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Pharmacokinetics and pharmacokinetic/ pharmacodynamic integration of marbofloxacin after intravenous and intramuscular administration in beagle dogs Sileshi Yohannes, Elias Gebru Awji, Seung-Jin Lee & Seung-Chun Park To cite this article: Sileshi Yohannes, Elias Gebru Awji, Seung-Jin Lee & Seung-Chun Park (2015) Pharmacokinetics and pharmacokinetic/pharmacodynamic integration of marbofloxacin after intravenous and intramuscular administration in beagle dogs, Xenobiotica, 45:3, 264-269, DOI: 10.3109/00498254.2014.969794 To link to this article: http://dx.doi.org/10.3109/00498254.2014.969794
Published online: 03 Dec 2014.
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Date: 23 August 2017, At: 23:05
http://informahealthcare.com/xen ISSN: 0049-8254 (print), 1366-5928 (electronic) Xenobiotica, 2015; 45(3): 264–269 ! 2014 Informa UK Ltd. DOI: 10.3109/00498254.2014.969794
RESEARCH ARTICLE
Pharmacokinetics and pharmacokinetic/pharmacodynamic integration of marbofloxacin after intravenous and intramuscular administration in beagle dogs Sileshi Yohannes1, Elias Gebru Awji2, Seung-Jin Lee1, and Seung-Chun Park1 Downloaded by [Australian Catholic University] at 23:05 23 August 2017
1
Laboratory of Pharmacokinetics and Pharmacodynamics, Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Kyungpook National University, Daegu, Republic of Korea and 2Lovelace Respiratory Research Institute, NM, USA Abstract
Keywords
1. The aim of the present study was to determine the PKs of marbofloxacin in beagle dogs after intravenous (i.v.) and intramuscular (i.m.) administration, the ex vivo and in vitro PK/PD indices of marbofloxacin against clinical isolates of Staphylococcus pseudintermedius, and the ex vivo AUC/MIC ratios associated with different levels of antibacterial activity. 2. After i.v. of marbofloxacin (2 mg/kg), the mean ± SEM values of AUC, t1/2b, Vss, and CL were 8.47 ± 3.51 h mg/mL, 8.08 ± 6.25 h, 2.32 ± 1.00 L/kg and 0.23 ± 0.06 L/kg/h and corresponding values after intramuscular injection were 11.37 ± 3.07 h mg/mL, 7.51 ± 3.70, 1.80 ± 0.90 L/kg and 0.17 ± 0.04 L/kg/h. After i.m. administration, a Cmax of 1.76 ± 0.09 mg/mL was achieved at Tmax of 0.47 ± 0.08 h. The ex-vivo AUC/MIC ratios required to produce bacteriostasis, bactericidal action and elimination of S. pseudintermedius were 65.03, 97.02 and 136.84 h. 3. The in vivo AUC/MIC ratios obtained after i.v. and i.m. administration of 2 mg/kg marbofloxacin (67.76 ± 1.23 and 91.18 ± 2.61) were below the ex vivo AUC/MIC ratios required for bactericidal activity and bacterial elimination (97.02 ± 9.24 2 mg/kg and 136.21 ± 7.58), suggesting that the recommended daily dosage (2 mg/kg) may not suffice to kill and eradicate S. pseudintermedius strains encountered in clinical area.
Beagle dogs, drug resistance, marbofloxacin, PK/PD, staphylococcus pseudintermedius History Received 22 August 2014 Revised 22 September 2014 Accepted 23 September 2014 Published online 3 December 2014
Introduction Marbofloxacin is a third generation fluoroquinolone that exhibits concentration dependent bactericidal activity against gram-negative and gram-positive bacteria (Aliabadi et al., 2003). It has shown higher in vitro activity against pathogenic strains isolated from urinary tract infections, respiratory tract infections, dermatological infections and otitis in pets (Meunier et al., 2004) and produced greater than 80% clinical efficacy in the treatment of superficial and deep bacterial pyoderma (Horspool et al., 2004), which is the most commonly diagnosed skin problem in dogs mainly caused by S. pseudintermedius, a coagulase positive normal resident bacterium of the skin. Although marbofloxacin is a frequently prescribed drug with high clinical efficacy against various bacterial infections, including canine pyoderma, fluoroquinolone resistance in S. pseudintermedius is an emerging problem (Perreten et al., 2010; Yoo et al., 2010). This undermines the successful treatment outcomes of fluoroquinolones and adds upon the Address for correspondence: Seung-Chun Park, Laboratory of Pharmacokinetics and Pharmacodynamics, Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Kyungpook National University, Daegu, Republic of Korea. E-mail: [email protected]
existing challenge in selecting the most appropriate antimicrobial therapy against methicillin resistant S. pseudintermedius (MRSP) infections, which is highly disseminated in dogs and cats (Kadlec et al., 2010; Perreten et al., 2010). Furthermore, the possibility of S. pseudintermedius human infection (Van Hoovels et al., 2006) means that fluoroquinolone resistance in this bacterium poses public health hazard. Thus, there is an overriding need to select dosage regimens based on current approaches such as PK/PD integration and modeling to minimize the selection of resistant mutants of bacteria and extend the useful life of antimicrobial agents. The pharmacokinetics of marbofloxacin has been investigated in different animals and it demonstrated an almost 100% bioavailability, higher concentration in plasma and peripheral tissue in goats (Waxman et al., 2001), cows (Schneider et al., 2004), cats (Albarellos et al., 2005), sheep (Sidhu et al., 2010) and dogs (Heinen, 2002). However, the integration of PK data with the time course activity (ex vivo) of marbofloxacin against S. pseudintermedius has not been studied in dogs. Consequently, integrating PK data with PD data (ex vivo and in vitro) helps to establish the PK/PD indices (AUC/MIC, Cmax/MIC) that predict efficacy and minimize resistance development (Gloede et al., 2010; Toutain & Lees, 2004). In addition, since there are
Pharmacokinetic of marbofloxacin in dogs
DOI: 10.3109/00498254.2014.969794
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contradicting values in PK/PD indices that correlate with prevention of resistant mutant selection and efficacy (Klepser et al., 2001; Madaras-Kelly et al., 2000), determining PK/PD indices specific to a particular pathogen and antimicrobial agent has received great attention. Therefore, we performed this study (i) to characterize pharmacokinetics of marbofloxacin following intravenous (i.v.) and intramuscular (i.m.) administration at a dose rate of 2 mg/kg body weight in beagle dogs, (ii) to explore ex vivo and in vivo PK/PD indices using S. pseudintermedius as a model bacterium, and (iii) by using the inhibitory sigmoid Emax model to establish ex vivo AUC/ MIC ratios that could produce bacteriostasis, bactericidal action and elimination of bacteria.
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Corp., St. Louis, MO). The serum concentration-time data of marbofloxacin obtained from i.v. and i.m. administration best fitted a two-compartment open model based on Akaike information criteria (AIC) values. Mean absorption time (MRT) for i.m. administration was calculated as the difference between MRT intramuscular and MRT intravenous. The absolute bioavailability (F) following intramuscular administration was calculated as the ratio of AUC0–24 h from i.v. dosage to the AUC0–24 h from i.m. dosage. F% ¼
AUCi:m ð100Þ AUCi:v
ð1Þ
Materials and methods
Where, AUCi.m and AUCi.v are mean area under concentration time curve after i.v. and i.m. administration.
Chemicals
Pharmacodynamics
Pure standard of marbofloxacin (Sigma Aldrich, St Louis, MO), methanol and acetonitrile (ACS/HPLC certified, B&J Brand) and potassium phosphate monobasic (ACS reagent, Sigma 99.0% purity) were used.
