J. vet. Pharmacol. Therap. 35, 580–587. doi: 10.1111/j.1365-2885.2012.01377.x.
Pharmacokinetics of ketamine and propofol combination administered as ketofol via continuous infusion in cats A. ZONCA* G. RAVASIO
M. GALLO* C. MONTESISSA S. CARLI* R. VILLA* & P. CAGNARDI* *Department of Veterinary Science and Technologies for Food Safety, Universita` degli Studi di Milano, Milan, Italy; Department of Veterinary Clinical Sciences, Universita` degli Studi di Milano, Milan, Italy; Department of Public Health, Comparative Pathology and Veterinary Hygiene, Agripolis, Universita` degli Studi di Padova, Viale dell’Universita`, Padua, Italy
Zonca, A., Ravasio, G., Gallo, M., Montesissa, C., Carli, S., Villa, R., Cagnardi, P. Pharmacokinetics of ketamine and propofol combination administered as ketofol via continuous infusion in cats. J. vet. Pharmacol. Therap. 35, 580–587. The pharmacokinetics of the extemporaneous combination of low doses of ketamine and propofol, known as ‘ketofol’, frequently used for emergency procedures in humans to achieve safe sedation and analgesia was studied in cats. The study was performed to assess propofol, ketamine and norketamine kinetics in six female cats that received ketamine and propofol (1:1 ratio) as a loading dose (2 mg ⁄ kg each, IV) followed by a continuous infusion (10 mg ⁄ kg ⁄ h each, IV, 25 min of length). Blood samples were collected during the infusion period and up to 24 h afterwards. Drug quantification was achieved by HPLC analysis using UV-visible detection for ketamine and fluorimetric detection for propofol. The pharmacokinetic parameters were deduced by a two-compartment bolus plus infusion model for propofol and ketamine and a monocompartmental model for norketamine. Additional data were derived by a noncompartmental analysis. Propofol and ketamine were quantifiable in most animals until 24 and 8 h after the end of infusion, respectively. Propofol showed a long elimination half-life (t1 ⁄ 2k2 7.55 ± 9.86 h), whereas ketamine was characterized by shorter half-life (t1 ⁄ 2k2 4 ± 3.4 h) owing to its rapid biotransformation into norketamine. The clinical significance of propofol’s long elimination half-life and low clearance is negligible when the drug is administered as short-term and lowdosage infusion. The concurrent administration of ketamine and propofol in cats did not produce adverse effects although it was not possible to exclude interference in the metabolism. (Paper received 13 July 2011; accepted for publication 21 December 2011) Dr Annalisa Zonca, Department of Veterinary Science and Technologies for Food Safety, Universita` degli Studi di Milano, Via Celoria 10, 20133 Milan, Italy. E-mail:
[email protected]
INTRODUCTION Ketamine, a phencyclidine derivative, is a rapid-acting general intravenous anaesthetic widely used in veterinary medicine that has been recently re-evaluated as an analgesic drug in humans (Aroni et al., 2009). Propofol (2,6 diisopropyl-phenol) is a shortacting injectable hypnotic agent for the induction and maintenance of general anaesthesia during minor surgical procedures (Duke, 1995). The extemporaneous combination of a low dose of ketamine and propofol in the same syringe, generally known as ‘ketofol’, is frequently used for emergency procedures in humans to achieve safe sedation and analgesia (Loh & Dalen, 2007; Arora, 2008). The combination is reported to maintain effective sedation and a more stable hemodynamic and respiratory profile, 580
owing to the opposing dose-dependent cardiovascular effects (Arora, 2008), although the real benefits are debated (Green et al., 2011). The perioperative administration of a low dose of ketamine may improve the postoperative analgesia and comfort in animals as well as in humans (Roytblat et al., 1993; Wagner et al., 2002). The clinical efficacy of both propofol and ketamine has been widely studied and demonstrated in many species under different conditions (Lerche et al., 2000; Wagner et al., 2002; Ilkiw et al., 2003; Pascoe et al., 2006). Pharmacokinetic studies following infusion administration have been carried out with propofol in dogs (Nolan & Reid, 1993) and with ketamine and propofol in sheep and ponies (Correia et al., 1996; Nolan et al., 1996). The pharmacokinetic behaviours of ketamine (Hanna et al., 1988) 2012 Blackwell Publishing Ltd
The effect of ketofol on cats 581
and propofol (Adam et al., 1980; Cleale et al., 2009) in cats have been investigated as single IV bolus administration, but no pharmacokinetic data are available regarding infusion of the combination of the two drugs. The aim of the work was to describe the pharmacokinetic aspects of the continuous infusion of ketamine and propofol administered simultaneously as ketofol in cats undergoing surgery. Whole-blood samples were used to quantify propofol, whereas ketamine and norketamine were extracted both from plasma and from red blood cell (RBC) samples, to investigate differences in parent compound and metabolite concentrations in these tissues.
