Reasons for performing study: To compare the pharmacokinetics of the fourth generation cephalosporin, cefquinome, in neonatal foals, 6-week-old foals and mature New Forest ponies in order to recommend appropriate dosage regimens for use of this drug.
Methods: Cefquinome was administered i.v. at 1 mg/kg bwt twice a day (q. 12 h), 1 mg/kg bwt 3 times a day (q. 8 h) or 4.5 mg/kg bwt q. 12 h to each age group (n = 6). Plasma cefquinome concentrations were analysed using high-performance liquid chromatography combined with electrospray tandem mass spectrometry.
Results: Both foal age groups had comparable pharmacokinetic data except for the volume of distribution at a steady-state (Vss), total body clearance (ClB) and mean residence time (MRT). Both ClB and MRT decreased as the age of the foals increased. Values of area under the curve increased, in a dose dependent manner, with significant increases for all age groups following administration of 4.5 mg/kg bwt q. 12 h. Total body clearance did not have comparable dose dependency.
Conclusions: Cefquinome can be given at a dose of 1 mg/kg bwt q. 12 h for the treatment of infections caused by susceptible pathogens with MIC<0.125 µg/ml. A higher dose of 4.5 mg/kg bwt q. 12 h is recommended for the treatment of bacterial pathogens with minimal inhibitory concentration (MIC) 0.125–0.5 µg/ml
Potential relevance: Commonly used dosing regimens should be critically evaluated in neonatal foals due to the higher volume of distribution of less lipophilic drugs in this age group.
Escherichia coli sepsis has been considered to be a major cause of equine neonatal death. However, although enteric Gram-negative bacteria remain the most common bacterial isolates found in bacteraemic foals, there has been an increase in the proportion of Gram-positive isolates in recent years, including Staphylococcus aureus, Streptococcus spp. and others (Koterba et al. 1984; Wilson and Madigan 1989; Marsh and Palmer 2001; Corley et al. 2007; Russell et al. 2008; Sanchez et al. 2008). Consequently, in critical clinical cases broad-spectrum antimicrobial drugs are used, at least until a specific microbiological diagnosis is established.
Cefquinome (Cobactan)a is a fourth generation cephalosporin that contains an aminothiazolyl group and an iminomethoxy group on the β-acyl side chain with a bicyclic pyridinium group attached at C-3, making it similar to cefpirome. This antimicrobial structure leads to an extended spectrum of antimicrobial activity with improved inhibition of Gram-positive bacteria and improved β-lactamase stability compared with older β-lactam antimicrobials (Neu 1986; Bryskier 1997). Cefquinome has excellent activity in vitro against most Enterobacteriaceae and haemolytic streptococci and good activity against methicillin-susceptible S. aureus strains. Cefquinome has moderate to low activity against methicillin-resistant S. aureus and Pseudomonas aeruginosa (Thomas et al. 2006) and very limited activity against Gram-negative anaerobes (Limbert et al. 1991; Chin et al. 1992; Bottner et al. 1995).
Cefquinome has been developed for veterinary use and is registered for use in cattle, pigs and horses in most European countries. Cefquinome's broad spectrum of activity covers many equine pathogens including: Streptococcus equi sp. zooepidemicus (S. zooepidemicus), Staphylococcus aureus/S. intermedius, Actinobacillus equuli, E. coli and other enterobacteriaceae (Thomas et al. 2006). Cefquinome is approved in horses to treat respiratory infections caused by S. zooepidemicus at a dose of 1 mg/kg bwt q. 24 h i.m. or i.v. and to treat foals with E. coli sepsis at a dose of 1 mg/kg bwt q. 12 h i.m. or i.v.
The pharmacokinetics of cefquinome in mice, dogs, pigs and calves have been reported (Limbert et al. 1991). For horses, the only data currently available is drawn from technical information presented during the process of drug licensing with the European Medicines Agency. After i.v. administration of cefquinome, at a dosage of 1 mg/kg bwt to mature horses, the maximal plasma concentration was 11.08 mg/l, the plasma clearance was estimated to be 1.73 ml/min/kg bwt, and the elimination half-life was 2.17 h (EMEA 2003). Pharmacokinetic data for very young animals and neonatal foals are not available.
In neonatal animals, the disposition of drugs may differ from that of mature individuals of the same species (Caprile and Short 1987; Gardner et al. 1993; Baggot 1994; Gardner and Papich 2001) due mainly to differences in body composition and the immaturity of the hepatic and renal excretion mechanisms. Consequently, specific dosage adjustments are needed for foals. The objective of the current study was to compare the pharmacokinetics between 2 age groups of foals and their mares in order to develop dosage regimens for neonatal and 6-week-old animals.