Bacteria
Animals and experimental design A two period cross over pharmacokinetics study of marbofloxacin was performed after intravenous (i.v.) and intramuscular (i.m.) administration in six clinically healthy male beagle dogs weighing (mean ± SD) 9.5 ± 1.42 kg. Animals were randomly divided in to i.v. and i.m. groups each containing three dogs. Each animal was housed in an individual pen and supplied antibiotic-free commercial feed and ad libitum clean water. In two phases with a 15-day washout period, all animals received 2 mg/kg body weight i.v. or i.m. dosage of marbofloxacin. Blood samples (2 ml) were collected before and at 0.15, 0.25, 0.5, 0.75, 1, 2, 4, 6, 9, 12 and 24 h after i.v. and i.m. administration and then centrifuged at 2000 g for 15 min. The serum samples were stored at 20 C until analyses. The study was approved by the Bioethical Committee of Kyungpook National University. HPLC analysis Before HPLC analysis the serum samples were deproteinated by 20% cold trichloroacetic acid solution in acetonitrile. Marbofloxacin concentrations in serum were assayed using Agilent 1100 series HPLC system (Santa Clara, CA) comprising 4.6 250 mm, 5 mm column. An isocratic mobile phase composed of HPLC grade acetonitrile and ACS reagent potassium phosphate monobasic (0.05 M, PH, 2.9) of ratio (80:20% v/v) was used. The UV detection wavelength and column temperature were 295 nm and 30 C respectively. The analytical recovery of marbofloxacin in serum was 97.05 ± 3.62. The quantification and detection limits were 0.042 and 0.012 mg/ml, respectively. The standard curve was linear (r2 > 0.99) over the concentration range of 0.01–10 mg/ml. The coefficients of variation (inter-day and intra-day) were below 10%. Pharmacokinetic analysis Pharmacokinetic analysis of marbofloxacin was performed using Phoenix WinNonlin 6.0 software program (Pharsight
Staphylococcus pseudintermedius isolates of dogs that visited Kyungpook National University Veterinary Hospital (Awji et al., 2012) and a quality control strain of S. aureus (ATCC 25923) were used. MIC and MBC determination The minimum inhibitory concentrations (MICs) of marbofloxacin against clinical isolates of S. pseudintermedius and S. aureus (ATCC 29213) were determined according to the guideline of clinical and laboratory standards institute (CLSI, 2007). In brief, tested strains stored in beads at 70 C were reactivated by streaking on Tryptic Soy Agar (TSA) (Becton Dicknson and Co., Sparks, MD) and incubated at 37 C for 24 h. After 24 h incubation, 4–5 colonies were directly suspended in Mueller-Hinton broth (MHB) to obtain 0.5 McFarland turbidity standards. Marbofloxacin at twice the required final concentration was serially diluted two-fold in 96 well plates to obtain concentrations ranging from 32 to 0.0625 mg/ml. Bacterial suspension (100 ml) with inoculum of approximately 5 105 cfu/ml was added into serially diluted drug concentrations in 96 well plates and incubated at 37 C for 24 h. After 24 h incubation, the lowest concentration that inhibits visible growth of microorganism was taken as MIC. Samples (20 ml) from all concentrations that showed no visible growth in 96-well plates were spotted on TSA and incubated at 37 C for 24 h and then the lowest concentration that inhibited growth on agar plates was considered as minimum bactericidal concentration (MBC). Ex vivo activity of marbofloxacin Serum samples from beagle dogs obtained at 1, 2, 4, 6, 9, 12 and 24 h after i.m. administration of marbofloxacin were used to determine ex vivo antibacterial activity against clinical isolate S. pseudintermedius. Serum samples from beagle dogs that did not receive marbofloxacin were used as control. Freshly grown eight to ten colonies of S. pseudintermedius were inoculated in to 9 ml MHB and incubated overnight at 37 C. A 100 ml overnight culture was added to 1 ml of serum samples to give a final inculum of approximately 106 cfu/ml and incubated at 37 C in shaking incubator at 150 rpm.
S. Yohannes et al.
Xenobiotica, 2015; 45(3): 264–269
Aliquots of bacterial suspension (100 ml) were withdrawn from each culture tubes before and at 1, 3, 6, 9, 12 and 24 h after incubation at 37 C and then subjected to tenfold serial dilution in 0.1% agar saline. Twenty microliters of suspensions were plated on to TSA. Once dry the plates were incubated at 37 C for 24 h and viable counts of samples taken at each time points were determined as cfu/ml. PK/PD integration
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Data obtained from in vitro MIC and MBC were integrated with pharmacokinetic data after both i.v. and i.m. administration. The relationship for serum ex-vivo AUC24 h/MIC and log10 cfu/ml difference of initial inoculum bacterial count and count after 24 h incubation was established using the sigmoid Emax model, described by the equation below:
E ¼ E0 þ
ðEmax E0 ÞCeN ðECN 50þCeN Þ
ð2Þ
where, E is the antibacterial effect measured as change in log10 cfu/ml in the serum sample after 24 h incubation compared to the initial inoculum, E0 is the difference in bacterial count (log10 cfu/ml) after 24 h incubation in beagle dog serum compared to the initial inoculum (0 h), Emax is the maximum effect of marbofloxacin (log10 cfu/ml reduction) after 24 h incubation in beagle dog serum compared to the initial inoculum, EC50 is the AUC24 h/MIC of marbofloxacin producing 50% of the maximal antibacterial effect, Ce is the AUC24 h/MIC of marbofloxacin in the effect compartment (ex vivo site), i.e. serum concentration multiplied by incubation time (24 h)/MICin vitro, and N is the Hill coefficient which describes the steepness of the AUC24 h/MIC-effect relationship. The antibacterial effect of marbofloxacin in serum was quantified by calculation of ex vivo AUC24 h/MIC values for bacteriostatic action, reduction of bacterial count by 50%, bactericidal action and bacterial elimination. Values of AUC24 h/MIC for bacteriostatic and bactericidal actions were defined as those which produced E ¼ 0 (no change in bacterial count after 24 h incubation) and E ¼ 3 (a 3 log reduction of the original inoculum count after 24 h incubation), respectively. An AUC24 h/MIC for bacterial elimination was the lowest value which provided the maximal antibacterial effect that is a reduction in bacterial count to the limit of detection (100 cfu/ml). Ex vivo AUC0–24 h/MIC values derived from sigmoid Emax equation either for bactericidal activity or elimination of bacteria after 24 h incubation were used to calculate optimal dosage using the equation described below: Dose ðper dayÞ ¼
AUIC MIC CL fu F 24 h
ð3Þ
where, AUIC: is ex-vivo AUC0–4 h/MIC for bactericidal activity or elimination obtained for serum; CL: is marbofloxacin clearance per day in dogs; fu: is the fraction of unbound drug; F: is bioavailability of marbofloxacin after i.v. or i.m. administration in beagle dogs.
Results Pharmacokinetics Mean ± SEM serum concentration time curves of marbofloxacin after single dose (2 mg/kg body weight) i.v. and i.m. administration are shown in Figure 1. Mean ± SEM pharmacokinetic parameters of marbofloxacin after single dose (2 mg/kg body weight) i.v. and i.m. administration are shown in Table 1. The values for major PK parameters, such as AUC, t1/2b, Vss, CLB and MRT were essentially comparable between the i.v. and i.m. routes, which were 8.47 ± 3.51 h mg/mL, 8.08 ± 6.25 h, 2.32 ± 1.00 L/kg, 0.23 ± 0.06 L/kg/h and 9.85 ± 7.88 h after i.v. administration and 11.37 ± 3.07 h mg/mL, 7.51 ± 3.70 h, 1.80 ± 0.90 L/kg, 0.17 ± 0.04 L/kg/h and 11.12 ± 6.02 h after i.m. injection. After i.m. administration a complete and rapid absorption was observed with mean absolute bioavailability and mean absorption time (MAT) of 107% and 1.25 h, respectively. After i.m administration, the peak serum concentration (Mean ± SEM) of 1.76 ± 0.09 mg/mL was achieved at Tmax of 0.47 ± 0.08 h. Pharmacokinetic/pharmacodynamic integration Marbofloxacin showed similar MICs (0.125 mg/ml) in both serum and broth against clinical isolate of S. pseudointermedius. The PK/PD parameters obtained from integration of PK data with in vitro MIC and MBC against S. pseudintermedius are presented in Table 2. In vivo AUC/MIC and AUC/MBC ratios of marbofloxacin against S. pseudintermedius after i.v. and i.m. administration were 67.76, 16.94 h and 91.18, 23.86 h, respectively. The time where the plasma concentration exceeds MIC (T > MIC) and MBC (T > MBC) after i.m. administration were 9.89 and 10.5 h, respectively. The Cmax/ MIC and Cmax/MBC ratios after i.m. administration of marbofloxacin were 13.04 and 3.4, respectively. Ex vivo antibacterial activity of marbofloxacin Ex-vivo antibacterial activity of marbofloxacin against S. pseudintermedius was determined in serum samples collected before and at 1, 2, 4, 6, 9, 12, 24 h after i.m administration (Table 3). The mean marbofloxacin concentrations for the indicated time points were 1.304, 0.908, 0.709, 0.590, 0.506, 0.338 and 0.044 mg/ml. Ex-vivo antibacterial 10 Marbofloxacinconcentraon (µg/mL)
266
1
0.1
0.01 0
5
10
15
20
Time (hour)
Figure 1. Serum concentration versus time profiles of marbofloxacin after i.v. (m) and i.m. (g) administration with 2 mg/kg of bodyweight in beagle dogs (n ¼ 6). Bars represent standard deviations.