MATERIALS AND METHODS Experimental design The pharmacokinetic study was carried out on six client-owned female cats randomly selected among the 15 enrolled in an extensive trial (Ravasio et al., 2011) for the clinical evaluation of the extemporaneous combination of ketamine and propofol. The study was approved by the Ethical Committee of the University of Milan, and all the owners were informed of the whole procedure and were asked to sign a written consent before enrolment of their cats. The six female cats, ageing 1–2 years and weighing between 2.7 and 4 kg b.w., were admitted to the Department of Veterinary Clinical Sciences for ovariectomy according to standard surgical procedures. All animals were judged healthy (ASA status I) on the basis of physical examination and of routine blood tests results. The animals received an IV loading dose of a mixture of 2 mg ⁄ kg b.w. of ketamine (Ketavet 100; Intervet Production, Segrate, Milan, Italy) plus 2 mg ⁄ kg b.w. of propofol (Propovet; Esteve Veterinaria, Milan, Italy), followed by the IV infusion of the same mixture at the dose of 10 mg ⁄ kg ⁄ h each for 25 min. During surgery and until the end of ketofol administration, Ringer’s lactated solution was infused (5 mL ⁄ kg ⁄ h, IV) through a cephalic catheter and physiologic parameters were recorded. Blood samples for drug quantification were collected through a jugular catheter before treatment (t0) at the end of the loading dose and at pre-established times during the infusion (5, 15 and 25 min). Following the completion of drug infusion, samples were collected at 10, 30 min, 1, 2, 4, 6, 8, 12 and 24 h, so that the total sampling time was about 24.4 h. Whole-blood samples (about 2 mL) were collected in heparinized tubes and immediately divided into two parts: one was immediately refrigerated at +4 C until propofol assay, while the other was centrifuged (1200 g, 10 min) to separate plasma and RBCs and maintained at )20 C until ketamine and norketamine assays. HPLC analysis Propofol-spiked solutions for the calibration curve were prepared by diluting the original stock solution (1 mg ⁄ mL) of propofol (2,6-Diisopropylphenol 97%; Sigma Aldrich, Milan, Italy) in cat 2012 Blackwell Publishing Ltd
blank whole blood, to reach concentrations ranging from 0.01 to 5 lg ⁄ mL. The HPLC analysis was performed according to Plummer (1987) with slight modifications. Briefly, 0.5 mL of fortified blood or sample was added with 0.5 mL of 0.1 M sodium dihydrogen orthophosphate solution and 3 mL of cyclohexane. The mixture was centrifuged (1100 g, 5 min), and the supernatant was transferred into tubes containing 50 lL of tetramethylammonium hydroxide and evaporated to dryness by a centrifugal evaporator. The residue was dissolved with 200 lL of mobile phase, and 50 lL of the upper layer was injected into HPLC for drug analysis. The mobile phase consisted of a mixture of 0.1% trifluoroacetic acid solution and acetonitrile (40:60) at a flow rate of 1.5 mL ⁄ min. The HPLC (Series 200; Perkin Elmer, Milan, Italy) was equipped with fluorescence detection (LC 240; Perkin Elmer) set at excitation and emission wavelengths of 276 and 310 nm, respectively. Drug separation was achieved by a C18 column (LiChrospher 100 RP-18, 125 mm · 4; Merck, Darmstadt, Germany) maintained at 20 C by a peltier column oven. Ketamine and norketamine assays were carried out according to methods described elsewhere (Gross et al., 1999; Roncada et al., 2007). Calibration curves were prepared as previously described by spiking feline blank plasma and RBC with stock solutions of ketamine and norketamine HCl (1 mg ⁄ mL in methanol, LGC