum and prostatic tissue. Journal of Chromatography 493, 337–346. Varma, R., Ahmad, A.H., Sharma, L.D., Aggarwal, P., Ahuja, V., 2003. Pharmacokinetics of enrofloxacin and its active metabolite ciprofloxacin in cows following single dose intravenous administration. Journal of Veterinary, Pharmacology and Therapeutics 26, 303–305. Villa, R., Prandini, E., Caloni, F., Carli, S., 1997. Serum protein binding of some sulphonamides, quinolones and fluoroquinolones in farm and domestic animals. Journal of Veterinary, Pharmacology and Therapeutics 20 (Suppl. 1), 21–86. Yamaoka, K., Nakagawa, T., Uno, T., 1978. Application of Akaike’s information criterion (AIC) in the evaluation of linear pharmacokinetic equations. Journal of Pharmacokinetics and Biopharmaceutics 6, 165–175.
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Comparative pharmacokinetics of enrofloxacin and ciprofloxacin in lactating dairy cows and beef steers following intravenous administration of enrofloxacin O.R. Idowu *, J.O. Peggins, R. Cullison, J. von Bredow US Food and Drug Administration, Center for Veterinary Medicine, 8401 Muirkirk Road, Laurel, MD 20708, USA
a r t i c l e
i n f o
Article history: Accepted 31 December 2009
Keywords: Enrofloxacin Ciprofloxacin Pharmacokinetics Lactating cows Steers
a b s t r a c t The comparative pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin were investigated in lactating cows and beef steers. The plasma elimination half-life of either enrofloxacin or ciprofloxacin was shorter in cows than in steers. The overall production of ciprofloxacin was slightly higher in steers than in cows (metabolite ratio: 64% and 59%, respectively). There was no significant difference in plasma protein binding of enrofloxacin between cows (percent bound: 59.4%) and steers (percent bound: 60.8%). Ciprofloxacin was more extensively bound to plasma proteins in steers (percent bound: 49.6%) than in cows (percent bound: 33.8%). The steady state volume of distribution of enrofloxacin is comparable in cows (1.55 L/kg) and steers (1.59 L/kg). Within either bovine class, plasma elimination half-life of enrofloxacin and ciprofloxacin are comparable, while plasma protein binding was higher for enrofloxacin than for ciprofloxacin. Ciprofloxacin was more concentrated in milk than enrofloxacin. Published by Elsevier Ltd.
1. Introduction Fluoroquinolones (FQs) are antibacterial agents related to nalidixic acid. FQs are used in both human and veterinary medicine to treat a variety of infections (Campoli-Richards et al., 1988; Neu, 1988; Abadia et al., 1995; Brown, 1996). Enrofloxacin was developed exclusively for use in animals. Like other FQs, enrofloxacin exhibits a broad spectrum of antibacterial activity, against both Gram-positive and Gram-negative bacteria, in diseased animals. In the United States, enrofloxacin is approved for use in beef cattle and calves (excluding veal calves), chickens and turkeys not laying eggs for human consumption, and in cats and dogs. Ciprofloxacin is a major, active metabolite of enrofloxacin in different species and is formed by the de-ethylation of enrofloxacin. Ciprofloxacin was the first of the two compounds to be developed and is only approved for use in humans (Tyczkowska et al., 1989; Flammer et al., 1991). The present study was planned to characterize the pharmacokinetics of enrofloxacin and its major metabolite, ciprofloxacin, in lactating dairy cattle and beef steers after intravenous administration of enrofloxacin. The pharmacokinetics of enrofloxacin in lactating Ayshire cows have been reported (Kaartinen et al., 1995). However, the study may not have yielded a true picture of the pharmacokinetics of enrofloxacin in lactating cows because the data presented were based on a microbiological assay of enrofloxacin in serum which measured both enrofloxacin and its active * Corresponding author. Tel.: +1 91 2402768215; fax: +1 91 2402768118. E-mail address: [email protected] (O.R. Idowu). 0034-5288/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.rvsc.2009.12.019
metabolites, including ciprofloxacin which is known to be more active than enrofloxacin. The microbiological assay based approach also did not allow the separate characterization of the pharmacokinetics of ciprofloxacin formed from enrofloxacin. A short communication on the pharmacokinetics of enrofloxacin and ciprofloxacin in non-lactating Jersey cows was published during the present study (Varma et al., 2003). The analytical method used during this study could only measure the plasma concentrations of ciprofloxacin and enrofloxacin for 12 h following drug administration. There are no prior investigations of the pharmacokinetics of enrofloxacin or ciprofloxacin in beef steers. The purpose of the present study was to compare the pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin in lactating cows and beef steers in order to establish if there are differences in the disposition of the two compounds between these two bovine production classes. 2. Materials and methods 2.1. Animals Six lactating Holstein dairy cows (584–727 kg) were obtained from the USDA herd. Six Angus steers (159–213 kg) were obtained from a commercial sale barn. Animals assigned to the study were identified with a number 1–6. This study was approved by the FDA Center for Veterinary Medicine Animal Care and Use Committee and was in compliance with their guidelines on care and use of animals.
