IV delivery of volatile anesthetics has many advantages over inhalational delivery, as demonstrated by >50 years of research.1–5a IV anesthesia induction is rapid because equilibration is circumvented between the anesthetic circuit and the lungs’ functional residual capacity. However, if the use of vaporizers could be eliminated, the direct and indirect costs of anesthesia could be reduced. Previous reports on IV use of inhalational anesthetics, either accidentally in humans6,7 or in experimental animals,8,9 have described severe damage to pulmonary vessels and death in most cases. Although emulsified volatile anesthetics with a lipid element can induce anesthesia without pulmonary complications, they require a relatively large volume of lipids, limiting their use in clinical practice.4,5,10 Recently, a novel 20% fluoropolymer-based emulsion of sevoflurane was tested for the induction of anesthesia and was effective.11 However, this formulation caused allergic reactions and was associated with hypotension and histamine release when tested in dogs.12 Jee et al.13 are currently developing a new fluoropolymer-based emulsion. In this study, taking into account these characteristics, we prepared a 20% sevoflurane lipid emulsion using caprylic triglycerides instead of long-chain triglycerides for the emulsion. IV administration of halothane and isoflurane can be monitored using a respiratory gas monitor because the end-expiratory concentration changes in parallel with anesthetic partial pressure in the blood.4,14,15 Clinically, it is difficult to monitor the actual blood concentration of drugs, as the predictive model development is complex.16 If inhaled anesthetics could be used IV, we could use real-time monitoring with a gas monitor because the primary route of elimination of IV sevoflurane is through the lungs.
The purpose of this study was to investigate the effectiveness (i.e., ED50, LD50) and the safety (i.e., allergic and/or anaphylactic reaction) of the novel emulsified sevoflurane in rats and to verify the stability of this emulsion for clinical practice.
Purified egg yolk lecithins were supplied by QP Corp. (Chofu, Tokyo, Japan) and consisted of phosphatidylcholine (80.0%), phosphatidylethanolamine (17.9%), lysophosphatidylcholine (0.2%), sphingomyelin (0.1%), and other components (1.8%). Glycerol was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Salinhes®, containing 6% hydroxyethyl starch and normal saline, was purchased from Fresenius Kabi Japan K.K., Tokyo, Japan. After sodium chloride contained in Salinhes was removed by dialysis, the hydroxyethyl starch solution was used as a stabilizing agent. Sevoflurane was provided by Maruishi Pharmaceutical Co. Ltd. (Osaka, Japan). Caprylic triglyceride (COCONARD RK) was purchased from the Kao Corporation (Tokyo, Japan).
An emulsion was prepared using a previously described method.17 However, we made one slight modification: purified egg yolk phosphatides at 1.2% (w/w) concentration were dispersed in 2.5% (w/w) glycerol solution containing 1.0% (w/w) hydroxyethyl starch. For the water phase, we used a high-speed disperser (HG-2, SMT Co. Ltd., Tokyo, Japan).
The 1:2 mixture of caprylic triglyceride/sevoflurane (1,1,1,3,3,3-hexafluoro-2-[fluoromethoxy]propane) was used as the oil phase. Agitation is required when preparing oil/water emulsions containing 20% (w/w) sevoflurane. The mixture of the oil phase and water phase was first emulsified for 5 minutes to prepare the coarse dispersion with a disperser. It was then passed through a high-pressure homogenizer (LAB2000, APV Gaulin Inc., Everett, MA) to obtain finer particles at 1200 bar (kgf/cm2). The resulting emulsions were stored in sealed vials until use.
All experiments were conducted according to the institutional ethical guidelines for animal experiments and the safety guidelines of the National Defense Medical College. These experiments were approved by the Animal Research Committee at the National Defense Medical College.