Standards S.r.l., Sesto San Giovanni, Milan, Italy) in the range 0.01–10 lg ⁄ mL. All plasma or RBC samples (0.5 mL) had 1 mL of 1 M NaOH solution added and then extracted into 4 mL of diethyl ether. After centrifugation (2500 g, 10 min), the analytes were back-extracted into sulphuric acid (250 lL of 0.025 M solution) and evaporated to form a residue. The residue was reconstituted with 100 lL of mobile phase, composed of 0.01 M sodium dihydrogen phosphate solution (at pH 2.36 with H3PO4) and acetonitrile (A:B = 82:18). The separation was achieved by HPLC analysis (Series 200, Perkin Elmer) equipped by Ultracarb 5 ODS, 150 · 4.6 column (Phenomenex, Castel Maggiore, Bologna, Italy), and the analytes were revealed by the UV-visible detector set at 215 nm of wavelength. The mobile phase flow rate was 1 mL ⁄ min, and the volume of injection was 70 lL. The calibration graphs were prepared by plotting the peak area of each analyte against the concentrations of drug added. All analytical methods were subject to intralaboratory validation according to the European legislation (EudraLex, 2005). All salts and solvents were of HPLC quality grade (Panreac, Cinisello Balsamo, Milan, Italy). Pharmacokinetic analysis The pharmacokinetic analyses were deduced from blood concentration–time data using a dedicated software (Phoenix Win NonLin 6.1; Pharsight Corporation, St. Louis, MO, USA) that allows compartmental and noncompartmental analysis of the experimental data. Minimum information criterion estimates (MAICE, Yamaoka et al., 1978) were used to choose the model best fitting the data. All data points were weighted by the inverse square of the fitted value. Blood and plasma concen-
582 A. Zonca et al.
trations of propofol and ketamine were fitted by a twocompartment model designed for IV bolus plus constant infusion, whereas a monocompartmental analysis was performed for norketamine plasma and RBC concentrations. All data (propofol, ketamine and norketamine in RBC and plasma) were also fitted by a noncompartmental analysis for infusion administration. The distribution half-life (t1 ⁄ 2k1) and terminal half-life (t1 ⁄ 2k2) were calculated as ln2 ⁄ kn. The volume of distribution in the central compartment (V1) was calculated as follows: V1 ¼ Dose=C0 where Dose is dose of drugs and C0 is the extrapolated serum concentration at time 0, whereas the volume of distribution in the peripheral compartment (V2) is V2 ¼ Vdss V1 where Vdss is calculated as V1*[(k12 + k21) ⁄ k21]. Body clearance (ClB) was calculated as ClB ¼ ln2 V1 =K10
HL
where K10_HL is the half-life of the constant of elimination from the central compartment (K10). The mean residence time (MRT) calculated by the noncompartmental model was corrected for the infusion length (TI), that is, MRT = [(AUMC/AUC) - (TI/2)], where the area under the serum concentration–time curve (AUC) and the area under the first moment curve (AUMC) are calculated by the trapezoidal method (Gibaldi & Perrier, 1982). The context-sensitive half-time (CST) was calculated by extrapolation from the individual curves of propofol and ketamine concentrations for each cat. Statistical analysis Pharmacokinetic parameters are reported as means (±SD). Harmonic means with pseudo-standard deviations were calculated for half-lives using a jack-knife technique (Lam et al., 1985). Differences in ketamine and norketamine concentrations between plasma and RBC and those of the related pharmacokinetic parameters were compared by Wilcoxon matched-pairs t-test (GraphPad InStat version 3.0.1, GraphPad Software, Inc., San Diego, CA, USA). Differences with P < 0.05 were considered significant.
