Rui Li, Wen-sheng Zhang, Jin Liu, Min Tang, Ying-ying Yang & Nan-Fu Luo

Veterinary Anaesthesia and Analgesia, 2012, 39, 373 384 doi:10.1111/j.1467-2995.2012.00733.x RESEARCH PAPER Minimum infusion rates and recovery times from different durations of continuous infusion of fospropofol, a prodrug of propofol, in rabbits: a comparison with propofol emulsion Rui Li, Wen-sheng Zhang, Jin Liu, Min Tang, Ying-ying Yang & Nan-Fu Luo Department of Anesthesiology and Translational Neuroscience Center, West China Hospital, Sichuan University, Chengdu, Sichuan, China Correspondence: Jin Liu, Department of Anesthesiology, West China Hospital, Sichuan University, No.37, Guo-xue-xiang, Chengdu, Sichuan 610041, China. E-mail: scujinliu@gmail.com Abstract bjective To explore, in rabbits, the minimum infusion rates (MIR) required and recovery time from long duration ( 8 hours) continuous infusion of fospropofol disodium, a novel water-soluble prodrug of propofol, and compare it with propofol. Study design Prospective, randomized, blinded experimental trial. Animals Ninety-six adult laboratory rabbits, mean ± SD weight 2.20 ± 0.15 kg. Methods Stage 1. 16 rabbits were assigned to receive fospropofol disodium or propofol to measure MIR, using an up-and-down method with response to tail-clamping stimulus (TCS). Stage 2. Eighty rabbits were allocated to group F (fospropofol disodium) or group P (propofol), and further subdivided (n = 10 in each subgroup) according to infusion time (2, 4, 6 or 8 hours), to groups F 2h, F 4h,F 6h,F 8h and P 2h,P 4h,P 6h,P 8h. Fospropofol or propofol were infused, and tail clamping applied to maintain the same depth of anaesthesia until infusion was completed. Times to recover righting reflex (RR), to respond to TCS, and total recovery to different durations of continuous infusion of two anaesthetic drugs were noted. Respiratory and pulse rates and oxygen saturation were analyzed. The plasma concentrations of fospropofol disodium, the active metabolite propofol (propofol F ) and propofol emulsion were measured with respect to loss and recovery of RR and TCS. Results MIR of fospropofol disodium was 2.0 mg kg )1 minute )1, and MIR of propofol was 0.9 mg kg )1 minute )1. Times in minutes to total recovery from anaesthesia in groups F and P were as follows, F 2h 15 ± 3; F 4h 26 ± 4; F 6h 52 ± 6; F 8h 84 ± 10; and P 2h 10 ± 1; P 4h 19 ± 7; P 6h 36 ± 7; P 8h 48 ± 5. Conclusions and clinical relevance After continuous intravenous infusion in rabbits ( 8 hours), fospropofol disodium and propofol both show an extension of recovery time with increasing infusion time, fospropofol disodium showing a significantly greater prolongation compared to propofol emulsion when infusion time increases to 6 and 8 hours. Keywords continuous intravenous infusion, fospropofol disodium, propofol, rabbits, recovery time. Introduction Propofol is a lipophilic anaesthetic that is used widely for induction and for maintenance of anaesthesia in humans and animals. The most commonly used preparations are formulated lipid emulsions, the drawbacks of which include inherent emulsion instability, injection pain, a need for 373

antimicrobial agents to prevent sepsis, and a concern of hyperlipidemia-related side effects (Richard et al. 2008). Fospropofol disodium chemically described as a phosphono--methyl-2, 6-diisopropylphenol, disodium salt (C 13 H 19 5 PNa 2 ) is a novel water-soluble, short-acting prodrug of propofol and is intended to eliminate the disadvantages associated with the lipid emulsion formulation of propofol (Schywalsky et al. 2003). Fospropofol disodium undergoes enzymolysis by endothelial cell surface alkaline phosphatases liberating propofol (hereafter referred to as propofol F ) as an active metabolite together with phosphate and formaldehyde (Fig. 1), and formaldehyde is converted to formate rapidly (Banaszczyk et al. 2002). Propofol has acquired worldwide acceptance, because of its rapid onset, short duration of action and clinical effect, and the quality and rate of recovery (Glen 1980; Aeschbacher & Webb 1993a). Its pharmacokinetics in most species are such as to make it the preferred agent for continuous infusion and it can maintain stable anaesthesia for prolonged periods but still have rapid recoveries (Richard et al. 2008). However, the use of continuous infusion of propofol in rabbits can be associated with slow recovery (Aeschbacher & Webb 1993b). The prodrug fospropofol disodium has a slower onset, a longer half-life and a longer duration of action than propofol emulsion (Fechner et al. 2004). The complex pharmacodynamics during long-term continuous infusion makes it more difficult to predict the duration of drug effect easily when the infusion is discontinued (Sano et al. 2006). n the basis of the pharmacokinetic behaviour of fospropofol disodium (Fechner et al. 2003, 2004; Schywalsky et al. 2003), we assumed that fospropofol disodium would have progressively even longer recovery times with increasing continuous infusion time compared to propofol emulsion. This current study was designed to evaluate and compare the recovery time of the two anaesthetic drugs after continuous intravenous (IV) infusion in rabbits. A secondary goal of this study was to compare arterial plasma concentrations of propofol with respect to physiologic reflexes during anaesthesia. Material and methods After Institutional Animal Care and Use Committee (Chengdu, Sichuan, China) approval, 96 adult Japanese White rabbits of both sexes, weighing 2.02 2.54 kg were enrolled in the present study. All rabbits were determined to be healthy as judged by physical examination. Before and during the study, the rabbits were housed in stainless steel cages (width depth height = 85 50 40 cm), providing 0.4 square metre per animal. The rabbits were fed a commercial pelleted diet and received supplementary hay and fresh vegetables