Anesthetic Effect and Toxic Evaluation
We studied 69 male Sprague-Dawley rats, 6 to 8 weeks of age and weighing 180 to 270 g (Japan SLC, Inc., Hamamatsu, Japan) to evaluate the effectiveness of the emulsion. First, we evaluated the median effective dose (ED50) for loss of righting reflex (LORR). The rats were tested on multiple days but were injected with sevoflurane only weekly to prevent effects related to accumulation. Each rat was weighed at baseline for consistency and then allowed to breathe pure oxygen spontaneously for 3 minutes in an induction chamber for preoxygenation. In experiments to determine the ED50, rats were allocated randomly to 6 groups consisting of 9 rats each and 20% emulsified sevoflurane was injected as a single bolus via the tail vein at a rate of 1 mL/min. The doses of emulsified sevoflurane in the 6 groups were 0.40, 0.45, 0.50, 0.56, 0.63, and 0.71 mL/kg. Immediately after injection of the emulsified sevoflurane, rats were placed in a supine position in the open-air so that they could not reinhale sevoflurane. LORR for >5 seconds, which is approximately half of the preliminary LORR at a minimal dose, was considered to be successful induction of anesthesia. The time for loss of LORR to return was also recorded. Rats were observed for 30 minutes and evaluated during weight checks to determine whether there were any complications.
Second, we investigated the lethal dose (LD50) to verify effects related to the peak concentration. Each rat was tested on a single day only. The LD50 was determined by clinical signs of death, such as pupil dilation or permanent cardiopulmonary arrest. Rats were allocated randomly to 7 groups consisting of 6 rats each and injected with the emulsified sevoflurane in the same manner. The doses of emulsified sevoflurane used in the 7 groups were 0.95, 1.0, 1.06, 1.12, 1.19, 1.26, and 1.33 mL/kg. In the same manner as we evaluated the ED50, each rat was weighed and preoxygenated. When respiratory arrest was induced after administration of 20% emulsified sevoflurane, ventilation was assisted with a facemask to prevent death because of respiratory depression. For the control group, an emulsion that did not contain sevoflurane was injected into 6 rats. The volume of this control emulsion was 1.33 mL/kg, which was equal to the maximal dose of the emulsion in the LD50 experiment. The rats were immobilized in a suitable container with their tail protruding, and a 1-mL syringe connected to a 27-g needle was used for the tail vein injection. The needle was inserted deeply, and the emulsion was administered at a preset and constant speed after backflow of blood was confirmed, indicating the needle was in the vein. If the emulsion could not be injected smoothly, for example, owing to movement of the rat, the rat was excluded from the study. It was also required that the rat remains in the container and not be removed immediately after injection. Emulsions were transferred to the syringe just before injection and were used within 1 month after being prepared.
Allergic and/or Anaphylactic Evaluation
Eight male Sprague-Dawley rats (8 weeks of age and weighing 236–270 g) with a jugular vein catheter surgically implanted before purchase were studied (Japan SLC, Inc.) to evaluate the presence of allergy and/or anaphylactic reaction by continuous infusion of emulsion. The rats were tested on a single day, and catheters were heparinized every day to avoid coagulation during the experiment. Rats were observed for 1 week intermittently to evaluate the potential adverse effects induced by drug administration.
Each rat was weighed, and prevital data were measured (blood pressure, heart rate, and respiratory rate). Blood pressure and heart rate were measured from the tail by a non-preheating monitor (MK-2000; Muromachi Kikai Co. Ltd., Tokyo, Japan). Respiratory rate was counted by 15-second measurements. Prevital data were measured 3 times to minimize the variability of each individual. The emulsion set by syringe-pump (TE-312; Terumo Co. Ltd., Tokyo, Japan) was connected to the catheter via extension tube (X1-100; Top Co. Ltd., Tokyo, Japan). By preliminary estimation, emulsion administration was started for sevoflurane at a rate of 4 mg/kg/h and was increased (or decreased) at 0.5 mg/kg/h every 5 minutes to until no pedal and/or tail reflexes were observed, but respiratory depression did not occur (“good condition”). The vital data and presence of adverse effects were evaluated for 30 minutes from the point of good condition (“time 0” in Fig. 1). The administration rate was titrated appropriately to maintain good condition during each experiment (Fig. 1D). The rats were observed carefully for 1 week after the experiment.