RESULTS Results of the analytical methods’ validation are reported in Table 1. There was a linear relationship (r2 value >0.98) between drugs’ concentrations and peaks’ area over the range investigated (0.01–5 lg ⁄ mL for propofol and to 10 lg ⁄ mL for ketamine and norketamine). The limit of quantification (LOQ) was set at 0.01 lg ⁄ mL for propofol in whole blood and for ketamine and norketamine in plasma and RBC. The intraday repeatability was measured as coefficient of variation (CV%) on six replicates of three concentrations, whereas the accuracy (%) was measured as closeness to the concentration added on the same replicates. The propofol samples in whole blood were found to be stable at +4 C for maximum 1 week, whereas ketamine and norketamine samples were stable in frozen samples for 6 months. No adverse effects were observed during the treatments. The total dose administered was 6.16 mg ⁄ kg for both drugs, calculated as sum of the loading dose (2 mg ⁄ kg) plus the infusion dose adjusted for the length of the infusion (10 mg ⁄ kg ⁄ h for 25 min, i.e. 4.16 mg ⁄ kg). To describe the correlation found between plasma and RBC concentrations, the profiles of ketamine and its metabolite are reported as mean values in Fig. 1. No significant differences were found between plasma and RBC drugs’ concentrations and pharmacokinetics (P > 0.05). Because of the overlapping concentrations of ketamine and norketamine in plasma and RBC samples, we are reporting here results only from plasma analysis. Figure 2 shows mean propofol whole-blood concentrations together with mean ketamine and norketamine plasma concentrations. Immediately after the loading dose, the mean propofol concentration was 2.33 ± 1.52 lg ⁄ mL; then, after a slight decrease (1.98 ± 0.22 lg ⁄ mL at 5 min), the blood concentrations gradually increased, so that at the end of the infusion period, the mean drug concentration was 3.93 ± 3.08 lg ⁄ mL (Fig. 2a). Twenty-four hours after the end of the infusion, propofol was still quantifiable in five of six cats with a mean value of 0.07 ± 0.06 lg ⁄ mL (Fig. 2b). Mean plasma ketamine concentration during the infusion period followed a behaviour similar to propofol. After the loading dose, mean drug concentration was 4.35 ± 1.66 lg ⁄ mL; then, after a slight decrease (3.71 ± 1.51 lg ⁄ mL after 5 min of infusion), the mean ketamine concentration was maintained at about 4.2 lg ⁄ mL for the whole infusion time (Fig. 2a). The drug
Table 1. Intralaboratory validation of analytical methods for propofol, ketamine and norketamine quantification in whole-blood, plasma and RBC samples Parameter (units) LOQ (lg ⁄ mL) Recovery (%) Intraday repeatability (CV%) Accuracy (%)
Propofol whole blood 0.01 73 ± 10 0.2–0.16 0.19–4.45
Ketamine plasma
Norketamine plasma
0.01 82 ± 6 6.1–24.6 )0.2–3
0.01 75 ± 5 7.6–16 )0.29–0.48
Ketamine RBC 0.01 82 ± 4 3.8–26.3 )0.35–6
Norketamine RBC 0.01 78 ± 8 13–21 )4.48–0.08
Recovery is reported as mean ± SD; intraday repeatability and accuracy are reported as range values. CV, coefficient of variation; RBC, red blood cells; LOQ, limit of quantification. 2012 Blackwell Publishing Ltd
(mg/mL)
The effect of ketofol on cats 583
Fig. 1. Semi-logarithmic plot of mean ketamine and norketamine concentrations (squares and triangles, respectively) in plasma (dotted line) and in RBC (solid line) samples until 24 h after the drugs administration (loading dose plus infusion of 25 min). Standard deviations are omitted for clarity.
Time (h)
(mg/mL)
10.00
(a)
1.00
Propofol Ketamine Norketamine
0.10
0.01 0.00
10.00
0.06
0.12
0.18
0.24
0.30
0.36
0.42
(b)
Propofol Ketamine Norketamine
(mg/mL)
Fig. 2. Semi logarithmic plot of mean (±SD) propofol (solid line, circle), ketamine (dotted line, square) and norketamine (dotted line, triangle) concentrations after ketofol administration as IV loading dose of 2 mg ⁄ kg b.w. of ketamine and propofol followed by the IV infusion at the dose of 10 mg ⁄ kg ⁄ h of ketamine and propofol. (a) shows mean concentrations (±SD) from the loading dose (t = 0.02 h) until the end of the infusion (25 min, 0.42 h). (b) shows mean concentrations (±SD) from the end of drugs administration up to 24 h after the administration (24.42 h).
1.00
0.10
0.01 0.00
was quantifiable for 6 h after the end of the infusion in all animals (mean value of 0.16 ± 0.13 lg ⁄ mL), whereas after 24 h, it was still detectable in only one subject (0.09 lg ⁄ mL) (Fig. 2b). Norketamine was quantified in all animals immediately 2012 Blackwell Publishing Ltd
5.00
10.00
15.00
20.00
25.00
after the loading dose with a mean concentration of 0.11 ± 0.06 lg ⁄ mL (Fig. 2a). Peak plasma concentration (1.76 ± 0.66 lg ⁄ mL) was achieved at about 10 min after the end of the infusion. Then, the metabolite concentrations slowly
584 A. Zonca et al.
decreased, and it was still quantifiable in two of six animals (0.27 ± 0.33 lg ⁄ mL) at 24 h (Fig. 2b). The pharmacokinetic parameters were derived from a twocompartment IV bolus plus constant infusion model for propofol and ketamine (whole-blood and plasma data, respectively), whereas data for plasma norketamine were derived from the monocompartmental analysis. Additional parameters (AUC, Cmax, Tmax, MRT) from noncompartmental analysis were also reported to complete data. All results are reported in Table 2.