daily. Water was always available. Food but not water was withheld for 12 hours prior to anaesthesia. The rabbits were kept under controlled environmental conditions, (temperature 20 22 C, humidity (60 ± 5%), 18 air changes per hour and a 12 hours day and night cycle). All animals underwent a period of acclimation of 48 hours before the study. All procedures associated with this study were performed in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. Preparation of fospropofol disodium Fospropofol disodium was manufactured by Yichang Humanwell Pharmaceutical Co., Ltd. (China) according to the formula developed by our laboratory (Li et al. 2009). The quality control system and the initial stability of fospropofol disodium had been approved by the State Food and Drug Administration (SFDA, China). In this formulation, 500 mg of lyophilized (freeze-dried) P alkaline phosphatase H P H H Fospropofol disodium Propofol F Phosphate Formaldehyde Figure 1 Chemical structure of fospropofol disodium and the scheme of its enzymolysis to propofol F. 374 Ó 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 39, 373 384

fospropofol disodium (China) was stored in a glass ampoule under aseptic conditions. Experimental protocol The current study was divided into two stages. Stage 1. Determination of MIR Stage 1 was measurement of minimum infusion rate (MIR) (ku et al. 2005) in 16 rabbits, assigned randomly to two groups to receive either fospropofol disodium or propofol (Diprivan; AstraZeneca, UK). n the day of study, the rabbits were placed in a restraining cage which allowed for more comfortable positioning while still preventing gross movements. A 22 gauge catheter (Spacelabs Medical, Inc., WA, USA) was inserted into the ear-marginal vein for the infusion of anaesthetic drugs and dextrose in lactated Ringer s solution (Qingshan Pharmaceutical Corp., China). Fospropofol disodium or propofol then was infused continuously using a TCI-I syringe pump (Slgo, China), the rabbits were placed in right recumbency above a warm air blanket (Rerwardst Technology Co., Ltd, China). Rectal temperature was monitored continuously and maintained within 0.5 C of physiologic values (39 C). Dextrose in lactated Ringer s solution was infused at the rate of 8 ml kg )1 hour )1, using a precision infusion pump, in order to maintain fluid balance. All the rabbits received 3 L minute )1 oxygen through a facemask throughout the procedure. Pulse oxygen saturation (Sp 2 ) and pulse rate (PR) were monitored by pulse oximetry using a 150B3 monitor (Philips, China) with the sensor on the tail. When the infusion was discontinued, the rabbit was moved to a warmed recovery cage (temperature was maintained at 24 26 C), and monitored continuously until fully recovered from anaesthesia. Any adverse event observed during or after drug administration was documented. After 15 minutes of acclimatization and stabilization of baseline measurements, the continuous infusion of either fospropofol disodium or propofol was initiated Both fospropofol disodium and propofol were targeted to produce an equipotent MIR. MIR for the two IV anaesthetics was defined as the infusion rate of IV anaesthetic preventing gross purposeful movement to a noxious stimulus in 50% of subjects (ku et al. 2005). MIR of fospropofol disodium initially was assumed to be 2.0 mg kg )1 minute )1 according to a previous pilot study (Li et al. 2009). From the molecular weights of 332.24 for fospropofol disodium and 178.27 for propofol, it was anticipated that 1 mg of fospropofol disodium would liberate 0.54 mg of propofol. MIR of propofol therefore was hypothesized to be 1.08 mg kg )1 minute )1. Minimum infusion rates were measured by judging the gross purposeful movement response to noxious stimulus, while increasing or decreasing 10% of the previous anaesthetic infusion rate (Bettschart-Wolfensberger et al. 2003; ku et al. 2005). The noxious stimulus (TCS) was a tail clamp, which consisted of the application of a 20 cm rubber-covered clamp (Pilling Instruments, PA, USA), 2 cm distal from the root of the tail, then clamped to the second ratchet. Each stimulus was applied for 60 seconds. Responses to TCS were judged by either positive or negative. The response was judged as positive only when gross purposeful movement (leg escape movement or head elevation) was observed, and was judged as negative when there was no gross purposeful movement. In addition, response to TCS was judged as positive when a spontaneous movement was observed before the TCS. Infusion rate was increased when a positive response was detected and decreased when negative response was detected. After maintaining the assumed MIR for 40 minutes, response to TCS was judged for the first time. The infusion rate was increased or decreased according to the response from positive to negative or negative to positive. Then responses to a TCS were judged every 25 minutes for both drugs. A change of the response from positive to negative or negative to positive was defined as a pair, and the stimulation was repeated at different infusion rates until three pairs of responses were recorded. MIR was determined as the average of these three mean values (ku et al. 2005). Stage 2. Maintenance of a stable plane of anaesthesia Stage 2 compared some cardiopulmonary measurements during, and the recovery times from prolonged infusions of the two drugs, and also measured blood drug concentrations at set goals of anaesthesia and recovery. Eighty rabbits were allocated randomly to one of two groups (ensuring that there were an equal number of male and female rabbits per group) to receive fospropofol disodium (group F) or propofol (group P) with different infusion times. According to Ó 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 39, 373 384 375