Pharmacokinetic of marbofloxacin in dogs
DOI: 10.3109/00498254.2014.969794
Table 1. Pharmacokinetic parameters of marbofloxacin in serum (mean ± SEM, n ¼ 6) after a single dose (2 mg/kg boy weight) i.v. and i.m. administration in beagle dogs.
Table 3. Ex vivo AUC24 h/MIC and AUC24 h/MBC (Mean ± SEM, n ¼ 6) after i.m. administration (2 mg/kg body weight) of marbofloxacin in beagle dogs.
Mean ± SEM
AUC AUC0–1 AUMC A B K10_HL K01_HL t1/2a t1/2b MRT Vss V1_F V2_F K10 K12 K21 CL_F CLD2_F Tmax Cmax MAT F (%)
PK/PD integration parameters
Units
i.v.
i.m.
h g/mL h mg/mL h h mg/mL 1/h 1/h h h h h h L/kg L/kg L/kg 1/h 1/h 1/h L/kg//h L/kg//h h mg/mL h –
8.47 ± 3.51 10.22 ± 3.62 83.57 ± 0.22 1.91 ± 0.54 0.08 ± 0.06 1.80 ± 0.79 – 0.36 ± 0.10 8.08 ± 6.25 9.85 ± 7.88 2.32 ± 1.00 0.60 ± 0.04 1.72 ± 0.60 0.38 ± 0.16 1.18 ± 0.26 0.42 ± 3.51 0.23 ± 0.06 0.80 ± 0.11 – – – –
11.37 ± 3.07 11.03 ± 3.12 – 2.71 ± 1.50 0.09 ± 0.04 1.95 ± 0.17 0.32 ± 0.16 0.25 ± 0.14 7.51 ± 3.70 11.12 ± 6.02 1.80 ± 0.90 0.49 ± 0.08 1.22 ± 0.80 0.35 ± 0.17 1.74 ± 0.78 0.70 ± 0.68 0.17 ± 0.04 0.86 ± 0.75 0.47 ± 0.08 1.76 ± 0.09 1.25 ± 0.51 107 ± 4.03
AUC, area under concentration versus time curve; AUC0–1, area under concentration versus time curve from zero to infinity; AUMC, area under the first moment curve; a, distribution rate constant; b, elimination rate constant; K10_HL, elimination half life; K01_HL, half life of absorption; t1/2(a), distribution half life; t1/2(b), elimination half life; A, distribution phase intercept; B, elimination phase intercept; MRT, mean residence time; Vss, volume of distribution at steady state; V1/F, volume of distribution of central compartment; V2/F, volume of distribution of peripheral compartment; K10, central compartment elimination rate constant; K12, rate constant for passage from central to peripheral compartment; K21, rate constant for passage from peripheral to central compartment; CL_F, total body clearance; CLD2_F, inter compartmental clearance; Tmax, time of maximum concentration; Cmax, maximum concentration; MAT, mean absorption time; F(%), percent absolute bioavailability.
Table 2. In vivo PK/PD integration of marbofloxacin (mean ± SEM, n ¼ 6) after i.m administration (2 mg/kg body weight) of marbofloxacin in beagle dogs. S. pseudintermedius PK/PD parameters
i.v.
i.m.
AUC24 h/MIC (h) AUC24 h/MBC (h) T>MIC T>MBC Cmax/MIC Cmax/MBC
67.76 ± 1.23 16.94 ± 0.86 9.83 ± 1.72 9.89 ± 1.89 – –
91.18 ± 2.61 23.86 ± 0.65 15.50 ± 6.68 10.50 ± 2.50 13.04 ± 1.03 3.40 ± 1.03
AUC0–24 h/MIC, ratio of AUC to MIC; AUC0–24 h/MBC, ratio of AUC to MIC; T > MIC, time that concentration of marbofloxacin exceeds MIC; T>MBC, time that concentration of marbofloxacin exceeds MBC; Cmax/MIC, ratio of peak serum concentration to MIC; Cmax/MBC, ratio of peak serum concentration to MBC.
activity of marbofloxacin against S. pseudintermedius in serum after i.m. administration is indicated in Figure 2. Log cfu/ml reduction to the level of lower detection limit (100 cfu/ml) was observed in samples collected at 1 and 2 h after i.m. administration. Marbofloxacin concentration
Sampling time (h)
AUC24 h/MIC
AUC24 h/MBC
1 2 4 6 9 12 24
250.39 ± 24.81 174.35 ± 7.35 136.21 ± 8.18 113.43 ± 7.37 97.26 ± 11.87 65.02 ± 11.90 11.88 ± 3.69
62.59 ± 6.20 43.58 ± 1.83 34.05 ± 2.04 28.35 ± 1.84 24.31 ± 2.96 16.25 ± 3.96 2.97 ± 1.97
AUC0–24 h/MIC and AUC0–24 h/MBC, ratios of serum AUC0–24 h in samples collected between 1 and 24 h after i.m. administration of marbofloxacin in beagle dogs to MIC and MBC in serum respectively.
10 0 Log10 (CFU/mL)
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PK parameters
267
8
1 2
6
4 4
6 9
2
12 24
0 0
6
12
18
24
Time (h)
Figure 2. Ex-vivo antibacterial activity of marbofloxaacin against S. pseudointermedius in serum after i.m. administration at a dose rate of 2 mg/kg. Values are means (n ¼ 6). Samples were harvested at predetermined times between 0 and 24 h. Table 4. PK/PD integration of ex-vivo serum marbofloxacin (Mean ± SEM, n ¼ 6) after i.m. administration (2 mg/kg body weight) of marbofloxacin in beagle dogs. Parameters Log Emax (cfu/ml) Log E0 (cfu/ml) Log Emax – LogE0 (cfu/ml) AUC24 h/MIC for bacteriostatic action AUC24 h/MIC50 AUC24 h/MIC for bactericidal action AUC24 h/MIC for bacterial elimination Slope (N)
Mean ± SEM 2.77 ± 0.10 4.04 ± 0.01 6.80 ± 0.01 65.03 ± 11.53 70.74 ± 3.69 97.02 ± 9.24 136.21 ± 7.58 5.44 ± 0.78
Emax difference in log of number of bacteria (cfu/ml) in sample incubated with marbofloxacin between time 0 and 24 h, when the detection limit (100 cfu/ml) is reached. E0, difference in log of number of bacteria (cfu/ml) in control sample (absence of marbofloxacin) between time 0 and 24 h; AUC0–24 h/MIC50, AUC24 h/MIC of marbofloxacin producing 50% of the maximum antibacterial effect; N, the hill coefficient.
in serum collected from 4 to 9 h after i.m. administration showed bactericidal activity, whereas marbofloxacin in serum samples collected at 12 h after i.m. dose had bacteriostatic effect. No inhibitory effect of marbofloxacin against S. pseudintermedius was observed in 24 h serum samples. As shown in Table 4 and Figure 3, the values of AUC/MIC ratio needed for bacteriostatic, reduction in bacterial count by
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S. Yohannes et al.
Figure 3. Sigmoid Emax relationship for bacterial count (log cfu/ml) versus ex vivo AUC24 h/MIC for S.pseudointermedius in serum of beagle dogs. The curve represents the line of predicted values and the circles are values of beagle dogs.
