J. vet. Pharmacol. Therap. 30, 353–357, doi: 10.1111/j.1365-2885.2007.00860.x.
Pharmacokinetic studies on tobramycin in horses H. HUBENOV* D. BAKALOV* S. KRASTEV* S. YANEV A. HARITOVA & L. LASHEV *Department of Surgery, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria; Department of Drug Toxicology, Institute of Physiology, Bulgarian Academy of Sciences, Sofia, Bulgaria; Department of Pharmacology, Veterinary Physiology and Physiological Chemistry, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria
Hubenov, H., Bakalov, D., Krastev, S., Yanev, S., Haritova, A., Lashev, L. Pharmacokinetic studies on tobramycin in horses. J. vet. Pharmacol. Therap. 30, 353–357. The objective of the study was to evaluate the pharmacokinetics of tobramycin in plasma and urine in the horse (n ¼ 7) after intravenous administration of a dose of 4 mg/kg b.w. Plasma tobramycin concentrations were assayed microbiologically and by means of HPLC analyses. Pharmacokinetic parameters, calculated on the basis of concentrations determined with the microbiological assay were not statistically different from those obtained when data from HPLC analysis were used, but the microbiological assay was more sensitive in the detection of low plasma and urine values. The values of the total body clearance (ClB) were 101.4 ± 30.1 and 130.0 ± 49.9 mL/kg/h, respectively. The overall extraction ratio was 2.9%. The determined capacity of elimination of tobramycin in horses was similar to those for other aminoglycosides. Within 24 h after treatment, 57.6 ± 12.2% of injected antibiotic was excreted in the urine. (Paper received 22 January 2007; accepted for publication 25 March 2007) Lubomir Lashev, Department of Pharmacology, Physiology of Animals and Physiological Chemistry, Faculty of Veterinary Medicine, Trakia University, Student’s Campus, 6000 Stara Zagora, Bulgaria. E-mail:
[email protected]
INTRODUCTION Aminoglycosides are a group of antimicrobial drugs with a high activity against pathogenic gram-negative microorganisms, responsible for diverse bacterial infections (Brown & Riviere, 1991). In equines, they are extensively used in the treatment of infections caused by Pseudomonas spp. and Enterobacteriaceae strains (Prescott & Baggot, 1993; Martin-Jimenez et al., 1998). In the available literature, reports regarding the pharmacokinetics of tobramycin in the horse are lacking, and data were confined to other aminoglycosides, such as gentamicin and amikacin (Orsini et al., 1985; Wichtel et al., 1992; Green & Conlon, 1993; Martin-Jimenez et al., 1998). In addition, only very few pharmacokinetic studies with tobramycin have been reported from other domestic species (Jernigan et al., 1988; Hadi et al., 1994). However, considering that as a broad-spectrum aminoglycoside, tobramycin exhibits higher activity against several gram-negative microorganisms than gentamicin (Braveny et al., 1980; Shawar et al., 1990; Gonzalez & Spencer, 1998; Wilson, 2001), and a lower toxicity (Donta & Lambke, 1985) its clinical applications seems to be attractive. Therefore, information about the pharmacokinetics of tobramycin in horses is necessary to evaluate the possible advantages of this drug over other widely used aminoglycosides in equine medicine. The clinical outcome of a therapy with aminoglycosides, being allocated to the group of drugs with a concentrationdependent killing activity, is correlated with ratio between the maximum plasma concentration (Cmax) and the minimum 2007 The Authors. Journal compilation 2007 Blackwell Publishing Ltd
inhibitory concentrations (MIC) (Riviere & Spoo, 1995; McKellar et al., 2004) measured in bacterial type strains or field isolates. Additional information on the variability of kinetic data can be obtained from, population studies, allowing to refine and optimize further the dosing regime by considering factors, such as (sub)species, age, gender, body weight, and health status (Martin-Jimenez et al., 1998). Therefore, the objective of the present study was to evaluate the main pharmacokinetic parameters of tobramycin in plasma and urine of the horse after intravenous (i.v.) administration, as a first step towards population pharmacokinetics.