the infusion time, group F and group P both were randomly divided further into four subgroups of 2, 4, 6 and 8 hours respectively (groups of F 2h,F 4h, F 6h,F 8h and P 2h,P 4h,P 6h,P 8h ). During the study, the order of running the different infusion times was randomized. All anaesthetic procedures were performed by the same experienced anaesthetists who were unaware of the treatment (fospropofol or propofol) group. Handling, catheterization, application of infusions, fluid administration, positioning, administration of oxygen, maintenance of rectal temperature, and monitoring were as described above for Stage 1. Additionally the middle ear artery was cannulated with a 14 gauge catheter (Spacelabs Medical, Inc., WA, USA) for blood sample collection. Respiratory rate (f R ) was determined by observing and counting thorax expansions. Recordings of f R, Sp 2 and PR were made prior to (t = 0) and during the infusion of fospropofol disodium or propofol (at 30 and 60 minutes and subsequently every 60 minutes until the infusion schedule was completed). Any adverse event observed during or after drug administration was documented. After 15 minutes of acclimatization and stabilization of baseline measurements, the continuous infusion of either fospropofol disodium or propofol was initiated. When the infusion was discontinued, the rabbit was moved to a warmed recovery cage (temperature was maintained at 24 26 C), and monitored continuously until fully recovered from anaesthesia. nset and recovery from anaesthesia were assessed and measured using the TCS and righting reflex (RR). nce the infusion commenced, RR was tested at 1 minute intervals, and the time to loss (T LR ) recorded. nce the RR was lost, the TCS was applied at 5 minutes intervals, and time to loss (T LT ) recorded. Similarly after the infusion schedule was completed, responses to TCS were judged every 5 minutes, time to return (T RT ) recorded, then RR tested every minute until this returned (T RR ). Full recovery time was defined as the total time elapsed from when the anaesthetic was discontinued to when the rabbit was upright, and making purposeful movements. During anaesthesia, TCS was employed to ensure that anaesthetic depth remained stable. After maintaining MIR (as measured in Stage 1) for 40 minutes, the response to TCS was judged for the first time; if positive infusion rate was increased incrementally (10% increment of the previous infusion rate). nce a negative TCS response had been achieved, infusion rate was decreased (10% increment of the previous infusion rate). If Sp 2 fell below 90% for more than 1 minute, the infusion rate was decreased without adjustment to the TCS schedule. The TCS response test was repeated every 25 minutes until the infusion was completed. Throughout induction, anaesthesia and recovery the same investigating anaesthetist, who was unaware of the treatment group, interpreted the responses to TCS and RR. Sample acquisition, handling, and processing Arterial blood samples (1 ml) from each of the eight subgroups were collected in Eppendorf tubes at the loss and recovery of RR and TCS after the start and the end of the infusion. The plasma concentration at the time of loss (C LR ) and recovery (C RR )ofrr and plasma concentration at the time of loss (C LT ) and recovery (C RT ) of response to TCS were recorded. In group F, samples were collected in Eppendorf tubes prefilled with heparin (Spacelabs Medical, Inc., WA, USA) and sodium ortho-vanadate (SV) (Zhongyuan Chemical Co., Ltd., China) at 10 mg ml )1 to inhibit in vitro conversion of fospropofol disodium to propofol by alkaline phosphatase enzymes. After centrifugation (1850 g, 10 minutes), plasma samples were stored at )70 C and were analyzed within 1 month (Fechner et al. 2003, 2004). Analysis of plasma fospropofol disodium was performed using high performance liquid chromatography-electrospray ionization mass spectrometry (Agilent 1100, CA, USA). The analytical range of this method was 55 40,000 ng ml )1, with coefficients of variation of 5.7% in the concentration range. Analyses of plasma propofol F and propofol were performed using a gas chromatography mass spectrometer (Agilent 6890). The analytical range of this method was 20 294,000 ng ml )1 with coefficients of variation of 3.6% in the concentration range. The lower limit of quantification of propofol was 5 ng ml )1. Intra-assay accuracy values were 100.3 102.7% of the nominal value. Inter-assay accuracy values were 98.5 114.6% of the nominal value. Euthanasia Following complete recovery from anaesthesia, rabbits were euthanased using an overdose of pentobarbital sodium (100 mg kg )1 IV). 376 Ó 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 39, 373 384

Statistics Data are presented as mean ± SD unless otherwise stated. All analyses were performed using SPSS 18.0 (SPSS Inc., IL, USA). The Kolmogorov Smirnov test supported the assumption of normal distribution. Homogeneity of variance was tested by the Levene s test. Comparisons of weight and MIR between the two groups were performed by the Student s t-test. Comparisons of gender and survival rates between the two groups were performed by chi-squared test. A repeated measures multivariate analysis of variance (MANVA) was used to compare differences in the measured variables during this phase of the protocol. If a significant effect was indicated, a post hoc test was performed. Statistical significance was defined as p<0.05. The sample size for this study was determined using measures of variance from a preliminary experiment and previous investigations (ku et al. 2005; Li et al. 2009; Martinez et al. 2009; Allweiler et al. 2010). The criterion for significance alpha was set at 0.05. Ten rabbits in each group would be sufficient to provide an 80% power to detect a significant difference between the fospropofol and propofol groups. Results The rabbits were significantly slower to sleep after receiving fospropofol