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All animals were catheterized in the jugular vein the morning of dosing. Enrofloxacin (BaytrilÒ, 100 mg/ml, Bayer Corporation, Agriculture Division, Shawnee Mission, Kansas, USA) was administered (5 mg/kg body weight) as an I.V. bolus via the indwelling jugular catheter. Following dosing, blood samples were collected periodically via a needle, and heparin-treated syringe. Blood samples were collected at 0.17, 0.33, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 11, 13, 15, 17, 23, 29 and 35 h after administration of enrofloxacin. Plasma was separated by centrifuging the blood sample at 3500 rpm (2460g) for 15 min at 4 °C. Plasma samples were stored at 80 °C until analyzed. Each dairy cow was also milked periodically, starting at 2 h post-dosing and continuing until 131 h post-dosing. The milk was weighed and the weights recorded on data sheets. Aliquots of milk were taken and stored at 80 °C until analyzed. 2.3. HPLC analysis Concentrations of enrofloxacin and ciprofloxacin were measured in plasma and milk using reverse-phase high performance liquid chromatography (HPLC) with either fluorometric or mass spectrometric detection. Details of the method validation studies are described elsewhere (Idowu and Peggins, 2004). Briefly, a 200 lL aliquot of internal standard solution of sarafloxacin (concentration 1 ng/ml) was placed in a 15 mL, glass centrifuge tube using an adjustable pipette, followed by 1 mL of plasma (or milk) sample containing enrofloxacin and ciprofloxacin. For plasma sample, 100 lL of concentrated o-phosphoric acid was added (50 lL for milk sample). After shaking the mixture briefly (about 5 s) on a vortex-mixer, 2 mL of acetonitrile was added. The centrifuge tube was capped, and shaken at high speed on a vortex mixer for about 10 s. After centrifuging the mixture for 5 min at 4000 rpm and 4 °C, the supernatant was decanted into another 15 mL glass centrifuge tube and 3 mL of dichloromethane was added. The mixture was shaken on a vortex-mixer for about 10 s at high speed and then centrifuged for 5 min at 4000 rpm (2840g) and 4 °C. For chromatography, the upper, aqueous layer was transferred into an autosampler vial, using a Pasteur pipet. The aqueous plasma (or milk) extracts were analyzed for ciprofloxacin and enrofloxacin using the following HPLC conditions: Chromatographic system: Hewlett–Packard Series 1100. Column: PLRP-S 100A 5 l 150 4.6 mm (Phenomenex, Inc., Torrance, CA). Fluorescence detector: Hewlett–Packard 1046A; kexc: 280 nm; kemi: 460 nm. Mobile phase: isocratic: 20% aqueous formic acid:methanol:acetonitrile (75:13:12, v/v/v); flow rate 1 mL/min. Injection volume: 150 lL. The aqueous plasma (or milk) extracts were analyzed for enrofloxacin using the following HPLC– MS conditions: LC/MS SYSTEM: API2000 LC–MS/MS. Column – C8, 2.0 30 mm; 3.5 l particle size. Mobile phase: gradient of 0.1% formic acid (mobile phase A) and a 50:50 (v/v) mixture of acetonitrile and methanol (mobile phase B). The following mobile phase gradient was used: 0–0.5 min: 80% mobile phase A: 20% mobile phase B; 0.5–3.0 min: 30% mobile phase A: 70% mobile phase B. Total run time was about 6 min, including the time for re-equilibration of the column. Mass spectrometer: APCI source; MS/MS (Multiple Reaction Monitoring) mode. Ciprofloxacin levels in plasma were determined by HPLC with fluorescence detection, while enrofloxacin levels in plasma were determined by HPLC with mass spectrometric detection. Milk levels of both drugs were determined by HPLC with mass spectrometric detection.