Table 2. Pharmacokinetic parameters (mean ± SD) of propofol (whole blood), ketamine and norketamine (plasma) after IV loading dose of ketamine plus propofol followed by IV infusion of the mixture (ketofol) for 25 min Parameter (units)
Propofol
Ketamine
Loading dose IV 2 (mg ⁄ kg) Infusion dose IV 10 (mg ⁄ kg ⁄ h) Two-compartment analysis t1 ⁄ 2k1 (h) 0.67 ± 0.33* t1 ⁄ 2k2 (h) 7.55 ± 9.86* k10 (1 ⁄ h) 0.51 ± 0.36 k12 (1 ⁄ h) 0.43 ± 0.23 k21 (1 ⁄ h) 0.20 ± 0.15 t1 ⁄ 2k10 (h) 1.38 ± 1.07* V1 (mL ⁄ kg) 1916.25 ± 1187.56 V2 (mL ⁄ kg) 5821.37 ± 6644.76 ClB (mL ⁄ h ⁄ kg) 692.36 ± 326.18 Noncompartmental analysis AUC(0–last) (hÆlg ⁄ mL) 9.35 ± 4.16 Cmax (lg ⁄ mL) 4.11 ± 2.95 Tmax (h) 0.53 ± 0.08 MRT(0–last) (h) 9.71 ± 7.91
2 10
0.95 ± 0.12* 4.04 ± 3.38* 1.14 ± 0.43 1.89 ± 2.63 0.68 ± 0.90 0.62 ± 0.23* 821.85 ± 439.58 2363.54 ± 1947.84 824.77 ± 306.98 7.62 5.73 0.14 3.59
± ± ± ±
2.07 1.83 0.17 3.3
Norketamine Monocompartmental analysis AUC(0–¥) (hÆlg ⁄ mL) Cmax (lg ⁄ mL) Tmax (h) t1 ⁄ 2k10 (h) Noncompartmental analysis AUC(0–last) (hÆlg ⁄ mL) Cmax (lg ⁄ mL) Tmax (h) MRT(0–last) (h)
15.06 1.54 1.21 2.91
± ± ± ±
8.78 0.43 0.48 3.9*
12.42 1.96 1.92 5.11
± ± ± ±
5.28 0.49 2.32 3.12
*Harmonic mean ± pseudo-SD; t½k1: distribution half-time; t½k2: elimination half-time; k10: the rate at which the drug leaves the system from the central compartment (the elimination rate); k12: the rate at which the drug passes from central to peripheral compartment; k21: the rate at which the drug passes from peripheral to central compartment; t1 ⁄ 2k10: the half-life associated with the rate constant k10; V1, volume of distribution in central compartment; V2, volume of distribution in peripheral compartment; ClB, body clearance; AUC(0–last), area under serum concentration–time curve from 0 to last measurable concentration; AUC(0–¥), area under serum concentration–time curve from 0 extrapolated to infinity; Cmax, maximum concentration; Tmax, time to reach Cmax; MRT, mean residence time.
DISCUSSION The validated analytical methods were adequate to quantify propofol, ketamine and its metabolite (norketamine) in the tissues investigated with good recovery, accuracy and precision (Table 1). The extraction of ketamine and norketamine from plasma was more easily achievable than from RBC samples. The good correlation observed between plasma and RBC samples has confirmed what has previously been reported by other authors of dog and horse studies (Roncada et al., 2005, 2007). Given the easier handling of plasma samples and the similarity of results obtained from both plasma and RBC, we believe that the quantification of ketamine and norketamine in plasma samples may therefore be preferable. The doses of propofol and ketamine administered in this study were equal to those adopted by Ilkiw et al. (2003) using total intravenous anaesthesia (TIVA) in cats. In our animals, ketamine and propofol concentrations during infusion reached a plateau, even if a slight increase in propofol amounts was recorded at the end of the infusion, as shown in Fig. 2a. Because of the adequateness of the anaesthesia (Ravasio et al., 2011), this plateau of concentrations can be considered effective for TIVA in cats. The extubation time in all cats undergoing ketofol administration (n = 8) was about 7 min, and recovery was completed within 30 min (Ravasio et al., 2011); thus, the shortterm (