disodium, compared with rabbits that received propofol, even although such a low infusion rate was started. Compared to propofol, induction from fospropofol disodium anaesthesia regimens was smooth and gradual, with rabbits exhibiting slow, narcotized movement. Stage 1. Determination of MIR There were no significant differences in the weight (2.20 ± 0.18 kg versus 2.21 ± 0.20 kg), and gender (female/male, 4/4 versus 4/4) between group F and group P. A summary of infusion rate (IR), time after changing infusion time (T), response to TCS (R) and mean value of the infusion rates for the pair of responses (MVI) are shown in Tables 1 and 2. There was little inter-individual difference in MIR in the two groups. MIR of each rabbit in group F was in the range Table 1 Measurement of the Minimum Infusion Rate (MIR) of fospropofol disodium in eight rabbits N. F1 N. F2 N. F3 N. F4 N. F5 N. F6 N. F7 N. F8 IR T R MI IR T R MI IR T R MI IR T R MI IR T R MI IR T R MI IR T R MI IR T R MI 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.2 40 + 2.2 40 + 1.8 40 ) 2.2 40 + 1.8 40 ) 1.8 40 ) 2.2 40 + 2.2 40 + 1.9 1.9 1.9 2.4 25 + 2.4 25 + 2.0 25 + 2.4 25 + 2.0 25 + 2.0 25 + 2.4 25 + 2.4 25 + 2.3 2.3 1.9 2.3 2.3 2.2 25 ) 2.6 25 + 2.2 25 + 2.2 25 ) 2.2 25 + 1.8 25 ) 2.2 25 ) 2.2 25 ) 2.5 2.1 2.1 1.9 2.0 25 ) 2.4 25 ) 2.0 25 ) 2.0 25 ) 2.0 25 ) 2.0 25 + 2.0 25 ) 2.0 25 ) 2.1 1.8 25 ) 2.2 25 ) 1.8 25 ) 2.2 25 + 1.8 25 ) 1.8 25 ) 1.8 25 ) 1.9 1.9 2.1 1.9 1.9 1.9 2.0 25 + 2.0 25 ) 2.0 25 + 2.0 25 ) 2.0 25 + 2.0 25 + 2.0 25 + 1.9 1.9 1.9 1.8 25 ) 1.8 25 ) 1.8 25 ) 1.8 25 ) 1.9 2.0 25 + 1.9 1.8 25 ) MIR F1 = 2.0 MIR F2 = 2.1 MIR F3 = 2.0 MIR F4 = 2.2 MIR F5 = 2.0 MIR F6 = 1.9 MIR F7 = 2.0 MIR F8 = 2.0 N. F: individual rabbit number; IR: infusion rate of fospropofol disodium (mg kg )1 minute )1 ); T: time after changing infusion time (minute); R: response to tail-clamping stimulus (+/)); MI: mean value of the infusion rates for the pair of responses (mg kg )1 minute )1 ); MIR F : average of three MIs of each rabbit in fospropofol disodium group. Ó 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 39, 373 384 377

Table 2 Measurement of the Minimum Infusion Rate (MIR) of propofol in eight rabbits N. P1 N. P2 N. P3 N. P4 N. P5 N. P6 N. P7 N. P8 IR T R MI IR T R MI IR T R MI IR T R MI IR T R MI IR T R MI IR T R MI IR T R MI 1.08 1.08 1.08 1.08 1.08 1.08 1.08 1.08 1.19 40 + 0.97 40 ) 0.97 40 ) 0.97 40 ) 0.97 40 ) 1.19 40 + 0.97 40 ) 0.97 40 ) 1.14 1.14 1.08 25 ) 0.86 25 ) 0.86 25 ) 0.86 25 ) 0.86 25 ) 1.08 25 ) 0.86 25 ) 0.86 25 ) 0.92 0.92 0.92 0.92 0.97 25 ) 0.97 25 + 0.97 25 + 0.97 25 + 0.75 25 ) 0.97 25 ) 0.97 25 + 0.75 25 ) 1.03 0.92 0.92 0.92 0.81 1.03 0.92 0.81 1.08 25 + 0.86 25 ) 0.86 25 ) 0.86 25 ) 0.86 25 + 1.08 25 + 0.86 25 ) 0.86 25 + 1.03 0.81 1.03 0.81 0.97 25 ) 0.75 25 ) 0.75 25 ) 0.75 25 ) 0.75 25 ) 0.97 25 ) 0.75 25 ) 0.75 25 ) 0.81 0.81 0.81 0.81 0.64 25 ) 0.86 25 + 0.86 25 + 0.86 25 + 0.64 25 ) 0.86 25 + 0.70 0.70 0.75 25 + 0.75 25 + MIR P1 = 1.06 MIR P2 = 0.85 MIR P3 = 0.88 MIR P4 = 0.88 MIR P5 = 0.81 MIR P6 = 1.06 MIR P7 = 0.85 MIR P8 = 0.81 N.P: individual rabbit number; IR: infusion rate of propofol (mg kg )1 minute )1 ); T: time after changing infusion time (minute); R: response to tail-clamping stimulus (+/)); MI: mean value of the infusion rates for the pair of responses (mg kg )1 minute )1 ); MIR P : average of three MIs of each rabbit in propofol group. of 1.8 to 2.6 mg kg )1 minute )1, and MIR in group P was in the range of 0.64 to 1.19 mg kg )1 minute )1. For fospropofol disodium, mean value of the eight MIRs was 2.0 mg kg )1 minute )1, and mean value of the eight MIRs was 0.9 mg kg )1 minute )1 for propofol. MIRs were statistically different between group F and group P (p = 0.012). Stage 2. Comparison of recovery time between Group F and Group P f the 80 rabbits enrolled in the study, one female rabbit in group F 8h died during the recovery phase. Group F had a 97.5% survival rate, and group P had a 100% survival rate. Survival rates were not statistically different between groups F and P. There were no statistically significant differences with respect to the sex and weight characteristics between groups F and P. The results from 79 rabbits contributed to the final data. The summary of comparison of full recovery time between group F and group P is presented in Table 3. There were statistically significant differences with respect to the recovery time between group F and group P (MANVA, p = 0.000). There was a trend towards significantly prolonged recovery time with increasing infusion time in both groups. Post hoc tests Table 3 Comparison of recovery time between Group F and Group P Infusion time (hours) 2 4 6 8 T Fospropofol (minutes) 15 ± 3 26 ± 4 52 ± 6 ## 84 ± 10 ## T Propofol (minutes) 10 ± 1 19 ± 7 36 ± 7** 48 ± 5** T Fospropofol ) T Propofol (minutes) 5 ± 3 7 ± 6 16 ± 9 36 ± 9 T Fospropofol : recovery time in group F; T Propofol : recovery time in group P; T Fospropofol ) T Propofol : the difference of recovery time between the two groups with the same infusion time. Values are expressed as mean ± SD. Recovery time = time from end of infusion to when the rabbit was upright and making purposeful movements. *p < 0.05, **p < 0.01, versus group F; # p < 0.05, ## p < 0.01, versus group P. showed that the recovery time was significantly slower in group F 8h when compared with group F 2h (p = 0.000), group F 4h (p = 0.000) and group F 6h (p = 0.000). The recovery time was significantly slower in group P 8h when compared with group P 2h (p = 0.001), group P 4h (p = 0.001) and group P 6h (p = 0.000). There were statistically significant differences with respect to the recovery time between 378 Ó 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 39, 373 384