Centricon-10 (Millipore) centrifugal filter (cut off 10,000 Da) device and centrifuged at 5000g for 9 h in Sorval RC-5 refrigerated centrifuge at 10 °C. A 500 lL aliquot of the plasma ultra-filtrate was placed in a test tube containing 200 lL of the sarafloxacin internal standard solution followed by 50 lL of concentrated ophosphoric acid. The mixture was mixed and then transferred to an autosampler vial, for analysis using the HPLC method described above. A set of plasma samples from one of the cows and a set of samples from one of the steers were used for the plasma protein binding studies. 2.5. Data analysis The plasma concentration–time data were analyzed using WinNonlin (Pharsight Corp., Cary, NC, USA), a recursive nonlinear pharmacokinetic curve fitting program. The most appropriate model was selected based on the Akaike information criteria (Yamaoka et al., 1978). The data for each animal were fit using a simple sum of 2 or 3 exponentials model. The dose event was modeled as a first order, bolus input. Statistical analysis of pharmacokinetic parameter estimates was performed using a two-tailed nonparametric t-test. Differences between means were considered significant where p 6 0.05.
3. Results The plasma concentration vs time curves for enrofloxacin and ciprofloxacin are shown in Fig. 1. Pharmacokinetic parameters, calculated from the plasma concentration–time data for enrofloxacin in lactating dairy cows, are shown in Table 1. These are terminal half-life (t1/2); mean residence time (MRT); volume of distribution (Vd); steady state volume of distribution (Vss); total body clearance (Cl); and area under the curve (zero to infinity) (AUC0–1). Similarly, pharmacokinetic parameters calculated from the plasma concentration–time data for enrofloxacin in beef steers are shown in Table 2. Pharmacokinetic parameters calculated from the plasma concentration–time data for ciprofloxacin in lactating dairy cows are shown in Table 3, while the pharmacokinetic parameters for ciprofloxacin in beef steers are shown in Table 4. A comparison of the pharmacokinetic parameters for enrofloxacin between the two groups of animals is shown in Table 5, while a comparison of the pharmacokinetic parameters for ciprofloxacin between the two groups of animals is shown in Table 6.
10000
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2.2. Animal dosing and sample collection
Cipro in Cows Cipro in Steers Enro in Cows Enro in Steers
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2.4. Protein binding Protein binding was measured by ultra-filtration. A set of plasma samples from a cow (or a steer) was transferred to
5
10
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Time (h) Fig. 1. Plasma enrofloxacin and ciprofloxacin levels in lactating dairy cows and steers after after I.V. administration of enrofloxacin to dairy cows.
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Table 1 Pharmacokinetic data for enrofloxacin in lactating dairy cows following intravenous administration of enrofloxacin.
Cow 1 Cow 2 Cow 3 Cow 4 Cow 5 Cow 6 Mean ± S.E.
T1/2 (h)
MRT (h)
Vss (L/kg)
Vd (L/kg)
Cl (L/h/kg)
AUC0–1 (lgh/mL)
4.63 4.31 2.05 4.07 4.6 2.48 3.69 ± 0.41
1.23 1.05 0.66 1.21 0.96 1.18 1.05 ± 0.08
2.37 1.69 0.63 1.66 1.29 1.73 1.56 ± 0.21
1.01 0.86 0.13 1.22 0.60 0.94 0.79 ± 0.16
1.93 1.61 0.96 1.38 1.35 1.46 1.45 ± 0.12
2.59 3.11 5.23 3.63 3.71 3.43 3.62 ± 0.36
Table 2 Pharmacokinetic data for enrofloxacin in beef steers following intravenous administration of enrofloxacin.