MATERIALS AND METHODS Drug Tobramycin sulfate (activity 690 UI, provided by Actavis Ltd, Sofia, Bulgaria) was used as a 20% w/v aqueous solution, prepared ex tempore. Animals Six clinically healthy horses from both genders, at the age of 1.5–12 years, weighing 250–585 kg were included in the trial. The complete pharmacokinetic studies were performed in seven animals, but because of deviations in the creatinine values in blood and urine before and after tobramycin treatment, one 353
354 H. Hubenov et al.
horse was excluded from kinetic analyses. The animals were fed commercial food 4–5 h after injection of the antibiotic and had free access to water. Experimental design The drug tobramycin was administered intravenously in v. jugularis. The treatments were given between 08:00 and 09:00 h in the morning. Blood samples were collected from the opposite v. jugularis. Tobramycin was applied at a dose of 4 mg/kg b.w. Blood samples of 5 mL each were collected in heparinized tubes by venipuncture prior to treatment and at post-treatment hours 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 h. They were centrifuged within 1 h (1400 g, 10 min) and plasma was stored at )20 C until being analyzed. Antibiotic concentrations were determined within 2 days after sampling. Urine samples were collected from spontaneously excreted urine. Within the first 24 h, the entire urine production was measured, and thereafter, urine samples were taken only once daily, during the next 4 days. Samples were stored at conditions described for plasma and were analyzed within 24 h after the last sampling. Tobramycin concentrations in urine were determined after dilution of samples. Before, and the day after drug administration, various hematological and biochemical parameters, including the creatinine values in plasma and urine, were measured. Quantification of tobramycin concentrations in plasma and urine Microbiological method A meat peptone agar (National Centre of Infectious and Parasitic Diseases, Sofia, Bulgaria) and Bacillus subtilis ATCC 6633 reference tester strain for quantitative determination of tobramycin were used. Standard solutions were prepared in plasma and urine samples collected from untreated animals. The concentrations of standard solutions were 12.5, 10, 6.25, 3.12, 1.56, 0.78, 0.39, 0.195, 0.098 and 0.048 lg/mL tobramycin. The limit of detection (LOD) and limit of quantification (LOQ) values were 0.024 and 0.048 lg/mL, respectively. Linearity of standard curve was confirmed by the test of lack of fit (P ¼ 0.214) and the value of r was 0.9898 ± 0.006. The intraassay and interassay coefficients of variation were 9.90 ± 3.53 and 13.29 ± 2.58, respectively. The data indicate the suitability of the applied method. HPLC analysis The protocol for the HPLC analysis with precolumn derivatization and fluorescence detection (Lai F., Varian application note, number 5) was used with some modifications for analysis of tobramycin in plasma. The Waters reverse phase liquid chromatography system was equipped with a quaternary pump 626E, guard-column Polar-RP, 4.0 · 20 mm, 5 lm, Phenomenex (Torrance, CA, USA); an analytical column Synergi Polar-RP ˚ , 4.6 · 150 mm, 5 lm, Phenomenex, with a working 80 A temperature of 25 C. Mobile phase consisted of acetonitrile/
water (48/52), containing 20 mM potassium phosphate buffer (pH 6.5), and a flow rate of 2.0 mL/min. The injection volume was 50 lL. Detection was conducted with a fluorescence detector 474 set at an excitation wavelength of kex ¼ 340 nm and emission of kem ¼ 450 nm. A Pentium 566 computer using Waters Empower software performed the instrument control and data recording. Prior to analysis, all plasma samples (200 lL) including the standards, controls and samples collected from the treated animals were deproteinated by adding 800 lL acetonitrile. After mixing, the sample was centrifuged at 12 880 g for 10 min. To a 200 lL aliquot of the supernatant, an equal volume of the derivatization reagent was added. Working solution of derivatization reagent contained 9.5 mL 0.4 M borate buffer (adjusted to pH 10.4 with KOH 450 g/L) mixed with 0.5 mL of a solution prepared from 200 mg OPA in 2 mL methanol plus 400 lL 2-mercaptoethanol. Thereafter, the samples were kept in the dark at room temperature for 30 min and were then subjected to HPLC analysis. The retention time of tobramycin was 11.8 min. In the concentration range between 0.5 and 20 lg/mL, a linear concentration between the peak area and the tested concentration was confirmed by analysis of linear regression (y ¼ 1298914x )6496699, R2 ¼ 0.9998). The LOQ of tobramycin was 0.2 lg/mL and the LOD was determined to be 0.1 lg/mL. Pharmacokinetic analysis The pharmacokinetic analysis was performed with the WinNonlin 4.0.1. (Pharsight Corporation, Mountain View, CA, USA) software. Plasma concentration curves vs. time were interpreted by a two compartmental model selected on the basis of Akaike’s criterion value. A bi-exponential equation was selected for the i.v. administration: CðtÞ ¼ A eat þ B ebt ;
ð1Þ
where C(t) represents the tobramycin plasma concentrations at any time t and a and b are the slopes of the first and second phases of tobramycin plasma disposition. The data points were weighted with the inverse of the squared fitted value. Accumulation factor (Ra) was estimated according to the following equation: Ra ¼
1 ; ð1 expbs Þ
ð2Þ
where b is the overall elimination rate constant of the drug and s is the dosage interval (Baggot, 2001). The overall extraction ratio was calculated according to Toutain and Bousquet-Me`lou (2004a). Statistical analysis Pharmacokinetic parameters were presented as mean ± standard deviation (SD). The Statistica 6.1 (Statistica for Windows; StatSoft, Inc., Tulsa, OK, USA, 1984–2002) software was used. Statistically significant differences of pharmacokinetic parameters estimated on the basis of concentrations measured by the 2007 The Authors. Journal compilation 2007 Blackwell Publishing Ltd
Pharmacokinetic studies on tobramycin in horses 355 Table 1. Pharmacokinetic parameters of tobramycin after single intravenous administration at 4 mg/kg b.w., to six horses: comparison of the results based on a microbiological method of detection and HPLC analyses
Concentration (µg/mL)
1000 100 10
Pharmacokinetic parameters (units)
1 0.1 0.01 0
2
4
6 Time (h)
8
10
12
Fig. 1. Plasma tobramycin concentrations in horses (n ¼ 6) after a single intravenous administration at 4 mg/kg as determined by a microbiological assay (r) and by HPLC analysis (n). The dots represent the measured concentrations and the lines indicate predicted concentrations.