groups F and P when infusion time increased to 6 hours (p = 0.012) and 8 hours (p =0.002), although the differences in recovery times between group F and group P were not significant when infusion time was 4 hours. An exploratory analysis of the data indicated that there was a trend towards a significantly increased difference of recovery time between the two groups with increasing infusion time. Group F 8h had the longest recovery times (84 ± 10 minutes). When infusion time increased to 8 hours, the recovery time of group P 8h was nearly half the recovery time in group F 8h (48 ± 5 minutes versus 84 ± 10 minutes, p = 0.000). There were no statistically significant differences in recovery time between genders. Comparison of response to RR and TCS between Group F and Group P The summary of response to RR and TCS between group F and group P is presented in Table 4. Time to lose the response to RR and TCS were both earlier in group P than in group F. There were statistically significant differences with respect to the time to lose the response to RR between group F and group P at the same infusion time (p = 0.001), although no statistical differences with the time to lose the response to TCS between the two groups. Time to recovery from TCS increased with increasing infusion time in both groups F and P after the infusion was discontinued. When infusion time increased to 6 and 8 hours, time to recovery from TCS in group P was approximately half the recovery time from TCS in group F (group P 6h (10 ± 3) minutes versus group F 6h (18 ± 4) minutes, p = 0.001; group P 8h (10 ± 4) minutes versus group F 8h (20 ± 6) minutes, p = 0.000). Comparison of arterial blood concentration of fospropofol and propofol between Group F and Group P Plasma concentrations with respect to physiologic reflexes (LR, LT, RT, RR) in group F and group P are summarized in Table 5. Plasma concentration of propofol was significantly lower than that of propofol F with the same infusion time. Plasma concentration (C RT, C RR ) of fospropofol disodium, propofol F or propofol did not increase proportionally with the infusion time. No statistically significant difference was found in C RT, C RR for groups F 2h, F 4h, F 6h and F 8h. No statistically significant difference was found in C RT, C RR between groups P 2h,P 4h,P 6h and P 8h. Comparison of respiratory rate, Sp 2 and pulse rate between Group F and Group P Respiratory rate, Sp 2 and PR are shown in Fig. 2. All 79 rabbits maintained spontaneous ventilation after infusion began. The baseline f R was rapid and shallow, and exceeded 95 breaths minute )1. When Table 4 Comparison of response to righting reflex and tail-clamping stimulus between Group F and Group P Fospropofol disodium Propofol Infusion time (hours) Infusion time (hours) 2 4 6 8 2 4 6 8 T LR (minutes) 19 ± 3 # 19 ± 3 # 18 ± 4 ## 17 ± 5 ## 8 ± 2* 9 ± 3* 8 ± 2** 6 ± 2** T LT (minutes) 76 ± 26 76 ± 18 75 ± 11 73 ± 13 65 ± 25 62 ± 25 62 ± 19 58 ± 25 T RT (minutes) 5 ± 2 14 ± 3 18 ± 4 ## 20 ± 6 ## 5 ± 1 8 ± 3 10 ± 3** 10 ± 4** T RR (minutes) 15 ± 3 26 ± 4 52 ± 6 ## 84 ± 10 ## 10 ± 1 19 ± 7 36 ± 7** 48 ± 5** Values are expressed as mean ± SD. T LR : time until loss of righting reflex; T LT : time until loss of response to tail-clamping stimulus; T RT : time until recovery of response to tail-clamping stimulus; T RR : time until recovery of righting reflex. When receiving anaesthetics, responses to righting reflex (RR) were judged every 1 minute for both drugs. Responses to tail-clamping stimulus (TCS) were judged after maintaining the MIR infusion for 40 minutes, then the following responses to a TCS were judged every 25 minutes for both drugs until the infusion schedule was completed. After the infusion schedule was completed, responses to TCS were judged every 5 minutes. nce the response to TCS was positive, responses to RR were not judged every 1 minute. *p < 0.05, **p < 0.01, versus group F; # p < 0.05, ## p < 0.01, versus group P. Ó 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 39, 373 384 379

Table 5 Comparison of arterial plasma concentration of fospropofol disodium, propofolf and propofol with respect to physiologic reflexes Fospropofol disodium PropofolF Propofol Infusion time (hours) Infusion time (hours) Infusion time (hours) 2 4 6 8 2 4 6 8 2 4 6 8 C LR (mg L )1 ) 196.5 ± 26.3 175.8 ± 36.8 143.8 ± 42.9 89.2 ± 27.1 5.6 ± 0.7 4.4 ± 0.8 4.2 ± 0.4 4.9 ± 0.7 1.8 ± 0.6 2.1 ± 0.5 1.4 ± 0.4 1.5 ± 0.3 CLT (mg L )1 ) 218.9 ± 32.2 280.5 ± 85.9 218.4 ± 40.8 151.7 ± 43.7 8.4 ± 1.3 9.9 ± 1.3 8.9 ± 0.9 6.8 ± 1.1 3.7 ± 2.0 4.8 ± 2.4 2.5 ± 1.0 2.4 ± 0.9 CRT (mg L )1 ) 12.3 ± 6.3 40.3 ± 40.1 7.0 ± 6.1 9.7 ± 5.1 5.2 ± 0.5 5.0 ± 0.6 6.6 ± 1.1 5.7 ± 2.3 1.5 ± 0.8 1.6 ± 0.8 2.5 ± 2.3 2.6 ± 2.4 C RR (mg L )1 ) 4.5 ± 1.1 3.1 ± 1.7 2.2 ± 1.8 3.5 ± 1.7 2.6 ± 0.3 2.8 ± 0.7 2.0 ± 1.0 2.0 ± 1.0 1.1 ± 0.4 1.2 ± 0.5 1.3 ± 0.8 1.3 ± 0.8 PropofolF: fospropofol disodium undergoes enzymolysis by endothelial cell surface alkaline phosphatases liberating propofolf as an active metabolite; CLR: blood concentration at the time of loss of righting reflex; C LT : blood concentration at the time of loss of response to tail-clamping stimulus; C RT : blood concentration at the time of recovery of response to tail-clamping stimulus; C RR : blood concentration at the time of recovery of righting reflex. Values are expressed as mean ± SD. the infusion started, each animal took several deep breaths. Respiratory effort reduced gradually with infusion, but became strong after withdrawal of the drugs, f R fluctuated between 36 98 breaths minute )1. Maximum degrees of depression of f R were 63% and 61% in groups F and P, respectively. Respiratory effort reduced earlier in group P than group F. Sp 2 decreased gradually during infusions, but never fell below 90%. There were no statistically significant differences in Sp 2 between groups F and P. After induction of anaesthesia, PR increased in all rabbits and remained at higher levels during the whole anaesthetic period. There