Steer 1 Steer 2 Steer 3 Steer 4 Steer 5 Steer 6 Mean ± S.E.
T1/2 (h)
MRT (h)
Vss (L/kg)
Vd (L/kg)
Cl (L/h/kg)
AUC0–1 (lgh/mL)
5.64 7.79 3.87 4.92 3.53 5.16 5.15 ± 0.57
2.54 2.40 2.04 2.33 2.23 2.09 2.27 ± 0.07
1.81 1.94 1.41 1.48 1.47 1.45 1.59 ± 0.08
0.90 0.96 0.72 0.86 1.00 1.19 0.94 ± 0.04
0.71 0.81 0.69 0.64 0.66 0.70 0.70 ± 0.02
7.00 6.18 7.22 7.88 7.61 7.19 7.18 ± 0.24
Table 3 Pharmacokinetic data for ciprofloxacin in lactating dairy cows following intravenous administration of enrofloxacin.
Cow 1 Cow 2 Cow 3 Cow 4 Cow 5 Cow 6 Mean ± S.E.
MRT (h)
T1/2 (h)
Cmax (lg/mL)
AUC0–1 (lgh/mL)
2.66 2.66 3.06 3.59 3.09 2.64 2.95 ± 0.15
2.07 2.60 3.35 3.99 3.44 2.32 2.96 ± 0.30
1.41 1.11 0.83 1.03 1.34 1.38 1.19 ± 0.09
1.92 1.93 2.10 2.45 2.31 2.14 2.14 ± 0.08
Table 4 Pharmacokinetic data for ciprofloxacin in beef steers following intravenous administration of enrofloxacin.
Steer 1 Steer 2 Steer 3 Steer 4 Steer 5 Steer 6 Mean ± S.E.
T1/2 (h)
Cmax (lg/mL)
Tmax (h)
AUC0–1 (lgh/mL)
5.46 17.9 2.80 3.63 3.44 12.4 7.60 ± 2.5
0.75 0.66 0.74 0.64 0.46 0.54 0.63 ± 0.04
0.88 0.65 0.91 0.89 1.17 0.99 0.92 ± 0.08
6.58 6.11 3.76 3.94 2.91 4.28 4.60 ± 0.57
Table 5 Comparison of pharmacokinetic parameters for enrofloxacin in lactating dairy cows and beef steers.
Dairy cows Beef steers
T1/2 (h)
MRT (h)
Cl (L/h/kg)
Vss (L/kg)
Vd (L/kg)
3.69 ± 0.41 5.15 ± 0.57
1.05 ± 0.08 2.27 ± 0.07
1.45 ± 0.12 0.70 ± 0.02
1.56 ± 0.21 1.59 ± 0.08
0.79 ± 0.16 0.94 ± 0.04
Table 6 Comparison of pharmacokinetic parameters for ciprofloxacin in lactating dairy cows and beef steers.
Dairy cows Beef steers
Half-life, t1/2 (h)
Cmax (lg/mL)
AUCciprofloxacin/AUCenrofloxacin
2.96 ± 0.3 7.60 ± 2.5
1.19 ± 0.09 0.630 ± 0.04
0.59 0.64
Plots of the ratio of the concentration of free (unbound) enrofloxacin to the total enrofloxacin concentration in plasma from cows and steers are shown in Fig. 2. Similar plots of the ratio of
the concentration of free (unbound) ciprofloxacin to the total ciprofloxacin concentration in plasma from cows and steers are shown in Fig. 3. In order to compare the protein binding of enrofloxacin to that of ciprofloxacin, a plot of the ratio of free (unbound) to total concentration for the two compounds in cow plasma is presented in Fig. 4. The protein binding data, expressed in terms percentage of enrofloxacin or ciprofloxacin bound to plasma proteins from the two bovine classes are summarized in Table 7. The variation of the milk concentration of enrofloxacin and ciprofloxacin with time following I.V. administration of enrofloxacin to lactating cows is illustrated in Fig. 5.