Stability of Emulsions
To investigate the stability of the emulsion, the particle size of the emulsified sevoflurane was measured on days 0, 7, 14, 28, 60, 240, and 365 after preparation. In addition, to investigate the stability of the emulsion after opening the vial, the concentration of the emulsified sevoflurane in 3 vials was analyzed immediately after opening the vial and at 3, 60, 120, and 180 minutes after transferring the emulsion from the vial to the syringe. The concentrations of the emulsified sevoflurane were determined by headspace gas chromatography (GC). The GC system used for the assay was a GC-2010 (Shimadzu Co. Ltd., Kyoto, Japan) with a Turbo Matrix 40 headspace sampler (PerkinElmer, Inc., Waltham, MA). The system was equipped with a fused silica capillary column (SUPELCO-WAX10 [SIGMA-ALDRICH Co. LLC., St. Louis, MO], 0.25 mm ID × 30 m), and the oven temperature was set at 60°C. Helium was used as a carrier gas, and “Split Mode” was selected. For quantification, the determination of the emulsified sevoflurane was calculated by standard addition method using standard samples of sevoflurane.
To enable sufficient statistical power in this study and reduce the risk of an underpowered false-negative result, we performed an a priori sample size calculation. A sample size of 9 rats per group was determined for the measurement of the ED50 (total n = 54) and 6 per group for the measurement of the LD50 (total n = 42). The number of rats per group was based on previous studies, and the final number of rats per group included extra rats to account for the potential failure of IV administration.11,13,18 To calculate the ED50 and LD50, we determined the number of rats with LORR or death, respectively, from the total number that received the emulsions. The ED50 and LD50 values were calculated using nonlinear regression, and data were fitted with a cumulative Gaussian model using GraphPad Prism (version 6.03; GraphPad Software, Inc., San Diego, CA). The fitting formula was as follows: z = (x − mean)/SD. Y = top × z distance (z), where x is scaled log10 of the dose of emulsion and Y is the response. The 95% confidence intervals (CIs) of ED50 and LD50 were also calculated. The therapeutic index (LD50/ED50), representing the safety of the drug, was also calculated. The values are presented as the mean ± SD.
Raw data of the dose-response relationship of emulsified sevoflurane are shown in Table 1. The dose-response curve for LORR by injection of 20% emulsified sevoflurane is shown in Figure 2. The ED50 was 0.47 mL/kg (95% CI, 0.46–0.48; R2 = 0.9966). The mean duration from the onset of anesthesia until recovery from LORR is shown in Figure 3. Anesthesia occurred before or immediately after completion of the bolus injection and typically continued for 10 to 20 seconds from the initiation of the injection. Within approximately 1 minute after the termination of injection, the rats had returned to grooming with no evidence of disorientation or residual sedation. No rats had adverse effects and/or died within 24 hours of the observation period. In addition, no rats lost weight at 24 hours after the injection of the emulsion. Figure 4 shows the dose-response curve for the LD50 of emulsified sevoflurane. The LD50 was 1.13 mL/kg (95% CI, 1.07–1.18; R2 = 0.9013). All rats that died in the LD50 experiment did so just after completion of the injection of the emulsified sevoflurane. The rats that did not die within 30 minutes after injection of emulsion had no complications during observation at 24 hours after the injection. Only 1 rat in the 0.95 mL/kg group did not die because of respiratory arrest during support ventilation using a facemask. All other rats that went into respiratory arrest were dead, despite ventilation. The therapeutic index (LD50/ED50) was 2.41 (95 CI%, 2.23–2.59). The emulsion that did not contain sevoflurane did not induce anesthesia, and no adverse effects were associated with its use. No adverse effects (i.e., rash, piloerection, edema, convulsion, and death) and no change in vital signs associated with anaphylactic reaction were observed in rats infused continuously with emulsion (Fig. 1). In addition, no rats had lost weight at 24 hours after infusion of the emulsion, and there was no evidence of adverse effects at the 1-week observation period. Figure 5 shows the particle size of the emulsion over 365 days (n = 5). After preparation of the emulsions, their particle size remained approximately 80 nm. Figure 6 shows the concentrations of the emulsified sevoflurane in 3 vials immediately after opening the vial and at 3, 60, 120, and 180 minutes after transferring the emulsion from the vial to the syringe. The mean concentrations were not <18% after 3 hours of opening the vial.