50%, bactericidal and elimination of bacteria in serum were 65.03, 70.74, 97.02 and 136.21, respectively. Based on the MIC (0.125 mg/ml) of marbofloxacin against S. pseudintermedius, the lowest calculated doses providing bactericidal and elimination of S. pseudintermedius were 2.1 and 2.9 mg/ kg/day, respectively.
Discussion Marbofloxacin has shown a complete absorption and bioavailability after i.m. administration in beagle dogs. Since bactericidal activity of fluoroquinolones is concentrationdependent (Aliabadi et al., 2003), the high bioavailability (>100%) and fast absorption (K01_HL, 0.32 ± 0.16) of marbofloxacin favors the in vivo bactericidal activity (Table 1). Although variation in bioavailability of a drug is expected among species, bioavailability of marbofloxacin observed in beagle dogs was comparable with values observed in several species, including sheep, goats and pigs (Ding et al., 2010; Marin et al., 2013; Sidhu et al., 2010; Waxman et al., 2001). The Cmax of marbofloxacin achieved after i.m. administration (Table 1) was higher than the MIC break point of fluoroquinolones recommended against most susceptible bacterial species (CLSI, 2007). This value was comparable with values reported in pigs and goats (Ding et al., 2010; Waxman et al., 2001), while it is slightly lower than that in rabbits (Cmax ¼ 2.04, Sidhu et al., 2010) and higher than that in sheep (Cmax ¼ 0.8, Marı´n et al., 2013). The longer elimination half-life of marbofloxacin observed in this study reflects the advantage of this drug in maintaining effective concentration in the body thereby allowing longer time for drug-pathogen interaction. The elimination half-life of marbofloxacin in beagle dogs after i.v. administration was comparable with values in pigs, rabbits, and goats (Ding et al 2010; Marı´n et al., 2013; Waxman et al., 2001) and longer than the value in sheep (3.96 h, Sidhu et al., 2010). For i.m. administration, the elimination half-life of marbofloxacin in the present study was in agreement with those values reported in goats, camels and rabbits (Larajeet al., 2006; Marı´n et al.,
Xenobiotica, 2015; 45(3): 264–269
2011; Waxman et al., 2001). However, it was shorter than values reported in pigs (Ding et al., 2010) and longer than values reported in horses and calves (Carretero et al., 2002; Ismail & El-Kattan, 2007). The high values of steady state volume of distribution (Vss) of marbofloxacin in beagle dogs (2.32 and 1.8 L/kg after i.v. and i.m. administration) suggest good tissue penetration of the drug in dogs. The higher K12/K21 ratio of marbofloxacin also indicates faster transport of marbofloxacin from central to peripheral compartment than redistribution from peripheral to central compartment. The higher peripheral distribution of marbofloxacin could enhance concentration dependent bactericidal activity of marbofloxacin at infection site (Walker, 2000). Plasma protein binding of drugs can affect the tissue distribution and hence activity of antimicrobial agents (Nix et al., 2004). However, marbofloxacin had similar MIC values against S. pseudintermedius both in serum and broth, suggesting low plasma protein binding of this drug. Similarly other fluoroquinolones, such as orbifloxacin were found to have low protein binding and similar MIC values in serum and growth media against several bacterial species (Elias et. al., 2009). The PK/PD modeling presents a better approach to dose titration studies for selecting rational dosage regimens in veterinary medicine (Toutain & Lees, 2004). In the current study, the PK data obtained from a single i.v. and i.m. administration of marbofloxacin in dogs was integrated with PD (in vitro and ex vivo) data using S. pseudintermedius as a model pathogen. Using the inhibitory sigmoid Emax model, the lowest ex-vivo AUC0–24 h/MIC ratios required for different levels of marbofloxacin activity against S. pseudintermedius were determined in serum of beagle dogs (Table 4). The in vivo AUC0–24 h/MIC ratio (91.18 h, Table 2) was lower than ex vivo AUC0–24 h/MIC ratio (97.02, Table 4) required for 3 log reduction of bacterial count (bactericidal action) against S. pseudintermedius, suggesting that the administered dose (2 mg/kg body weight per day) of marbofloxacin might not guarantee clinical efficacy against infections associated with S. pseudointermedius. Although there is no universal agreement on the time that drug concentration should remain above MIC (T > MIC), the percentage of time (%T ¼ 63.21) that marbofloxacin serum concentration exceeds MIC over the dosing interval (Table 2) might favor time-dependent activity of marbofloxacin against gram-positive bacteria (Aliabadi & Lees, 2002). For fluoroquinolones the ratios of AUC0–24 h/MIC and Cmax/MIC are predictive of bacterial eradication (Andes et al., 2002; Craig, 1998; Forrest et al., 1993). The ex vivo AUC0–24 h/MIC ratios of marbofloxacin required for bactericidal action and eradication of the clinical strain with MIC of 0.125 mg/ml were 97.02 ± 9.24 h and 136.21 ± 7.58 h, which are higher than the in vivo AUC24/MIC ratio (91.18 ± 2.61) achieved after a single i.m. administration of marbofloxacin (2 mg/kg). In clinical area, S. pseudintermedius strains have higher MIC values. For example, in our previous study, the MIC90 of S. pseudintermedius strains was found to be 0.5 mg/ml (Awji et al., 2012). These suggest that the conventional dose of marbofloxacin (2 mg/kg) may not suffice to kill and eradicate S. pseudintermedius strains encountered in clinical area.
DOI: 10.3109/00498254.2014.969794
Conclusions The results of the present study suggest that marbofloxacin administered i.m. in dogs has good pharmacokinetic profile in terms of high bioavailability, large volume of tissue distribution and prolonged elimination half-life. The PK/PD integration and modeling indicate that the conventional 2 mg/kg daily dose marbofloxacin may not achieve AUC0–24 h/MIC ratio required for bactericidal activity and bacterial elimination, and higher doses of marbofloxacin should be considered to combat clinical infections such as canine pyoderma caused by S. pseudintermedius. However, further study in both healthy and naturally diseased animals and disease model are recommended to establish these findings in a clinical setting.
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Acknowledgements This research was supported in part by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 2011-0021670) and in part by a grant from the Next-Generation BioGreen 21 Program (No. PJ009007), Rural Development Administration, Republic of South Korea.
Declaration of interest Authors declare no competing interest.