4. Discussion The plasma concentration–time profile following a single I.V. dose of enrofloxacin in all 6 lactating cows and five of the steers was best fitted by a 3-compartment open model. This is comparable with plasma concentration–time profile following a single I.V. dose of enrofloxacin in pregnant mid-lactating dairy cows (Malbe et al., 1996). This is in contrast with literature reports in which a 2-compartment model was found to provide the best fit for I.V. enrofloxacin plasma concentration–time data in some species, including non-lactating cows (Varma et al., 2003; Bregante et al., 1999) non-lactating sheep (Bregante et al., 1999), and lactating sheep (Haritova et al., 2003). The plasma concentration–time profile in one of the steers used in the present study was best fitted by a 2-compartment open model. A comparison of the pharmacokinetic parameters for enrofloxacin in the two classes of animals, as summarized in Table 5, show that the clearance of enrofloxacin was significantly faster in cows compared to steers. This is further reflected in the significantly longer elimination half-life and Mean Residence Time (MRT) in steers. There was no significant difference between cows and steers in the steady state volume of distribution, or volume of distribution. This suggests that the differences in half-life and clearance between cows and steers were not due to differences in the body distribution of enrofloxacin in the two animal classes. The clearance of enrofloxacin in lactating dairy cows observed in the present study (1.45 L/h/kg) is comparable with that reported for mid-lactating pregnant cows (1.26 L/ h/kg/; Malbe et al., 1996) and non-lactating cows (1.14 L/h/kg; Varma et al., 2003).
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0.5 0.45 0.4
Free/Total ratio
0.35 0.3 Cow
0.25
Steer 0.2 0.15 0.1 0.05 0 0.17 0.33 0.5
0.75
1
1.5
2
2.5
3 4 Time (h)
5
6
7
8
9
11
Fig. 2. Ratio of free to total plasma levels of enrofloxacin in lactating dairy cows and beef steers.
Table 7 Comparison of protein binding data for enrofloxacin and ciprofloxacin in lactating dairy cows and beef steers.
0.90
0.70
Percent plasma protein binding (mean ± S.E.)
0.60 Steer plasma Cow plasma
0.50
Enrofloxacin
Ciprofloxacin
60.8 ± 1.05 59.4 ± 1.48
49.6 ± 2.58 33.75 ± 1.73
0.40 Cow Steer
0.30 0.20
A comparison of the pharmacokinetic parameters for ciprofloxacin in the two classes of animals, as summarized in Table 6, show that the elimination half-life of ciprofloxacin was significantly longer in steers compared to cows. The mean plasma half-life of ciprofloxacin in steers is apparently skewed by the relatively high half-life values for two of the steers. However, even after excluding the data from these two steers from the calculations, the mean half-life of ciprofloxacin in the remaining 4 steers (3.83 ± 0.57 h)
0.10 0.00 1.00
2.00
3.00
5.00
7.00
9.00
Time (h) Fig. 3. Ratio of free to total plasma levels of ciprofloxacin in lactating dairy cows and beef steers.
Cow Enro Free/Total ratio
0.9
Cow Cipro Free/Total ratio 0.8 0.7
0.5 0.4 0.3 0.2 0.1
11
9
8
7
6
2. 5
2
1
1. 5
5
75
0.
0.
03
.3
0 0. 17
Free/Total ratio
0.6
5
0.50
4
0.17
3
Free/Total ratio
0.80
Time (h) Fig. 4. Ratio of free to total plasma levels of enrofloxacin and ciprofloxacin in dairy cows.
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10000
Milk Enrofloxacin
1000
Conc (ng/mL)
Mlk Ciprofloxacin
100
10
1
0
20
40
60
80
100
120
140
Time (h) Fig. 5. Enrofloxacin and ciprofloxacin levels in milk after I.V. administration of enrofloxacin to dairy cows.