Materials and methods
Nine 1–3-day-old (neonatal) New Forest Pony foals and their dams were used in this study. The foals were tested a second time when they reached the age of 6 weeks. The dams varied in age between 3–19 years. Each foal had been born at term and housed in a box stall with its dam during the course of the experiment. Mares were fed commercial corn pulp pellets twice a day and had ad libitum access to grass, hay, and water. Animals were considered to be healthy based on unremarkable clinical examination and an APGAR score >9 (Corley and Furr 2003). Packed cell volume, white blood cell count and total protein were within normal limits for all age groups. Throughout the study, all animals were monitored for any signs of an adverse reaction that might be related to the treatment throughout the study. The Committee of Animal Welfare at the Faculty of Veterinary Medicine, Utrecht University, the Netherlands approved this study protocol.
Drug administration and sampling
Cefquinome sulphate (Cobactan 4.5%)a was diluted with benzylalcohol to a concentration of 45 mg/ml and administered to each animal through a jugular vein catheter (18 gauge/1/1¾)b. Three dosing regimens were tested in groups of 6 animals using a Latin Square Model with a 24 h washout period between treatments. The following dosing regimens were assessed: 1 mg/kg bwt q. 12 h; 1 mg/kg bwt q. 8 h; and 4.5 mg/kg bwt q. 12 h. Each animal was used to test 2 of the 3 different dosing regimens. Dosing continued for 24 h in which a steady-state was reached according to the calculation of the accumulation factor (R). Blood samples were collected in heparinised tubes (Vacuette LH lithium Heparine)c from the opposite jugular vein through a catheter prior to drug administration and at 0.25, 0.5, 1, 2, 4, 8 and 12 h following drug administration. Blood samples were centrifuged for 3 min at 5000 ×g and plasma was separated and stored at -20°C for further analysis.
Determination of cefquinome plasma concentrations
In order to determine cefquinome concentrations, plasma samples were diluted and extracted with acetonitrile in water (50/50 v/v). Cefpirome served as the internal standard. The organic phase was collected and diluted with acetic acid prior to online solid phase extraction (column-switching), followed by high-performance liquid chromatography combined with electrospray tandem mass spectrometry using the method described by Maes et al. (2007)f. The lower limit of quantification for cefquinome was 0.015 µg/ml. Using horse serum as matrix, standard curves and validation of the assay was conducted and shown to be linear in the range 15–4000 ng/ml.
Pharmacokinetic software (WinNonlin version 5.2.1, 1 Pharsight and Tripos)e was used to determine the best-fit curves for plasma cefquinome concentration vs. time data. The data were evaluated using one- and 2-compartment models. The application of Akaike's information criteria was performed in order to determine the best fit for the plasma cefquinome concentration-time curve. A weighting factor of 1/yhatb was applied and the following pharmacokinetic parameters were calculated: α-distribution rate constant; β-elimination rate constant; area under the plasma concentration-time curves from 0 h to infinity (AUC0-∞); area of the volume of distribution (Vd); volume of distribution at a steady-state (Vss); mean residence time (MRT); and total body clearance (ClB) (Gardner and Papich 2001). These parameters were used to simulate plasma concentrations for repeated administration.
To verify that the recommended and applied doses for cefquinome adhered to pharmacokinetic/pharmacodynamic principles for a bactericidal, time-dependent antimicrobial the following formula was applied:
Where T>MIC is the time interval (in percent) during which the plasma-concentration is above or equal to the minimal inhibitory concentration (MIC)-values, D is the proposed dose, t1/2 the terminal elimination half-life, and τ is the dose interval (Turnidge 1998).
To confirm that a steady-state was reached, the accumulation ratio (R) was calculated using the formula where the accumulation ratio is based on minimum concentrations after first dose (C1(min)) and at steady-state (Css(min)) (Brocks and Mehvar 2010).
Pharmacokinetic parameters for age groups and applied dosage regimens are reported as mean ± s.d. Statistical analysis was performed using software Statistica 6.1 for Windowsf. The data from the 3 age groups were compared with ANOVA and a Bonferroni post hoc test. Values of P<0.01 were considered significant.
The administered doses of cefquinome were well tolerated in all animals and no clinical signs of adverse effects or intolerance were observed. Samples taken at t = 0 (i.e. prior to cefquinome application) were all below the limit of detection, thus confirming that the intertreatment washout period was sufficient for elimination of the drug.
Plasma concentration-time curves were best described by a 2-compartment model except in the neonatal foal group when treated with 4.5 mg/kg bwt q. 12 h and are presented in Figure 1. Mean pharmacokinetic parameters are presented in Table 1. As predicted, the area under the curve (AUC) was significantly increased in groups treated with 4.5 mg/kg bwt q. 12 h, compared to the lower dose.