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Forrest A, Nix DE, Ballow CH, et al. (1993). Pharmacodynamics of intravenous ciprofloxacin in seriously ill patients. Antimicrob Agents Chemother 37:1073–81. Gloede J, Scheerans C, Derendorf H, Kloft C. (2010). In vitro pharmacodynamic models to determine the effect of antibacterial drugs. J Antimicrob Chemother 65:186–201. Heinen E. (2002). Comparative serum pharmacokinetics of the fluoroquinolones enrofloxacin, difloxacin, marbofloxacin, and orbifloxacin in dogs after single oral administration. J Vet Pharmacol Ther 25:1–5. Horspool LJ, van Laar P, van den Bos R, Mawhinney I. (2004). Treatment of canine pyoderma with ibafloxacin and marbofloxacin: fluoroquinolones with different pharmacokinetic profiles. J Vet Pharmacol Ther 27:147–53. Ismail M, El-Kattan YA. (2007). Comparative pharmacokinetics of marbofloxacin in healthy and Mannheimia haemolytica infected calves. Res Vet Sci 82:398–404. Kadlec K, Schwarz S, Perreten V, et al. (2010). Molecular analysis of methicillin-resistant Staphylococcus pseudintermedius of feline origin from different European countries and North America. J Antimicrob Chemother 65:1826–8. Klepser ME, Ernst EJ, Petzold CR, et al. (2001). Comparative bactericidal activities of ciprofloxacin, levofloxacin, moxifloxacin, and trovafloxacin against Streptococcus pneumonia in a dynamic in vitro model. Antimicrob Agents Chemother 45:673–8. Larajeet R, Talmi A, Bounaga R, et al. (2006). Comparative pharmacokinetics of marbofloxacin after a single intramuscular administration at two dosages to camels (Camelus dromedaries). J Vet Pharmacol Ther 29:229–31. Madaras-Kelly KJ, Demasters TA. (2000). In vitro characterization of fluoroquinolone concentration/MIC antimicrobial activity and resistance while simulating clinical pharmacokinetics of levofloxacin, ofloxacin, or ciprofloxacin against Streptococcus pneumoniae. Diagn Microbiol Infect Dis 37:253–60. Marı´n P, Alamo LF, Escudero E, et al. (2013). Pharmacokinetics of marbofloxacin in rabbit after intravenous, intramuscular, and subcutaneous administration. Res Vet Sci 94:698–700. Meunier D, Acar JF, Martel JL, et al. (2004). A seven-year survey of susceptibility to marbofloxacin of pathogenic strains isolated from pets. Int J Antimicrob Agents 24:592–8. Nix DE, Matthias KR, Ferguson EC. (2004). Effect of ertapenem protein binding on killing of bacteria. Antimicrob Agents Chemother 48: 3419–24. Perreten V, Kadlec K, Schwarz S, et al. (2010). Clonal spread of methicillin-resistant Staphylococcus pseudintermedius in Europe and North America: an international multicentre study. J Antimicrob Chemother 65:1145–54. Schneider M, Valle M, Woehrle F, Boisrame B. (2004). Pharmacokinetics of marbofloxacin in lactating cows after repeated intramuscular administrations and pharmacodynamics against mastitis isolated strains. J Dairy Sci 87:202–11. Sidhu PK, Landoni MF, Aliabadi FS, Lees P. PK/PD integration and modeling of marbofloxacin in sheep. Res Vet Sci 88:34–41. Toutain PL, Lees P. (2004). Integration and modeling of pharmacokinetic and pharmacodynamic data to optimize dosage regimens in veterinary medicine. J Vet Pharmacol Ther 27:467–77. Van Hoovels L, Vankeerberghen A, Boel A, et al. (2006). First case of Staphylococcus pseudintermedius infection in a human. J Clin Microbiol 44:4609–12. Walker RD. (2000). The use of fluoroquinolones for companion animal antimicrobial therapy. Aust Vet J 78:84–90. Waxman S, Rodrı´guez C, Gonza´lez F, et al. (2001). Pharmacokinetic behavior of marbofloxacin after intravenous and intramuscular administrations in adult goats. J Vet Pharmacol Ther 4:375–8. Yoo JW, Lee KJ, Lee SY, et al. (2010). Antibiotic resistance profiles of Staphylococcus pseudintermedius isolates from canine patients in Korea. J Microbiol Biotechnol 20:1764–8.
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Pharmacokinetics and pharmacokinetic/ pharmacodynamic integration of marbofloxacin after intravenous and intramuscular administration in beagle dogs Sileshi Yohannes, Elias Gebru Awji, Seung-Jin Lee & Seung-Chun Park To cite this article: Sileshi Yohannes, Elias Gebru Awji, Seung-Jin Lee & Seung-Chun Park (2015) Pharmacokinetics and pharmacokinetic/pharmacodynamic integration of marbofloxacin after intravenous and intramuscular administration in beagle dogs, Xenobiotica, 45:3, 264-269, DOI: 10.3109/00498254.2014.969794 To link to this article: http://dx.doi.org/10.3109/00498254.2014.969794
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Date: 23 August 2017, At: 23:05
http://informahealthcare.com/xen ISSN: 0049-8254 (print), 1366-5928 (electronic) Xenobiotica, 2015; 45(3): 264–269 ! 2014 Informa UK Ltd. DOI: 10.3109/00498254.2014.969794
RESEARCH ARTICLE
Pharmacokinetics and pharmacokinetic/pharmacodynamic integration of marbofloxacin after intravenous and intramuscular administration in beagle dogs Sileshi Yohannes1, Elias Gebru Awji2, Seung-Jin Lee1, and Seung-Chun Park1 Downloaded by [Australian Catholic University] at 23:05 23 August 2017
1
Laboratory of Pharmacokinetics and Pharmacodynamics, Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Kyungpook National University, Daegu, Republic of Korea and 2Lovelace Respiratory Research Institute, NM, USA Abstract
Keywords
1. The aim of the present study was to determine the PKs of marbofloxacin in beagle dogs after intravenous (i.v.) and intramuscular (i.m.) administration, the ex vivo and in vitro PK/PD indices of marbofloxacin against clinical isolates of Staphylococcus pseudintermedius, and the ex vivo AUC/MIC ratios associated with different levels of antibacterial activity. 2. After i.v. of marbofloxacin (2 mg/kg), the mean ± SEM values of AUC, t1/2b, Vss, and CL were 8.47 ± 3.51 h mg/mL, 8.08 ± 6.25 h, 2.32 ± 1.00 L/kg and 0.23 ± 0.06 L/kg/h and corresponding values after intramuscular injection were 11.37 ± 3.07 h mg/mL, 7.51 ± 3.70, 1.80 ± 0.90 L/kg and 0.17 ± 0.04 L/kg/h. After i.m. administration, a Cmax of 1.76 ± 0.09 mg/mL was achieved at Tmax of 0.47 ± 0.08 h. The ex-vivo AUC/MIC ratios required to produce bacteriostasis, bactericidal action and elimination of S. pseudintermedius were 65.03, 97.02 and 136.84 h. 3. The in vivo AUC/MIC ratios obtained after i.v. and i.m. administration of 2 mg/kg marbofloxacin (67.76 ± 1.23 and 91.18 ± 2.61) were below the ex vivo AUC/MIC ratios required for bactericidal activity and bacterial elimination (97.02 ± 9.24 2 mg/kg and 136.21 ± 7.58), suggesting that the recommended daily dosage (2 mg/kg) may not suffice to kill and eradicate S. pseudintermedius strains encountered in clinical area.
Beagle dogs, drug resistance, marbofloxacin, PK/PD, staphylococcus pseudintermedius History Received 22 August 2014 Revised 22 September 2014 Accepted 23 September 2014 Published online 3 December 2014
Introduction Marbofloxacin is a third generation fluoroquinolone that exhibits concentration dependent bactericidal activity against gram-negative and gram-positive bacteria (Aliabadi et al., 2003). It has shown higher in vitro activity against pathogenic strains isolated from urinary tract infections, respiratory tract infections, dermatological infections and otitis in pets (Meunier et al., 2004) and produced greater than 80% clinical efficacy in the treatment of superficial and deep bacterial pyoderma (Horspool et al., 2004), which is the most commonly diagnosed skin problem in dogs mainly caused by S. pseudintermedius, a coagulase positive normal resident bacterium of the skin. Although marbofloxacin is a frequently prescribed drug with high clinical efficacy against various bacterial infections, including canine pyoderma, fluoroquinolone resistance in S. pseudintermedius is an emerging problem (Perreten et al., 2010; Yoo et al., 2010). This undermines the successful treatment outcomes of fluoroquinolones and adds upon the Address for correspondence: Seung-Chun Park, Laboratory of Pharmacokinetics and Pharmacodynamics, Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, Kyungpook National University, Daegu, Republic of Korea. E-mail: [email protected]
existing challenge in selecting the most appropriate antimicrobial therapy against methicillin resistant S. pseudintermedius (MRSP) infections, which is highly disseminated in dogs and cats (Kadlec et al., 2010; Perreten et al., 2010). Furthermore, the possibility of S. pseudintermedius human infection (Van Hoovels et al., 2006) means that fluoroquinolone resistance in this bacterium poses public health hazard. Thus, there is an overriding need to select dosage regimens based on current approaches such as PK/PD integration and modeling to minimize the selection of resistant mutants of bacteria and extend the useful life of antimicrobial agents. The pharmacokinetics of marbofloxacin has been investigated in different animals and it demonstrated an almost 100% bioavailability, higher concentration in plasma and peripheral tissue in goats (Waxman et al., 2001), cows (Schneider et al., 2004), cats (Albarellos et al., 2005), sheep (Sidhu et al., 2010) and dogs (Heinen, 2002). However, the integration of PK data with the time course activity (ex vivo) of marbofloxacin against S. pseudintermedius has not been studied in dogs. Consequently, integrating PK data with PD data (ex vivo and in vitro) helps to establish the PK/PD indices (AUC/MIC, Cmax/MIC) that predict efficacy and minimize resistance development (Gloede et al., 2010; Toutain & Lees, 2004). In addition, since there are
Pharmacokinetic of marbofloxacin in dogs
DOI: 10.3109/00498254.2014.969794
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contradicting values in PK/PD indices that correlate with prevention of resistant mutant selection and efficacy (Klepser et al., 2001; Madaras-Kelly et al., 2000), determining PK/PD indices specific to a particular pathogen and antimicrobial agent has received great attention. Therefore, we performed this study (i) to characterize pharmacokinetics of marbofloxacin following intravenous (i.v.) and intramuscular (i.m.) administration at a dose rate of 2 mg/kg body weight in beagle dogs, (ii) to explore ex vivo and in vivo PK/PD indices using S. pseudintermedius as a model bacterium, and (iii) by using the inhibitory sigmoid Emax model to establish ex vivo AUC/ MIC ratios that could produce bacteriostasis, bactericidal action and elimination of bacteria.