is still significantly longer than the mean half-life of ciprofloxacin lactating dairy cows. To compare the production levels of ciprofloxacin in the two animal classes the ratio of AUC (ciprofloxacin) to AUC (enrofloxacin) were calculated for the two classes. As shown in Table 6, the metabolic conversion of enrofloxacin to ciprofloxacin was 59% in dairy cows and 64% in beef steers. The greater overall production level of ciprofloxacin in steers may partly account for the longer persistence (longer half-life) of ciprofloxacin in steer plasma compared to lactating cows. The metabolic conversion of enrofloxacin to ciprofloxacin was 29% in mid-lactating pregnant cows (Malbe et al., 1996). The average metabolic conversion of enrofloxacin to ciprofloxacin in lactating Ayrshire cows was reported to be 35% by (Kaartinen et al., 1995). Compared to other animals, the metabolic conversion of enrofloxacin to ciprofloxacin, after I.V. administration of enrofloxacin, was 35% in sheep (Mengozzi et al., 1996), and 23% in female goats (Rao et al., 2002). As shown in Fig. 1, the maximum concentration of ciprofloxacin in lactating cows was attained very rapidly and Tmax (time to reach maximum concentration) had been attained before the first plasma sample was taken at 10 min post-dose. Ciprofloxacin has been reported to appear in plasma, at a relatively high concentration, within 2 min following I.V. administration of enrofloxacin to non-lactating Jersey cows, even though peak plasma concentration was attained 30 min post-dose (Varma et al., 2003). Data from the present study suggest a much more rapid metabolism of enrofloxacin to ciprofloxacin by the lactating dairy cow compared to the non-lactating cow. In contrast, the production of ciprofloxacin was relatively slower in the beef steers. This difference in the rate of production of ciprofloxacin between the two animal classes is also reflected in the significantly higher maximum plasma concentration (Cmax) of ciprofloxacin in dairy cows, as shown in Table 6. It should be noted, however, that Tmax for ciprofloxacin was attained in about an hour in the beef steers, and this is still later than the Tmax of 30 min reported for non-lactating cows (Varma et al., 2003). Thus it seems, in general, beef steers metabolize enrofloxacin more slowly than cows, even though the overall production of ciprofloxacin from enrofloxacin is slightly higher in steers than in lactating cows. The plasma protein binding data, illustrated in Figs. 2–4 (and summarized in Table 7) show that the extent of plasma protein binding of enrofloxacin was similar in both classes of animals (Fig. 2). This is confirmed with the data in Table 7 which shows there is no significant difference in the percentage of enrofloxacin
bound to plasma protein in lactating cows compared to steers. In contrast, the percentage of ciprofloxacin bound to plasma protein in steers is significantly higher than the percentage of ciprofloxacin bound to plasma protein in lactating cows (Fig. 3 and Table 7). The more extensive plasma protein binding of ciprofloxacin in steers may explain, in part, the longer elimination half-life of ciprofloxacin in steers compared to lactating cows. Within either bovine class, enrofloxacin was more extensively bound to plasma protein than ciprofloxacin (Fig. 4 and Table 7). As shown in Fig. 4, the free fraction of ciprofloxacin was over one and a half times greater than that of enrofloxacin. The in vivo percentage of bound enrofloxacin (59.4%) and bound ciprofloxacin (33.8%) reported in the present study is in close agreement with the respective values of 56% and 31% reported earlier, based on the in vitro incubation of enrofloxacin and ciprofloxacin with bovine plasma (Villa et al., 1997). The disposition of enrofloxacin and ciprofloxacin in milk following a single oral dose of enrofloxacin in lactating dairy cows is illustrated in Fig. 5, showing the rapid disappearance of enrofloxacin from milk and the higher concentration of ciprofloxacin in milk, compared to enrofloxacin. In the present study, enrofloxacin could not be detected in milk from any of the cows beyond 17 h postdose. In contrast, ciprofloxacin was detectable in milk from two of the cows 131 h post-dose. Only about 0.04% of the enrofloxacin dose appeared in milk. In contrast, 0.29–1.7% (0.74 ± 1.2%) of the enrofloxacin dose appeared in milk as ciprofloxacin. After intravenous injection of enrofloxacin in mid-lactating pregnant cows 0.04% of the total enrofloxacin dose was recovered in milk and another 0.8% was recovered as ciprofloxacin (Malbe et al., 1996). The greater concentration of ciprofloxacin in milk relative to enrofloxacin has also been observed in lactating rabbits (Aramayona et al., 1996). It has been suggested that the more rapid disappearance of enrofloxacin from milk relative to ciprofloxacin could be due to the slow metabolism of enrofloxacin to ciprofloxacin within the udder and the trapping of ciprofloxacin in milk due to its ionization in the relatively low pH environment of milk (Malbe et al., 1996). The protein binding data obtained in the present study also suggest that the greater concentration of ciprofloxacin in milk relative to enrofloxacin may reflect the lesser binding of ciprofloxacin to plasma proteins, and the corresponding higher concentration of transferable ciprofloxacin in plasma. Although, in absolute terms, more of ciprofloxacin was excreted into milk, the plasma levels of ciprofloxacin were higher than the plasma levels of enrofloxacin throughout most of the sampling period, as shown in Fig. 1. Therefore, clearance into milk does not appear to play a major role in the differences observed in the plasma kinetics of enrofloxacin and ciprofloxacin in cows. However, the greater plasma clearance of both enrofloxacin and ciprofloxacin in cows as compared to steers may, in part, be due to the excretion of the compounds into milk.