two analytical methods were determined with Wilcoxon test. Level of significance was set at P < 0.05.
Calculated using a microbiological method
t1/2a (h) t1/2b (h) ClB (mL/kg/h) AUC(0)¥) (lgÆh/mL) MRT (h) Vss (L/kg) Vd(area) (L/kg) k12 (h)1) k21 (h)1) k10 (h)1)
0.29 2.49 101.38 42.08 2.42 0.242 0.369 1.46 0.81 1.10
± ± ± ± ± ± ± ± ± ±
0.06 0.61 30.11 10.74 1.05 0.11 0.14 1.53 0.52 0.30
Calculated using an HPLC method 0.27 4.02 130.04 35.87 4.54 0.550 0.699 2.24 0.99 0.75
± ± ± ± ± ± ± ± ± ±
0.21 1.58 49.86 17.33 151 0.196 0.235 1.48 0.56 0.22
Data were presented as mean ± SD. t1/2a, distribution half-life; t1/2b, terminal elimination half-life; AUC0)¥, area under the plasma concentration–time curves from zero to infinity; Vd(area), area volume of distribution; Vss, steady-state volume of distribution; MRT, mean residence time; ClB, total body clearance; k12, k21, the distribution rate constants from central to peripheral compartment and back; k10, elimination rate constant from central compartment.
RESULTS
DISCUSSION The aim of this study was to describe the main pharmacokinetic parameters for the aminoglycoside tobramycin, which has not been studied in horses yet. Following i.v. injection, plasma concentrations were measured at different time points with two different methods, a microbiological assay and by chemical 2007 The Authors. Journal compilation 2007 Blackwell Publishing Ltd
1000 Concentration (µg/mL)
Plasma tobramycin concentrations, assayed microbiologically, were detected up to 12 h after i.v. administration. Because of the lower sensitivity of the HPLC method, measurable concentrations were detected only up to the 10t h after i.v. tobramycin administration in horses (Fig. 1). Until 4 h post-treatment, the antibiotic concentrations measured microbiologically were higher as those measured by HPLC, but thereafter declined to values lower than those measured by HPLC (Fig. 1). Mean values of pharmacokinetic parameters for tobramycin are presented in Table 1. Pharmacokinetic parameters, calculated on the basis of concentrations determined by microbiological assay were not statistically different from those obtained with the results from the HPLC analyses. Because of satisfactory validation parameters, and the higher sensitivity of the microbiological assay, the latter was used to determine urine concentrations of tobramycin. Figure 2 represents the measured urine concentrations of tobramycin in the samples from spontaneously excreted urine during 4 days after treatment. Up to 24 h after drug administration, 57.5 ± 12.2% of the dose was excreted in the urine. This is in agreement with the fact that high tobramycin concentrations were found in the next samples taken 24 h after drug administration. The overall extraction ratio, calculated according to Toutain and Bousquet-Me`lou (2004a,b), was 2.90%.