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Corp., St. Louis, MO). The serum concentration-time data of marbofloxacin obtained from i.v. and i.m. administration best fitted a two-compartment open model based on Akaike information criteria (AIC) values. Mean absorption time (MRT) for i.m. administration was calculated as the difference between MRT intramuscular and MRT intravenous. The absolute bioavailability (F) following intramuscular administration was calculated as the ratio of AUC0–24 h from i.v. dosage to the AUC0–24 h from i.m. dosage. F% ¼
AUCi:m ð100Þ AUCi:v
ð1Þ
Materials and methods
Where, AUCi.m and AUCi.v are mean area under concentration time curve after i.v. and i.m. administration.
Chemicals
Pharmacodynamics
Pure standard of marbofloxacin (Sigma Aldrich, St Louis, MO), methanol and acetonitrile (ACS/HPLC certified, B&J Brand) and potassium phosphate monobasic (ACS reagent, Sigma 99.0% purity) were used.
Bacteria
Animals and experimental design A two period cross over pharmacokinetics study of marbofloxacin was performed after intravenous (i.v.) and intramuscular (i.m.) administration in six clinically healthy male beagle dogs weighing (mean ± SD) 9.5 ± 1.42 kg. Animals were randomly divided in to i.v. and i.m. groups each containing three dogs. Each animal was housed in an individual pen and supplied antibiotic-free commercial feed and ad libitum clean water. In two phases with a 15-day washout period, all animals received 2 mg/kg body weight i.v. or i.m. dosage of marbofloxacin. Blood samples (2 ml) were collected before and at 0.15, 0.25, 0.5, 0.75, 1, 2, 4, 6, 9, 12 and 24 h after i.v. and i.m. administration and then centrifuged at 2000 g for 15 min. The serum samples were stored at 20 C until analyses. The study was approved by the Bioethical Committee of Kyungpook National University. HPLC analysis Before HPLC analysis the serum samples were deproteinated by 20% cold trichloroacetic acid solution in acetonitrile. Marbofloxacin concentrations in serum were assayed using Agilent 1100 series HPLC system (Santa Clara, CA) comprising 4.6 250 mm, 5 mm column. An isocratic mobile phase composed of HPLC grade acetonitrile and ACS reagent potassium phosphate monobasic (0.05 M, PH, 2.9) of ratio (80:20% v/v) was used. The UV detection wavelength and column temperature were 295 nm and 30 C respectively. The analytical recovery of marbofloxacin in serum was 97.05 ± 3.62. The quantification and detection limits were 0.042 and 0.012 mg/ml, respectively. The standard curve was linear (r2 > 0.99) over the concentration range of 0.01–10 mg/ml. The coefficients of variation (inter-day and intra-day) were below 10%. Pharmacokinetic analysis Pharmacokinetic analysis of marbofloxacin was performed using Phoenix WinNonlin 6.0 software program (Pharsight
Staphylococcus pseudintermedius isolates of dogs that visited Kyungpook National University Veterinary Hospital (Awji et al., 2012) and a quality control strain of S. aureus (ATCC 25923) were used. MIC and MBC determination The minimum inhibitory concentrations (MICs) of marbofloxacin against clinical isolates of S. pseudintermedius and S. aureus (ATCC 29213) were determined according to the guideline of clinical and laboratory standards institute (CLSI, 2007). In brief, tested strains stored in beads at 70 C were reactivated by streaking on Tryptic Soy Agar (TSA) (Becton Dicknson and Co., Sparks, MD) and incubated at 37 C for 24 h. After 24 h incubation, 4–5 colonies were directly suspended in Mueller-Hinton broth (MHB) to obtain 0.5 McFarland turbidity standards. Marbofloxacin at twice the required final concentration was serially diluted two-fold in 96 well plates to obtain concentrations ranging from 32 to 0.0625 mg/ml. Bacterial suspension (100 ml) with inoculum of approximately 5 105 cfu/ml was added into serially diluted drug concentrations in 96 well plates and incubated at 37 C for 24 h. After 24 h incubation, the lowest concentration that inhibits visible growth of microorganism was taken as MIC. Samples (20 ml) from all concentrations that showed no visible growth in 96-well plates were spotted on TSA and incubated at 37 C for 24 h and then the lowest concentration that inhibited growth on agar plates was considered as minimum bactericidal concentration (MBC). Ex vivo activity of marbofloxacin Serum samples from beagle dogs obtained at 1, 2, 4, 6, 9, 12 and 24 h after i.m. administration of marbofloxacin were used to determine ex vivo antibacterial activity against clinical isolate S. pseudintermedius. Serum samples from beagle dogs that did not receive marbofloxacin were used as control. Freshly grown eight to ten colonies of S. pseudintermedius were inoculated in to 9 ml MHB and incubated overnight at 37 C. A 100 ml overnight culture was added to 1 ml of serum samples to give a final inculum of approximately 106 cfu/ml and incubated at 37 C in shaking incubator at 150 rpm.
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Xenobiotica, 2015; 45(3): 264–269
Aliquots of bacterial suspension (100 ml) were withdrawn from each culture tubes before and at 1, 3, 6, 9, 12 and 24 h after incubation at 37 C and then subjected to tenfold serial dilution in 0.1% agar saline. Twenty microliters of suspensions were plated on to TSA. Once dry the plates were incubated at 37 C for 24 h and viable counts of samples taken at each time points were determined as cfu/ml. PK/PD integration
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Data obtained from in vitro MIC and MBC were integrated with pharmacokinetic data after both i.v. and i.m. administration. The relationship for serum ex-vivo AUC24 h/MIC and log10 cfu/ml difference of initial inoculum bacterial count and count after 24 h incubation was established using the sigmoid Emax model, described by the equation below:
E ¼ E0 þ
ðEmax E0 ÞCeN ðECN 50þCeN Þ
ð2Þ
where, E is the antibacterial effect measured as change in log10 cfu/ml in the serum sample after 24 h incubation compared to the initial inoculum, E0 is the difference in bacterial count (log10 cfu/ml) after 24 h incubation in beagle dog serum compared to the initial inoculum (0 h), Emax is the maximum effect of marbofloxacin (log10 cfu/ml reduction) after 24 h incubation in beagle dog serum compared to the initial inoculum, EC50 is the AUC24 h/MIC of marbofloxacin producing 50% of the maximal antibacterial effect, Ce is the AUC24 h/MIC of marbofloxacin in the effect compartment (ex vivo site), i.e. serum concentration multiplied by incubation time (24 h)/MICin vitro, and N is the Hill coefficient which describes the steepness of the AUC24 h/MIC-effect relationship. The antibacterial effect of marbofloxacin in serum was quantified by calculation of ex vivo AUC24 h/MIC values for bacteriostatic action, reduction of bacterial count by 50%, bactericidal action and bacterial elimination. Values of AUC24 h/MIC for bacteriostatic and bactericidal actions were defined as those which produced E ¼ 0 (no change in bacterial count after 24 h incubation) and E ¼ 3 (a 3 log reduction of the original inoculum count after 24 h incubation), respectively. An AUC24 h/MIC for bacterial elimination was the lowest value which provided the maximal antibacterial effect that is a reduction in bacterial count to the limit of detection (100 cfu/ml). Ex vivo AUC0–24 h/MIC values derived from sigmoid Emax equation either for bactericidal activity or elimination of bacteria after 24 h incubation were used to calculate optimal dosage using the equation described below: Dose ðper dayÞ ¼
AUIC MIC CL fu F 24 h
ð3Þ
where, AUIC: is ex-vivo AUC0–4 h/MIC for bactericidal activity or elimination obtained for serum; CL: is marbofloxacin clearance per day in dogs; fu: is the fraction of unbound drug; F: is bioavailability of marbofloxacin after i.v. or i.m. administration in beagle dogs.