5. Conclusions Pharmacokinetic parameters were determined for enrofloxacin and its metabolite, ciprofloxacin, following administration of enrofloxacin to 6 healthy lactating dairy cows and 6 healthy beef steers. The clearance of enrofloxacin was significantly faster in cows compared to steers. This is correlated with the significantly longer elimination half-life and Mean Residence Time in steers. No difference was detected between cows and steers in the steady state volume of distribution, indicating that whatever the reasons for the differences in half-life and clearance, there do not appear to be differences in distribution between the two classes. There was no difference in the extent of binding of enrofloxacin to plasma proteins in dairy cows and beef steers. Ciprofloxacin was more bound to plasma proteins in steer than in lactating cows. In either bovine
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class, plasma protein binding was higher for enrofloxacin than for ciprofloxacin. Enrofloxacin was apparently more rapidly metabolized to ciprofloxacin by the lactating dairy cow, although the extent of overall production of ciprofloxacin was higher in steers. Ciprofloxacin was more concentrated in milk than enrofloxacin. However, for most of the sampling period, the plasma levels of ciprofloxacin were higher than that of enrofloxacin. This suggests that for the dairy cow, clearance into milk does not appear to play a major role in the differences observed in the plasma kinetics of enrofloxacin and its metabolite, ciprofloxacin. In contrast, clearance into milk may play a role in the differences between steers and dairy cows in the pharmacokinetics of enrofloxacin and ciprofloxacin. Although only a very small proportion of the enrofloxacin dose appeared in milk as either unchanged enrofloxacin or as ciprofloxacin, the greater plasma clearance of both enrofloxacin and ciprofloxacin in cows as compared to steers may in part be due to the excretion of the compounds into milk. References Abadia, A.R., Aramayona, J.J., Muñoz, M.J., Pla-Delfina, J.M., Bregante, M.A., 1995. Ciprofloxacin pharmacokinetics in dogs following oral administration. Zentralblatt für Veterinärmedizin (A) 42, 505–511. Aramayona, J.J., Mora, J., Fraile, L.J., Garcia, M.A., Abadia, A.R., Bregante, M.A., 1996. Penetration of enrofloxacin and ciprofloxacin into breast milk, and pharmacokinetics of the drugs in lactating rabbits and neonatal offspring. American Journal of Veterinary Research 57, 547–553. Bregante, M.A., Saez, P., Aramayona, J.J., Fraile, L., Garcia, M.A., Solans, C., 1999. Comparative pharmacokinetics of enrofloxacin in mice, rats, rabbits, sheep and cows. American Journal of Veterinary Research 60, 1111–1116. Brown, S.A., 1996. Fluoroquinolones in animal health. Journal of Veterinary Pharmacology and Therapeutics 19, 1–14. Campoli-Richards, D.M., Monk, J.P., Price, A., Benfield, P., Todd, P.A., Ward, A., 1988. Ciprofloxacin. A review of its antibacterial activity, pharmacokinetic properties and therapeutic use. Drugs 35, 373–447.
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