In the current study, IV administration of a novel 20% sevoflurane formulation induced anesthesia without major complications. Furthermore, recovery from anesthesia was very rapid—within 60 seconds. The advantages of IV administration of volatile anesthetics are achieved by circumventing equilibration of the anesthesia circuit and lungs and include rapid onset of anesthesia and recovery without adverse events. In contrast to inhalation anesthesia, IV administration can also reduce the total dose of anesthetics, as it reduces surplus gases that do not reach the systemic circulation. In addition, IV administration of emulsions can be monitored using a real-time gas monitor.4,14,15 This is a significant advantage because it is difficult to monitor actual blood concentrations continuously during conventional IV anesthesia.16
Zhou et al.18 previously reported that lipid emulsions of halothane and isoflurane were supersaturated. According to their results, the saturated concentrations of sevoflurane in 20% and 30% Intralipid® (the lipid emulsion used; Sino-Swed Pharmaceutical Co. Ltd., Beijing, China) were only 2.07% and 3.46%, respectively. If these emulsions are used in humans, the lipid content introduced to the body would not be clinically acceptable. Recently, Huang et al.19 conducted a phase I clinical trial using 8% emulsified isoflurane containing 30% Intralipid. The emulsions produced rapid onset of unconsciousness in all volunteers followed by fast, predictable, and complete recovery. However, their use was associated with common adverse events, such as injection site pain and transient tachycardia. Although the IV administration of volatile anesthetics is currently being developed, it is necessary to devise a method to reduce the lipid content of the emulsions (i.e., higher relative anesthetic concentration).
There are 3 advantages of our emulsion compared with previous emulsions. First, the concentration of sevoflurane is increased to 20%. Increasing the concentration of sevoflurane contained in the emulsion can simultaneously decrease the lipid content. Second, all materials (e.g., purified egg yolk lecithins, glycerol, Salinhes, and panasate 800) contained in the emulsions can be administered to humans. However, further research is required because polysorbate 80, a component of the emulsion approved by the U.S. Food and Drug Administration, has been associated with histamine release and severe hypotension in dogs.20 Third, we used sevoflurane as an anesthetic drug-administered IV. It has been reported that the blood/gas partition coefficient can be increased approximately 3 times by the continuous infusion of an isoflurane emulsion.14,15 Therefore, sevoflurane favors continuous infusion, as the blood/gas partition coefficient of sevoflurane is smaller than that of isoflurane.
The therapeutic index, which is considered to represent the safety of the drug, was 2.41 (95% CI, 2.23–2.59) in our experiment. This value is comparable with fluoropolymer-based emulsified sevoflurane (therapeutic index, 2.6),11 propofol (therapeutic index, 3.1),18 and thiopental (therapeutic index, 2.2).21
In this study, we chose to use a bolus injection to evaluate the ED50 and LD50 of our emulsion because it can be used to measure the duration of anesthetic effects (i.e., LORR) and the peak concentration (i.e., lethal dose). Bolus injection was also used in previous reports investigating the emulsions.11,13,18 We also evaluated responses to painful stimulation (tail clamping for 3 seconds after anesthesia), which was performed separately from the ED50 experiment, and confirmed there was no dose-dependent response (50% no response volume of emulsion was 0.54 mL/kg [95% CI, 0.52–0.57; R2 = 0.9838], data not shown). Continuous infusion instead of a bolus injection was used to evaluate the safety of the emulsion to obtain more accurate vital data. To obtain accurate and constant data from rodents, especially when they are awake, is not easy and requires special techniques in response to the rodent’s character. Thus, the continuous infusion of emulsion might represent the future in terms of clinical practice. Figure 1 shows that rat vital signs were reduced slightly after continuous infusion of the emulsion because of the anesthetic effect. But rat vital signs were quite stable during the continuous infusion. If anaphylactic reaction had been induced by the emulsion, the vital signs would have been significantly altered.22–24 Although the depth of anesthesia was titrated to prevent pedal and/or tail reflex without respiratory depression, respiratory depression gradually occurred. This suggests that the administration rate was too fast: therefore, future studies should use a reduced administration rate. No adverse effects were observed in any of the experiments, indicating that the emulsified sevoflurane was not harmful to rats.