Results Pharmacokinetics Mean ± SEM serum concentration time curves of marbofloxacin after single dose (2 mg/kg body weight) i.v. and i.m. administration are shown in Figure 1. Mean ± SEM pharmacokinetic parameters of marbofloxacin after single dose (2 mg/kg body weight) i.v. and i.m. administration are shown in Table 1. The values for major PK parameters, such as AUC, t1/2b, Vss, CLB and MRT were essentially comparable between the i.v. and i.m. routes, which were 8.47 ± 3.51 h mg/mL, 8.08 ± 6.25 h, 2.32 ± 1.00 L/kg, 0.23 ± 0.06 L/kg/h and 9.85 ± 7.88 h after i.v. administration and 11.37 ± 3.07 h mg/mL, 7.51 ± 3.70 h, 1.80 ± 0.90 L/kg, 0.17 ± 0.04 L/kg/h and 11.12 ± 6.02 h after i.m. injection. After i.m. administration a complete and rapid absorption was observed with mean absolute bioavailability and mean absorption time (MAT) of 107% and 1.25 h, respectively. After i.m administration, the peak serum concentration (Mean ± SEM) of 1.76 ± 0.09 mg/mL was achieved at Tmax of 0.47 ± 0.08 h. Pharmacokinetic/pharmacodynamic integration Marbofloxacin showed similar MICs (0.125 mg/ml) in both serum and broth against clinical isolate of S. pseudointermedius. The PK/PD parameters obtained from integration of PK data with in vitro MIC and MBC against S. pseudintermedius are presented in Table 2. In vivo AUC/MIC and AUC/MBC ratios of marbofloxacin against S. pseudintermedius after i.v. and i.m. administration were 67.76, 16.94 h and 91.18, 23.86 h, respectively. The time where the plasma concentration exceeds MIC (T > MIC) and MBC (T > MBC) after i.m. administration were 9.89 and 10.5 h, respectively. The Cmax/ MIC and Cmax/MBC ratios after i.m. administration of marbofloxacin were 13.04 and 3.4, respectively. Ex vivo antibacterial activity of marbofloxacin Ex-vivo antibacterial activity of marbofloxacin against S. pseudintermedius was determined in serum samples collected before and at 1, 2, 4, 6, 9, 12, 24 h after i.m administration (Table 3). The mean marbofloxacin concentrations for the indicated time points were 1.304, 0.908, 0.709, 0.590, 0.506, 0.338 and 0.044 mg/ml. Ex-vivo antibacterial 10 Marbofloxacinconcentraon (µg/mL)
266
1
0.1
0.01 0
5
10
15
20
Time (hour)
Figure 1. Serum concentration versus time profiles of marbofloxacin after i.v. (m) and i.m. (g) administration with 2 mg/kg of bodyweight in beagle dogs (n ¼ 6). Bars represent standard deviations.
Pharmacokinetic of marbofloxacin in dogs
DOI: 10.3109/00498254.2014.969794
Table 1. Pharmacokinetic parameters of marbofloxacin in serum (mean ± SEM, n ¼ 6) after a single dose (2 mg/kg boy weight) i.v. and i.m. administration in beagle dogs.
Table 3. Ex vivo AUC24 h/MIC and AUC24 h/MBC (Mean ± SEM, n ¼ 6) after i.m. administration (2 mg/kg body weight) of marbofloxacin in beagle dogs.
Mean ± SEM
AUC AUC0–1 AUMC A B K10_HL K01_HL t1/2a t1/2b MRT Vss V1_F V2_F K10 K12 K21 CL_F CLD2_F Tmax Cmax MAT F (%)
PK/PD integration parameters
Units
i.v.
i.m.
h g/mL h mg/mL h h mg/mL 1/h 1/h h h h h h L/kg L/kg L/kg 1/h 1/h 1/h L/kg//h L/kg//h h mg/mL h –
8.47 ± 3.51 10.22 ± 3.62 83.57 ± 0.22 1.91 ± 0.54 0.08 ± 0.06 1.80 ± 0.79 – 0.36 ± 0.10 8.08 ± 6.25 9.85 ± 7.88 2.32 ± 1.00 0.60 ± 0.04 1.72 ± 0.60 0.38 ± 0.16 1.18 ± 0.26 0.42 ± 3.51 0.23 ± 0.06 0.80 ± 0.11 – – – –
11.37 ± 3.07 11.03 ± 3.12 – 2.71 ± 1.50 0.09 ± 0.04 1.95 ± 0.17 0.32 ± 0.16 0.25 ± 0.14 7.51 ± 3.70 11.12 ± 6.02 1.80 ± 0.90 0.49 ± 0.08 1.22 ± 0.80 0.35 ± 0.17 1.74 ± 0.78 0.70 ± 0.68 0.17 ± 0.04 0.86 ± 0.75 0.47 ± 0.08 1.76 ± 0.09 1.25 ± 0.51 107 ± 4.03
AUC, area under concentration versus time curve; AUC0–1, area under concentration versus time curve from zero to infinity; AUMC, area under the first moment curve; a, distribution rate constant; b, elimination rate constant; K10_HL, elimination half life; K01_HL, half life of absorption; t1/2(a), distribution half life; t1/2(b), elimination half life; A, distribution phase intercept; B, elimination phase intercept; MRT, mean residence time; Vss, volume of distribution at steady state; V1/F, volume of distribution of central compartment; V2/F, volume of distribution of peripheral compartment; K10, central compartment elimination rate constant; K12, rate constant for passage from central to peripheral compartment; K21, rate constant for passage from peripheral to central compartment; CL_F, total body clearance; CLD2_F, inter compartmental clearance; Tmax, time of maximum concentration; Cmax, maximum concentration; MAT, mean absorption time; F(%), percent absolute bioavailability.
Table 2. In vivo PK/PD integration of marbofloxacin (mean ± SEM, n ¼ 6) after i.m administration (2 mg/kg body weight) of marbofloxacin in beagle dogs. S. pseudintermedius PK/PD parameters
i.v.
i.m.
AUC24 h/MIC (h) AUC24 h/MBC (h) T>MIC T>MBC Cmax/MIC Cmax/MBC
67.76 ± 1.23 16.94 ± 0.86 9.83 ± 1.72 9.89 ± 1.89 – –
91.18 ± 2.61 23.86 ± 0.65 15.50 ± 6.68 10.50 ± 2.50 13.04 ± 1.03 3.40 ± 1.03
AUC0–24 h/MIC, ratio of AUC to MIC; AUC0–24 h/MBC, ratio of AUC to MIC; T > MIC, time that concentration of marbofloxacin exceeds MIC; T>MBC, time that concentration of marbofloxacin exceeds MBC; Cmax/MIC, ratio of peak serum concentration to MIC; Cmax/MBC, ratio of peak serum concentration to MBC.
activity of marbofloxacin against S. pseudintermedius in serum after i.m. administration is indicated in Figure 2. Log cfu/ml reduction to the level of lower detection limit (100 cfu/ml) was observed in samples collected at 1 and 2 h after i.m. administration. Marbofloxacin concentration
Sampling time (h)
AUC24 h/MIC
AUC24 h/MBC
1 2 4 6 9 12 24
250.39 ± 24.81 174.35 ± 7.35 136.21 ± 8.18 113.43 ± 7.37 97.26 ± 11.87 65.02 ± 11.90 11.88 ± 3.69
62.59 ± 6.20 43.58 ± 1.83 34.05 ± 2.04 28.35 ± 1.84 24.31 ± 2.96 16.25 ± 3.96 2.97 ± 1.97
AUC0–24 h/MIC and AUC0–24 h/MBC, ratios of serum AUC0–24 h in samples collected between 1 and 24 h after i.m. administration of marbofloxacin in beagle dogs to MIC and MBC in serum respectively.