100 10 1 0.1 0.01 0
20
40
60
80
100
Time (h) Fig. 2. Urine tobramycin concentrations in horses (n ¼ 6) after a single intravenous administration at 4 mg/kg determined by microbiological assay. The dots represent the measured concentrations in the urine samples from all horses.
analysis (HPLC). The data of plasma tobramycin concentrations and the corresponding values of calculated pharmacokinetic parameters exhibited some differences. The variations in HPLCdetermined concentrations were more obvious, and were reflected in the values of the calculated kinetic parameters. Moreover, as there was no evidence for the presence of metabolites in the analyzed plasma samples during HPLC analysis, it was assumed that the microbiological assay is more sensitive and gives precise values. These findings are in agreement with the previous statement that aminoglycosides and tobramycin in particular, do not undergo a systemic degradation in detectable concentrations (Riviere & Spoo, 1995). This implies that the principal limitation of microbiological assays i.e. their inability to detect metabolites of antibiotics can be neglected here.
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Our data on the pharmacokinetics of tobramycin in horses are comparable to the available literature describing the kinetic behavior of related aminoglycosides, such as gentamicin and amikacin, in this animal species (Orsini et al., 1985; Wichtel et al., 1992; Green & Conlon, 1993; Martin-Jimenez et al., 1998). The systemic distribution of all these antibiotics is rapid, which is confirmed by the low t1/2a values, and the relatively high k12 value. The biological half-life of tobramycin is similar to respective values determined for gentamicin and amikacin in horses (Brown & Riviere, 1991). When tobramycin is given once daily (s ¼ 24 h) the accumulation factor is equal to 1, which suggests that accumulation is not to be expected (Toutain & Bousquet-Me`lou, 2004b). The determined capacity of elimination of tobramycin in horses was similar to those, calculated by us on the basis of available data for amikacin (2.7%) and kanamycin (2.6%) and higher than that of gentamicin (1.7– 2.2%), streptomycin (1.4%) and neomycin (2.4%). Comparing different animal species, it becomes obvious that camels (1.45%) and cats (1.5%) have apparently a low capacity of elimination, whereas higher values were detected in humans (2.3%) and horses (2.9% in our study) (Baggot et al., 1981; Orsini et al., 1985; Magdesian et al., 1998). The presence of high tobramycin concentrations in the urine until the end of the investigation probably reflects the existence of selective tissue accumulation in the kidneys, which is typical for aminoglycosides. Moreover, it can be assumed that the applied microbiological assay underestimates the real urine concentrations, which results in lower percentage of recovery of tobramycin in the urine in comparison with that in rats (Reinhard et al., 1994). There is no information available about the activity of tobramycin against pathogens isolated from horses, with exception of some studies with bacterial isolates associated with infectious keratitis in this animal species (Moore et al., 1995). MIC90 for susceptible pathogenic human and animal strains of Pseudomonas aeruginosa ranged between 0.5 and 8 lg/mL (Macia et al., 2006; Traczewski & Brown, 2006). The Cmax/MIC ratio has been shown to be the most useful PK-PD index for predicting the efficacy of aminoglycosides. The desirable value of Cmax/MIC ratio is >10, at which the risk for selection of resistance is considered to be minimal. The Cmax/MIC ratio for intravenously administered tobramycin in horses would be 95.6 ± 6.0 if the predicted plasma concentration at time 0 (47.81 lg/mL) and the cited MIC for strains Pseudomonas aeruginosa were used. The repeated application of a dose of 4 mg/kg at a dose interval of 24 h will result in the same concentration profile every day, as no accumulation takes place, with the exception of a possible trapping in proximal tubule cells. The latter, known from studies with other aminoglycosides cannot be entirely excluded. These data suggest that the used dose will allow achievement of Cmax/ MIC ratio higher than 10 for pathogen strain with MIC of 1 lg/mL. The expected Cp,ss(min) value is much lower than the safe maximum for the trough tobramycin concentrations of 2 lg/mL in human beings (Saleh-Mghir et al., 1992). Moreover, this dose was not associated with any significant changes in the investigated blood and urine biochemical parameters
(data not shown). However, these calculations are based on plasma concentrations only, and the MIC values for Pseudomonas aeruginosa isolated from horses need to established. Therefore, further studies are required to define the real breakpoint values for Cmax/MIC ratio for tobramycin not only in healthy horses, but also in diseased animals. For urine, a 5to 10-fold higher Cmax/MIC ratio was measured, because of the 5- to 10-fold higher urine concentrations of tobramycin. Validation of these findings in clinical trials is also needed, as an increase in MIC values in urine can occur, depending on several factors, including urine pH, which could be less alkaline after intense exercises or diseases (Garcia-Calvo et al., 2001; Lees, 2004). In conclusion, we presented here for the first time, a set of parameters describing the kinetic of tobramycin in healthy horses after i.v. injection. It can be concluded that in general, tobramycin followed the characteristic systemic behavior of aminoglycosides following a single i.v. administration in horses.
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