were statistically significant differences with respect to PR between group F and group P when infusion time increased to 300, 360, 420 and 480 minutes (p < 0.05). Discussion MIR is a standard measure that has been used to enable comparison of the recovery time and physiologic effect produced by equipotent doses of various injectable anaesthetic agents (Flaishon et al. 1997). Also, MIR is an important index for evaluating the potency and determining the adequate dose of IV anaesthetics for total IV anaesthesia (ku et al. 2005). As a concept comparable to minimum alveolar concentration (MAC), Cp 50 defined as the plasma concentration of IV anaesthetic preventing a positive response to stimulus in 50% of subjects, has been considered to be helpful in comparing anaesthetic potency and individual sensitivity to an anaesthetic (ku et al. 2005). However, we did not consider Cp 50 as suitable in the present study. Although MIR and MAC both reflect a concept of anaesthetic depth, whilst the concentration of inhalation anaesthetic agent in the blood closely parallels that in the alveoli, in contrast, the blood concentration of an injectable agent will depend on the total dose injected and the pharmacokinetics that govern redistribution and elimination of the agent. Thus, unlike MAC, MIR may be a timedependent variable (Flaishon et al. 1997; Bettschart-Wolfensberger et al. 2001 ku et al. 2005). A review of the literature indicated that there was no significant correlation between MIR and Cp 50 (ku et al. 2005). It is probably attributed to the difference between their physiological meanings. Taking propofol as an example, Cp 50 can be considered as the propofol concentration in blood required for changing the state of consciousness from alert to anaesthetic, whereas MIR can be 380 Ó 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 39, 373 384

Respiratory rate (breaths minute 1 ) 100 80 60 40 20 Fospropofol Propofol 0 60 120 180 240 300 360 420 480 Time (minutes) Sp 2 (%) 100 98 96 94 92 90 88 86 84 82 80 Fospropofol Propofol 0 60 120 180 240 300 360 420 480 Time (minutes) Pulse rate (beats minute 1 ) 300 280 260 240 220 Fospropofol Propofol 200 0 60 120 180 240 300 360 420 480 Time (minutes) Figure 2 Respiratory rates, Sp2, and pulse rates during continuous infusion of fospropofol disodium and propofol. Data presented are mean ± SD. Rabbits (n = 10 in each group) underwent fospropofol disodium or propofol infusion for 2, 4, 6, or 8 hours. Thus from times 0 120 n = 40; 120 240 n = 30; 240 360 n = 20 and 360 480 n = 10. considered as the dosage of propofol required in order to compensate for propofol removed from blood and maintain an anaesthetic state. It is presumed that Cp 50 mainly reflects the tolerance for propofol and MIR reflects not only the tolerance but also the removal rate of propofol from blood, so there is no significant correlation between the two. Finally, it is impracticable to measure plasma drug Ó 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 39, 373 384 381

concentration in real time. Thus, in the present study we chose to determine MIR using a practical approach to IV anaesthesia without measuring Cp 50. In the study reported here, none of the rabbits responded to TCS when fospropofol disodium was infused at rates of >2.6 mg kg )1 minute )1 or when propofol was infused at rates of >1.19 mg kg )1 minute )1. However, we found that most of the rabbits had some autonomic responses including an increase in f R, which would suggest that the depth of anaesthesia, or at least anti-nociception, was inadequate. To avoid administration of an overdose of anaesthetic drugs in rabbits, the anaesthetist initially should try to choose an anaesthetic depth that allows some rabbits to respond with movements to TCS at the time they initiate infusion of anaesthetic drugs. Movements in response to TCS were always gentle and easy to control, and it proved to be a relatively satisfactory method in the current study. To enable comparison of recovery time, this study utilized equivalent MIRs and equivalent maintenance of depth of anaesthesia. A pilot study had established that a stable plane of anaesthesia could be maintained when fospropofol disodium was infused at the rate of 2.0 mg kg )1 minute )1 for one hour (Li et al. 2009). According to available data from the literature, the half-lives of fospropofol disodium, propofol F and propofol following continuous IV infusion in the rabbit were 3 4 minutes, 5 6 minutes and 2 4 minutes, respectively (Fechner et al. 2003, 2004; Li et al. 2009). In order to reach steady-state concentrations in arterial blood, an infusion time lasting at least five half-lives is required. Although the prodrug fospropofol disodium leads to rapid liberation of active drug, propofol F shows a longer half-life, a delay in onset and a sustained duration of action when compared with propofol emulsion (Fechner et al. 2003, 2004; Li et al. 2009). In order to ensure propofol F plateau during continuous infusion, the measurement of MIR was initiated at 40 minutes after the start of infusion. Since continuous infusion of fospropofol disodium produced a longer time to peak propofol F plasma concentration, anaesthetic depth was monitored by repeating the TCS response test every 25 minutes for both agents until the infusion was completed. Fospropofol disodium can be accompanied by the development of transient pruritus during the initial infusion, particularly at the injection site. We found that the rabbits in group F kept scratching the ear which was being infused for about 3 5 minutes after initiation of infusion. Transient pruritus may be associated with slight increases in serum phosphorus levels after IV administration, but no clinically significant adverse effects from increased phosphorus levels have been observed (Fechner et al. 2004). MIR of propofol reported