Table 1. Mean ± s.d. cefquinome pharmacokinetic parameters in 3 different age groups of New Forest Ponies after the i.v. administration
1 mg/kg bwt q. 12 h
1 mg/kg bwt q. 8 h
4.5 mg/kg bwt q. 12 h
α= distribution rate constant; β= elimination rate constant; t1/2β= terminal elimination half-life; AUC0-∞= area under the serum concentration-time curves from 0 h to ∞; Vdarea, Vss= area volume of distribution, steady-state volume of distribution respectively; MRT = mean residence time, ClB= total body clearance; * statistically significant difference between the 3 dosing regimes within the same age group at level P<0.01. a,b,c statistically significant differences, at level P<0.01 between age groups treated with the same dose are shown with different characters.
A steady-state was reached, confirmed by the calculation of R = 1.04 ± 0.03, for all ages groups. In the 6-week-old foal group, a significantly increased clearance (ClB) was observed at a dosing regimen of 1 mg/kg bwt q. 12 h. As expected, the Vss was significantly increased in both neonatal and 6-week-old foals compared with that of the mare group. In the neonatal foal group, the MRT was significantly prolonged compared to the MRT estimated for the 6-week-old foal and mare groups. When dosed at 4.5 mg/kg bwt q. 12 h, clearance between age groups did not differ significantly. The Vss was significantly increased in both neonatal and 6-week-old foals compared with that of the mare group. The estimated MRT was significantly prolonged in the neonatal foal compared with that of the 6-week-old foal and mare groups.
Given the rapid distribution of the drug, and the limited number of blood samples taken during the early phase of distribution, plasma concentration-time curves in the neonatal group treated with 4.5 mg/kg bwt q. 12 h had to be described using a one-compartment model (Fig 1c).
The plasma-concentration time curves and kinetic parameters obtained were compared with MIC values from the most relevant pathogens (e.g. E. coli: 0.125 µg/ml and S. aureus: 0.5 µg/ml) in order to establish optimal dosing regimens. A plasma-concentration time curve simulation for repeated dosing in neonatal foals, comparing the 3 dosing regimens with respect to the MIC-value of S. aureus (0.5 µg/ml) is shown in Figure 2 (Thomas et al. 2006). The overall results from the comparison of the MIC values (pharmacokinetic data) and the calculated kinetic data (PK) are presented in Table 2. In neonates a dose of 1 mg/kg bwt q. 8 h resulted in T>MIC≥66.28% (± 5.49%) and in 6-week-old foals T>MIC≥48.53% (± 13.44 %) against pathogens with a MIC<0.5 µg/ml. In neonates, a dose of 4.5 mg/kg bwt q. 12 h resulted in T>MIC for 85.5% (±4.5%) of the dosing interval (MIC<0.5 µg/ml) and T>MIC≥65.13% (±5.49%) in 6-week-old foals.
Table 2. Calculated T>MIC for 3 dosage regimens of cefquinome based on estimated pharmacokinetic parameters in 3 different age groups of New Forest ponies
1 mg/kg bwt q. 12 h i.v.
1 mg/kg bwt q. 8 h i.v.
4.5 mg/kg bwt q. 12 h i.v.
MIC = minimal inhibitory concentration.
MIC 0.5 µg/ml
44.19 ± 3.66%
66.28 ± 5.49%
85.5 ± 4.5%
32.35 ± 8.96
48.53 ± 13.44%
65.13 ± 11.37%
41.94 ± 9.13%
62.92 ± 13.69%
78.60 ± 11.86%
MIC 0.125 µg/ml
82.27 ± 4.39%
123.40 ± 6.59%
123.58 ± 6.04%
62.56 ± 11.14%
93.85 ± 16.71%
95.34 ± 14.52%
75.73 ± 11.59%
113.60 ± 17.39%
112.39 ± 15.35%
Sepsis and pneumonia are common causes of morbidity and mortality in newborn foals (Cohen 1994). The horse is more susceptible to developing sepsis, compared with other large animals (Roy 2004). Kinetic parameters may differ between foals and mature horses due to the differences in these groups' body compositions and there is a need to review dosing strategies for antimicrobial drugs in foals. In the current study, different therapeutic regimens for cefquinome were compared in neonatal foals, 6-week-old foals and their dams. The disposition of cefquinome in horses is similar to that described in other animal species such as dogs, pigs and cattle. The Vss ranges from 0.2–0.24 l/kg bwt in mature animals (Limbert et al. 1991). In this study, the significantly higher Vss found in foals is presumed to reflect the higher percentage of body water in young animals (Cummings et al. 1990). Similar differences have been reported for the Vss of piglets (0.46 ± 0.10 l/kg bwt) compared with sows (0.12 l/kg bwt) (Block et al. 2005; Li et al. 2008) and a linear decrease in Vd during the first 3 months of life for ceftiofur in calves has been observed (Brown et al. 2006). The effects of age on total body clearance appear to be inconsistent as ClB is driven by both the volume of distribution and the renal capacity. Neonatal foals have a low clearance rate that increases by age 6 weeks. Complete maturation of renal function may take up to 4 weeks, with considerable improvement in functional parameters occurring within the first 5 days after birth (Baggot 1994). Thus, renal maturation may explain the significantly higher ClB in 6-week-old foals compared with that of the neonatal foals. Mares had the lowest volume of distribution and a low clearance rate, which may be partly explained by higher protein binding in mature animals. The total body clearance in horses is within the range of reported values for this animal species(0.11 ± 0.03 l/kg bwt/h) (EMEA 2003). Similar data have been found in dogs (0.19), pigs (0.158) and cattle (0.13 l/kg bwt/h) (Limbert et al. 1991; Allan and Thomas 2003).