10 0 Log10 (CFU/mL)
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PK parameters
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8
1 2
6
4 4
6 9
2
12 24
0 0
6
12
18
24
Time (h)
Figure 2. Ex-vivo antibacterial activity of marbofloxaacin against S. pseudointermedius in serum after i.m. administration at a dose rate of 2 mg/kg. Values are means (n ¼ 6). Samples were harvested at predetermined times between 0 and 24 h. Table 4. PK/PD integration of ex-vivo serum marbofloxacin (Mean ± SEM, n ¼ 6) after i.m. administration (2 mg/kg body weight) of marbofloxacin in beagle dogs. Parameters Log Emax (cfu/ml) Log E0 (cfu/ml) Log Emax – LogE0 (cfu/ml) AUC24 h/MIC for bacteriostatic action AUC24 h/MIC50 AUC24 h/MIC for bactericidal action AUC24 h/MIC for bacterial elimination Slope (N)
Mean ± SEM 2.77 ± 0.10 4.04 ± 0.01 6.80 ± 0.01 65.03 ± 11.53 70.74 ± 3.69 97.02 ± 9.24 136.21 ± 7.58 5.44 ± 0.78
Emax difference in log of number of bacteria (cfu/ml) in sample incubated with marbofloxacin between time 0 and 24 h, when the detection limit (100 cfu/ml) is reached. E0, difference in log of number of bacteria (cfu/ml) in control sample (absence of marbofloxacin) between time 0 and 24 h; AUC0–24 h/MIC50, AUC24 h/MIC of marbofloxacin producing 50% of the maximum antibacterial effect; N, the hill coefficient.
in serum collected from 4 to 9 h after i.m. administration showed bactericidal activity, whereas marbofloxacin in serum samples collected at 12 h after i.m. dose had bacteriostatic effect. No inhibitory effect of marbofloxacin against S. pseudintermedius was observed in 24 h serum samples. As shown in Table 4 and Figure 3, the values of AUC/MIC ratio needed for bacteriostatic, reduction in bacterial count by
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Figure 3. Sigmoid Emax relationship for bacterial count (log cfu/ml) versus ex vivo AUC24 h/MIC for S.pseudointermedius in serum of beagle dogs. The curve represents the line of predicted values and the circles are values of beagle dogs.
50%, bactericidal and elimination of bacteria in serum were 65.03, 70.74, 97.02 and 136.21, respectively. Based on the MIC (0.125 mg/ml) of marbofloxacin against S. pseudintermedius, the lowest calculated doses providing bactericidal and elimination of S. pseudintermedius were 2.1 and 2.9 mg/ kg/day, respectively.
Discussion Marbofloxacin has shown a complete absorption and bioavailability after i.m. administration in beagle dogs. Since bactericidal activity of fluoroquinolones is concentrationdependent (Aliabadi et al., 2003), the high bioavailability (>100%) and fast absorption (K01_HL, 0.32 ± 0.16) of marbofloxacin favors the in vivo bactericidal activity (Table 1). Although variation in bioavailability of a drug is expected among species, bioavailability of marbofloxacin observed in beagle dogs was comparable with values observed in several species, including sheep, goats and pigs (Ding et al., 2010; Marin et al., 2013; Sidhu et al., 2010; Waxman et al., 2001). The Cmax of marbofloxacin achieved after i.m. administration (Table 1) was higher than the MIC break point of fluoroquinolones recommended against most susceptible bacterial species (CLSI, 2007). This value was comparable with values reported in pigs and goats (Ding et al., 2010; Waxman et al., 2001), while it is slightly lower than that in rabbits (Cmax ¼ 2.04, Sidhu et al., 2010) and higher than that in sheep (Cmax ¼ 0.8, Marı´n et al., 2013). The longer elimination half-life of marbofloxacin observed in this study reflects the advantage of this drug in maintaining effective concentration in the body thereby allowing longer time for drug-pathogen interaction. The elimination half-life of marbofloxacin in beagle dogs after i.v. administration was comparable with values in pigs, rabbits, and goats (Ding et al 2010; Marı´n et al., 2013; Waxman et al., 2001) and longer than the value in sheep (3.96 h, Sidhu et al., 2010). For i.m. administration, the elimination half-life of marbofloxacin in the present study was in agreement with those values reported in goats, camels and rabbits (Larajeet al., 2006; Marı´n et al.,
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2011; Waxman et al., 2001). However, it was shorter than values reported in pigs (Ding et al., 2010) and longer than values reported in horses and calves (Carretero et al., 2002; Ismail & El-Kattan, 2007). The high values of steady state volume of distribution (Vss) of marbofloxacin in beagle dogs (2.32 and 1.8 L/kg after i.v. and i.m. administration) suggest good tissue penetration of the drug in dogs. The higher K12/K21 ratio of marbofloxacin also indicates faster transport of marbofloxacin from central to peripheral compartment than redistribution from peripheral to central compartment. The higher peripheral distribution of marbofloxacin could enhance concentration dependent bactericidal activity of marbofloxacin at infection site (Walker, 2000). Plasma protein binding of drugs can affect the tissue distribution and hence activity of antimicrobial agents (Nix et al., 2004). However, marbofloxacin had similar MIC values against S. pseudintermedius both in serum and broth, suggesting low plasma protein binding of this drug. Similarly other fluoroquinolones, such as orbifloxacin were found to have low protein binding and similar MIC values in serum and growth media against several bacterial species (Elias et. al., 2009). The PK/PD modeling presents a better approach to dose titration studies for selecting rational dosage regimens in veterinary medicine (Toutain & Lees, 2004). In the current study, the PK data obtained from a single i.v. and i.m. administration of marbofloxacin in dogs was integrated with PD (in vitro and ex vivo) data using S. pseudintermedius as a model pathogen. Using the inhibitory sigmoid Emax model, the lowest ex-vivo AUC0–24 h/MIC ratios required for different levels of marbofloxacin activity against S. pseudintermedius were determined in serum of beagle dogs (Table 4). The in vivo AUC0–24 h/MIC ratio (91.18 h, Table 2) was lower than ex vivo AUC0–24 h/MIC ratio (97.02, Table 4) required for 3 log reduction of bacterial count (bactericidal action) against S. pseudintermedius, suggesting that the administered dose (2 mg/kg body weight per day) of marbofloxacin might not guarantee clinical efficacy against infections associated with S. pseudointermedius. Although there is no universal agreement on the time that drug concentration should remain above MIC (T > MIC), the percentage of time (%T ¼ 63.21) that marbofloxacin serum concentration exceeds MIC over the dosing interval (Table 2) might favor time-dependent activity of marbofloxacin against gram-positive bacteria (Aliabadi & Lees, 2002). For fluoroquinolones the ratios of AUC0–24 h/MIC and Cmax/MIC are predictive of bacterial eradication (Andes et al., 2002; Craig, 1998; Forrest et al., 1993). The ex vivo AUC0–24 h/MIC ratios of marbofloxacin required for bactericidal action and eradication of the clinical strain with MIC of 0.125 mg/ml were 97.02 ± 9.24 h and 136.21 ± 7.58 h, which are higher than the in vivo AUC24/MIC ratio (91.18 ± 2.61) achieved after a single i.m. administration of marbofloxacin (2 mg/kg). In clinical area, S. pseudintermedius strains have higher MIC values. For example, in our previous study, the MIC90 of S. pseudintermedius strains was found to be 0.5 mg/ml (Awji et al., 2012). These suggest that the conventional dose of marbofloxacin (2 mg/kg) may not suffice to kill and eradicate S. pseudintermedius strains encountered in clinical area.
DOI: 10.3109/00498254.2014.969794
Conclusions The results of the present study suggest that marbofloxacin administered i.m. in dogs has good pharmacokinetic profile in terms of high bioavailability, large volume of tissue distribution and prolonged elimination half-life. The PK/PD integration and modeling indicate that the conventional 2 mg/kg daily dose marbofloxacin may not achieve AUC0–24 h/MIC ratio required for bactericidal activity and bacterial elimination, and higher doses of marbofloxacin should be considered to combat clinical infections such as canine pyoderma caused by S. pseudintermedius. However, further study in both healthy and naturally diseased animals and disease model are recommended to establish these findings in a clinical setting.
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Acknowledgements This research was supported in part by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 2011-0021670) and in part by a grant from the Next-Generation BioGreen 21 Program (No. PJ009007), Rural Development Administration, Republic of South Korea.
Declaration of interest Authors declare no competing interest.
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