Thermodynamically, an emulsion is an unstable system and does not exist in equilibrium. Therefore, evaluation of the stability of the emulsion is critical because it may eventually separate into water and oil. Stability of our emulsion was assessed by measuring particle size for 365 days. In addition, we measured the concentration of sevoflurane in the emulsions immediately after opening the vial and at 3, 60, 120, and 180 minutes after transferring the emulsion from the vial to the syringe. The particle size in the emulsions was stable over the 365 days of the study period (Fig. 5). After opening the vials, the mean concentrations of the emulsions were not <18% and showed only a 10% decrease from the original emulsion concentration (Fig. 6). In the current experiment, emulsions were administered within 3 hours after vials were opened and used within 1 month after the emulsions were prepared.
There were several limitations in this study. Although we confirmed that all rats were alive during the week after the ED50 experiment and continuous infusion, the long-term effects of emulsified sevoflurane remain unknown. Second, it may be insufficient to evaluate allergic and/or anaphylactic reaction in rats only, as allergic reactions have mostly been reported in dogs.20 Taken together, determination of the minimum alveolar concentration for continuous infusion under endotracheal intubation and further evaluation of allergic responses in dogs are necessary.
In conclusion, we prepared a novel 20% emulsified sevoflurane using caprylic triglycerides. As for both bolus injection and continuous infusion of the 20% emulsified sevoflurane, anesthesia was safely induced in rats without major complications. The toxicity of this emulsion is comparable with previous emulsions and other IV anesthetics. The emulsion was stable for over 365 days unopened and for 180 minutes after opening the vial. Further investigation of this emulsion is expected in the future.
Name: Toru Morohashi, MD.
Contribution: This author helped design the study, collect the data, and prepare the manuscript.
Attestation: Toru Morohashi attests to the integrity of the original data and the analysis reported in this manuscript.
Name: Sayako Itakura, MD.
Contribution: This author helped collect the data and analyze the data.
Attestation: Sayako Itakura is the archival author.
Name: Ken-ichi Shimokawa, PhD.
Contribution: This author helped collect the data and analyze emulsion of data.
Attestation: Ken-ichi Shimokawa is the archival author.
Name: Fumiyoshi Ishii, PhD.
Contribution: This author helped prepare the emulsion, collection emulsion of data, and prepare the manuscript about the emulsion.
Attestation: Fumiyoshi Ishii approved the final manuscript.
Name: Takehiko Ikeda, MD, PhD.
Contribution: This author helped advise the study design and revise the article critically for intellectual content.
Attestation: Takehiko Ikeda attests to the integrity of the original data and the analysis reported in this manuscript.
Name: Tomiei Kazama, MD, PhD.
Contribution: This author helped conduct the study.
Attestation: Tomiei Kazama approved the final manuscript.
This manuscript was handled by: Markus W. Hollmann, MD, PhD, DEAA.
We thank Kazuya Ishii, Kumiko Soga, Hironori Nagahara, Masayasu Takatsu, Yukihiko Watanabe (Maruishi Pharmaceutical Co. Ltd., Osaka, Japan), Yumiko Takaenoki (Department of Anesthesiology, National Defense Medical College, Tokorozawa, Saitama, Japan) and Kiyoko Takamiya (Department of Pharmacology, National Defense Medical College, Tokorozawa, Saitama, Japan) for excellent technical help in this study; and Dr. Kouichi Fukuda (Center for Laboratory Animal Science, National Defense Medical College, Tokorozawa, Saitama, Japan) for the assistance in animal administration.
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