In this study, total body clearance, and its secondary parameter elimination half-life, did not show dose-dependency, whereas AUC were significantly increased in the groups treated with 4.5 mg/kg bwt q. 12 h. Similar data, demonstrating a linear dose-dependent increase of AUC and the absence of other dose-related changes in pharmacokinetics of cefquinome, have been reported in dogs (Limbert et al. 1991).
Cefquinome is a β-lactam antimicrobial and acts as a time-dependent bactericidal drug (Thomas et al. 2006). The most important pharmacodynamic/pharmacokinetic parameter for this type of drug is the length of the time above the MIC90 value. It is generally recommended that the plasma concentrations should exceed MIC90 for at least 40% of the time between 2 consecutive applications. In critically ill patients, or patients with compromised immune responses, including neonates, MIC90 values should preferably be exceeded for 80% of the time between 2 consecutive applications (Toutain et al. 2002).
Provided that the patient's immune function is normal, cefquinome at a dose of 1 mg/kg bwt q. 12 h provides plasma concentrations higher than MIC for slightly longer than 80% of the dosing interval. Therefore, this dosage regimen meets pharmacokinetic–pharmacodynamic criteria predicting a successful therapy for susceptible bacteria with MIC≤0.125 µg/ml, although it must be that this prediction is based on PK parameters from healthy animals. This dosage regimen was effective and safe in the treatment in foals under field conditions with bacterial infections and sepsis due to E. coli, Clostridium perfringens and Staphylococcus spp. (Rohdich et al. 2009). With immune compromise, as can be expected in critically ill foals, or the encountered pathogen has a MIC value ≥0.5 µg/ml (e.g. some strains of S. aureus), a higher dose is recommended. The experimental data presented here shows that, in neonates, a dose of 4.5 mg/kg bwt q. 12 h ensured that T>MIC reached 80% for bacteria with MIC values ≥0.5 µg/ml. This higher dose ensures that desirable plasma concentrations required for the treatment of less susceptible pathogens in neonatal foals can be achieved with 2 treatments per day, which is less stressful for the foals and more practical than more frequent injections.
One important limitation of the current study is that the animals were healthy; hence changes in kinetic parameters due to disease conditions could not be estimated. In conclusion, age-dependent differences in the volume of distribution of cefquinome were observed when neonatal foals, 6-week-old foals and their dams were compared. Age also influenced related kinetic parameters such as body clearance and MRT. Comparison of the pharmacokinetic parameters with MIC90 values of various equine pathogens indicates that currently recommended dosing regimen of 1 mg/kg bwt q. 12 h are likely to be effective in the treatment of nonimmunocompromised foals affected by cefquinome-susceptible bacterial pathogens. In contrast, in clinical cases of life-threatening sepsis of neonatal foals, it is advisable to use a higher dose, for example 4.5 mg/kg bwt q. 12 h, unless it has been confirmed that the pathogen, has MIC90 values lower than 0.125 µg/ml. This recommendation is also valid in critically ill cases of other age groups.
Authors' declaration of interests
No conflicts of interest have been declared.
Source of funding
Utrecht University, the Netherlands.
The authors kindly acknowledge the support of the Intervet Innovation GmbH in the analysis of the plasma samples and would like to thank the horse owners and laboratory technicians for their efforts and support.
a Intervet-SP AH, Boxmeer, Alphen a/d Rijn, The Netherlands.
b Braun, Melsungen, Germany.
c Greiner Bio-one B.V., Alphen a/d Rijn, The Netherlands.
d Intervet Innovation GmbH, Schwabenheim, Germany.