here is similar to investigations in other studies. Aeschbacher & Webb (1993b), after injecting an initial bolus of propofol (7.3 mg kg )1 ), established a propofol infusion rate of 1.0 mg kg )1 minute )1 for a total of 8 hours. No transient apnoea was observed in the present study. In general, it was our impression that rabbits in group F were sleepier and slower to recover than those in group P with the same infusion time. During the recovery phase, rabbits that had received fospropofol disodium (6 hours, 8 hours groups) had a significant increase in mean recovery time compared to that for propofol emulsion. They also were slower to recover compared with rabbits that received propofol, even although these differences were not statistically significant when infusion time was 2 or 4 hours. Cessation of anaesthesia resulted in a period of immobility followed by a seemingly instantaneous return to consciousness and awareness with subsequent hyperactivity for 30 to 120 seconds in group P. Rabbits in group F seemed to re-narcotize and not to move at all for five to 30 minutes, prior to appearing normal and fully recovered. Recovery from fospropofol disodium anaesthesia regimens was smooth and gradual, with animals exhibiting slow, deliberate movements. Rabbits receiving fospropofol disodium clinically were slower to recover, which may be attributed to accumulation of fospropofol disodium. ur results are consistent with the previous theoretical assumption of a slower recovery after fospropofol disodium infusion. Loss or recovery of reflex responses (RR and TCS) occurred significantly faster in rabbits in group P than those in group F. When comparing the propofol concentration at equipotent doses of both drugs, we observed that the concentration for propofol F was much higher than for propofol emulsion. This is in accordance with previous work in rats (Dutta & Ebling 1998), where the dose of lipid-free propofol was twice the equipotent dose of lipid emulsion. The longer duration of unconsciousness may have been a consequence of the slower decrease of propfol F concentration after fospropofol disodium infusion. 382 Ó 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 39, 373 384

An increase in PR has been observed after induction and during maintenance with propofol in rabbits (Aeschbacher & Webb 1993b), and this was also the case for PR in this study in all groups. Depression of f R was the most common adverse effect after induction of anaesthesia in this study. The immediate initiation of oxygen supplementation alleviated any hypoxia and is recommended. Endotracheal intubation and assisted respiration might also be advisable (Aeschbacher & Webb 1993b). ne rabbit in group F 8h died during the recovery phase. The observations during the fospropofol disodium infusion and the evaluation of the necropsy reports offer several explanations for its death. The rabbit was infused with a high average maintenance dose of fospropofol disodium in an attempt to weaken its response to TCS. The overall effect was noted at recovery, when the rabbit was unable to maintain an upright posture and remained recumbent for 2 hours after anaesthesia was discontinued. The pharmacologic effects of anaesthetics increase with increasing infusion time, resulting in increased anaesthetic depth, and prolonged recovery times. Arterial blood pressure was not measured in this study, so any contribution by hypotension cannot be assessed. Fluid volume overload was unlikely to have caused the death of this animal. In this study, a mean total dose of anaesthetic drugs and Dextrose in lactated Ringer s solution resulted in a fluid infusion rate of 11 14 ml kg )1 hour )1. However, although not statistically significant, the mortality associated with group F is clinically relevant. The results of this study indicate that, under the conditions of this experiment, the IV infusion of fospropofol disodium alone is inadequate to provide safe, long-term anaesthesia in rabbits. An unexpected long recovery and respiratory depression during recovery phase are features that mitigate against the use of fospropofol disodium as a single agent without intubation or assisted respiration for prolonged anaesthetic periods in this species. Although it has been questioned whether the continuous infusion of propofol emulsion to rabbits is suitable (since wide individual differences in the response to propofol were observed in the recovery period in rabbits after long-term anaesthesia), injectable anaesthetic agents are a preferred method of anaesthesia for rabbits in the laboratory setting (Aeschbacher & Webb 1993b). The option of undertaking studies in rabbits is justified since these animals are widely used in biomedical research. Rabbits allow good handling and restraining and good collection and monitoring of vital signs. The parallel group design used required a relatively large sample size. However, we did not consider a crossover design appropriate in the present study. The reasons are: 1) Long-term infusion of the agents used could lead to drug tolerance which could affect the next treatment period in a crossover design; 2) Long-term infusion of fospropofol disodium could lead to carryover effects since it has a relatively long half-life; 3) A study conducted by Ypsilantis et al. (2007) reported that rabbits anaesthetized for a prolonged period with propofol developed a fatal Propofol Infusion Syndrome. With a parallel group design, the study avoided any possible cumulative toxicity due to the carryover effects from one treatment period to the next; and 4) In a crossover design, blocking results in the loss of some degrees of freedom and leads to a wider confidence interval on the difference between treatments (Cody & Slack 1992). Hence a parallel group design was considered more appropriate than a crossover design in this study. This study has several limitations. First, infusion rates of the two drugs were titrated using the response to a tail clamp stimulus (rather than a specific bispectral index value) to assess and reflect the current anaesthetic depth. Silva et al. (2011) demonstrated that, of the most recently introduced anaesthetic depth indices, the index of consciousness and the permutation entropy were the least variable and most reliable in reflecting different anaesthetic depths in a rabbit model. Second, although an equivalent model allows us to compare the differences in recovery time of these two agents, we failed to measure plasma drug concentration in real time for both drugs and use this to establish an appropriate pharmacokinetic model. Traditionally, the terminal elimination half-life has been used as a measure of offset of drug action. However, the length of anaesthetic administration influences the rate at which concentrations of anaesthetics decrease after their discontinuation. The contextsensitive half-life, the time to halving of the blood concentration after termination of drug administration by an infusion designed to maintain a constant concentration, has been proposed as a more useful measure of the pharmacokinetic offset of IV anaesthetics. This concept has clinically relevant implications in that it would allow a more accurate prediction of the recovery from long-term IV Ó 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 39, 373 384 383

infusion. However, even without an appropriate pharmacokinetic model, it is reasonable to suggest that the context-sensitive half-life of fospropofol disodium is significantly longer than propofol, based on the recovery of reflex responses reported in the present study. Third, the current study lacks the monitoring of physiologic effects (arterial blood pressure, blood gases and end tidal C 2 ). Data of respiratory rate, Sp 2 and pulse rate were not recorded during the recovery phase. In conclusion, we have compared the recovery time of fospropofol disodium and propofol in rabbits after different lengths of continuous infusion. Propofol and its prodrug, fospropofol disodium, both can produce anaesthesia using this species. We have shown that fospropofol disodium significantly prolongs recovery time, compared to propofol emulsion, when infusion times were increased to 6 and 8 hours. Acknowledgements This study was supported in part by grant No.2005CB522601 from the 973 Program, Beijing, China, and by grant No. 30700782 from the National Research Foundation of Nature Sciences, Beijing, China. References Aeschbacher G, Webb AI (1993a) Propofol in rabbits. 1. Determination of an induction dose. Lab Anim Sci 43, 324 327. Aeschbacher G, Webb AI (1993b) Propofol in rabbits. 2. Long-term anesthesia. Lab Anim Sci 43, 328 335. Allweiler S, Leach MC, Flecknell PA (2010) The use of propofol and sevoflurane for surgical anaesthesia in New Zealand White rabbits. Lab Anim 44, 113 117. Banaszczyk MG, Carlo AT, Millan V et al. (2002) Propofol phosphate, a water-soluble propofol prodrug: in vivo evaluation. Anesth Analg 95, 1285 1292. Bettschart-Wolfensberger R, Freeman SL, Jäggin-Schmucker N et al. (2001) Infusion of a combination of propofol and medetomidine for long-term anesthesia in ponies. Am J Vet Res 62, 500 507. Bettschart-Wolfensberger R, Bowen IM, Freeman SL et al. (2003) Medetomidine-ketamine anaesthesia induction followed by medetomidine-propofol in ponies: infusion rates and cardiopulmonary side effects. Equine Vet J 35, 308 313. Cody RL, Slack MK (1992) Crossover design in pharmacy research. Ann Pharmacother 26, 327 333. Dutta S, Ebling WF (1998) Formulation-dependent pharmacokinetics and pharmacodynamics of propofol in rats. J Pharm Pharmacol 50, 37 42. Fechner J, Ihmsen H, Hatterscheid D et al. (2003) Pharmacokinetics and clinical pharmacodynamics of the new propofol prodrug GPI 15715 in Volunteers. Anesthesiology 99, 303 313. Fechner J, Ihmsen H, Hatterscheid D et al. (2004) Comparative Pharmacokinetics and Pharmacodynamics of the new propofol prodrug GPI 15715 and propofol. Anesthesiology 101, 626 639. Flaishon R, Lang E, Sebel PS (1997) Textbook of Intravenous Anesthesia. The Williams &Wilkins Co., London, UK, pp. 545 563. Glen JB (1980) Animal studies of the anaesthetic activity of ICI 35868. Br J Anaesth 52, 731 741. Li R, Li J, Zhang WS et al. (2009) Pharmacokinetics of fospropofol disodium administered as a constant rate intravenous infusions in rabbits. J Sichuan Univ (Med Sci Edi) 40, 337 340 (in chinese). Martinez MA, Murison PJ, Love E (2009) Induction of anaesthesia with either midazolam or propofol in rabbits premedicated with fentanyl/fluanisone. Lab Rec 164, 803 806. ku K, hta M, Yamanaka T et al. (2005) The minimum infusion rate (MIR) of propofol for total intravenous anesthesia after premedication with xylazine in horses. J Vet Med Sci 67, 569 575. Richard EF, Danneman PJ, Brown M (2008) Anesthesia and Analgesia in Laboratory Animals (2nd edn). Academic Press, London, UK, pp. 38 42. Sano T, Nishimura R, Kanazawa H et al. (2006) Pharmacokinetics of fentanyl after single intravenous injection and constant rate infusion in dogs. Vet Anaesth Analg 33, 266 273. Schywalsky M, Ihmsen H, Tzabazis A et al. (2003) Pharmacokinetics and pharmacodynamics of the new propofol prodrug GPI 15715 in rats. Eur J Anaesthesiol 20, 182 190. Silva A, Ferreira DA, Venâncio C et al. (2011) Performance of electroencephalogram-derived parameters in prediction of depth of anaesthesia in a rabbit model. Br J Anaesth 106, 540 547. Ypsilantis P, Politou M, Mikroulis D et al. (2007) rgan toxicity and mortality in propofol-sedated rabbits under prolonged mechanical ventilation. Anesth Analg 105, 155 166. Received 19 April 2011; accepted 30 September 2011. 384 Ó 2012 Association of Veterinary Anaesthetists and the American College of Veterinary Anesthesiologists, 39, 373 384