|Year : 2014 | Volume
| Issue : 1 | Page : 16-21
A cyclodextrin formulation to improve use of the anesthetic tribromoethanol (Avertin ® )
Arlene McDowell1, Jessica A Fothergill1, Azeem Khan2, Natalie J Medlicott1
1 New Zealand's National School of Pharmacy, University of Otago, Dunedin, NewZealand
2 Deparment of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, United Kingdom
|Date of Submission||20-Nov-2013|
|Date of Decision||20-Nov-2013|
|Date of Acceptance||20-Nov-2013|
|Date of Web Publication||4-Jan-2014|
New Zealand's National School of Pharmacy, University of Otago, Dunedin
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: Efficacy and safety concerns have been raised in the literature with the use of tribromoethanol (TBE) (Avertin ® ) for anesthesia in rats and mice when administered by intraperitoneal (IP) injection. Despite the controversy, it remains in common usage as an anesthetic agent in laboratory rodents for short-term surgical procedures. Cyclodextrins have been shown to improve drug solubility and were investigated here as an improved anesthetic formulation for mice. Materials and Methods: The phase solubility of TBE with hydroxypropyl-β-cyclodextrin (HP-β-CD) was estimated. The efficacy of two anesthetic regimens was compared in this study; the conventional TBE formulation solubilized in tert-amyl alcohol and a HP-β-CD formulation containing TBE. Mice (n = 6) were administered the formulations by IP injection and the pharmacodynamic parameters of time to induction of anesthesia, duration of anesthesia and recovery time were measured using a combined reflex score (CRS). Results and Discussion: Phase solubility studies showed a linear increase in the solubility of TBE with increasing HP-β-CD concentration and suggested >1:1 binding of the drug in the cyclodextrin complex. At a dose of 260 mg/kg the standard TBE formulation appeared to produce deeper anesthesia than the cyclodextrin formulation, with a minimum average CRS of 1.8 compared with 5.2. No post-mortem pathology was observed in mice that received either the conventional or cyclodextrin formulation. Conclusion: The cyclodextrin TBE formulation did not conclusively provide an improved anesthetic response at a dose of 260 mg/kg compared with the conventional formulation. The improved solubility of TBE with HP-β-CD and the reduced variability in anesthetic response warrants the further investigation of this formulation. This study has also identified the value of using the anticholinergic atropine in association with TBE for anesthesia.
Keywords: Anesthetic, cyclodextrin, mice, solubility, tribromoethanol
|How to cite this article:|
McDowell A, Fothergill JA, Khan A, Medlicott NJ. A cyclodextrin formulation to improve use of the anesthetic tribromoethanol (Avertin ® ). J Pharm Bioall Sci 2014;6:16-21
|How to cite this URL:|
McDowell A, Fothergill JA, Khan A, Medlicott NJ. A cyclodextrin formulation to improve use of the anesthetic tribromoethanol (Avertin ® ). J Pharm Bioall Sci [serial online] 2014 [cited 2020 Sep 20];6:16-21. Available from: http://www.jpbsonline.org/text.asp?2014/6/1/16/124303
Tribromoethanol (TBE) is an injectable anesthetic used in biomedical research for short-term surgical procedures (5-30 min) involving laboratory rodents. TBE provides anesthetic effect through its action as a general central nervous system depressant and is metabolized in the liver by conjugation with glucuronic acid prior to excretion in the urine as TBE glucuronate.  It is commonly used in embryo transfer procedures to produce transgenic mice and has minimal effects on embryonic development and reproductive performance  and in studies using echocardiography in mice due to the modest cardiodepressive effects compared to other injectable anesthetics.  TBE is generally administered by the intraperitoneal (IP) route and is reported to produce good surgical anesthesia and muscle relaxation with rapid induction and recovery within 80 min.  The recommended dose for surgical anesthesia in mice is 240 mg/kg IP,  although the range used by researchers is between 125 mg/kg and 500 mg/kg,  TBE has been marketed under the trade names Avertin, Bromethol, Ethobrom, Narcolan and Narkolan (a 66.7% solution of pharmaceutical-grade TBE in tert-amyl alcohol), however these products are no longer manufactured,  so researchers must now formulate their own solution for use from non-pharmaceutical-grade TBE. Adverse effects reported with the use of the extemporaneous preparation of TBE include intestinal ileus and fibrous abdominal adhesions as well as increased morbidity, particularly if given subsequent doses. ,, TBE undergoes photodegradation to dibromoacetaldehyde and hydrobromic acid,  both irritants and some authors have attributed adverse effects to these degradation products. However, a study carried out by Lieggi et al. found no correlation between dibromoacetaldehyde concentration in the injection preparations and occurrence of adverse effects. The current formulation practice, which involves heating solid TBE in an organic co-solvent (tert-amyl alcohol), may be less than optimal with respect to drug solubility and stability and our research will address these limitations by investigating a new approach that may improve the utility of this anesthetic agent.
Cyclodextrins are cyclic oligosaccharides that possess a hydrophobic core and hydrophilic exterior and are used as water-soluble drug carriers for hydrophobic injectable drugs.  On mixing with suitably sized drugs in an aqueous solution, inclusion complexes are formed where hydrophilic water molecules drive inclusion of hydrophobic drug molecules within the cyclodextrin cavity. A dynamic equilibrium between the free drug, free cyclodextrin and drug: Cyclodextrin complex is established in solution.  These reversible inclusion complexes improve the drug's aqueous solubility, chemical stability and potentially reduces irritancy at the injection site. , Medlicott et al. demonstrated that encapsulation of melphalan with a hydroxypropyl-β-cyclodextrin (HP-β-CD) formulation resulted in less damage to venous endothelial cells in vitro compared with co-solvents that are typically used to improve solubility. Of the cyclodextrins available HP-β-CD is popular because it has high solubility and low toxicity when administered parenterally  and has been used previously to increase the aqueous solubility of anesthetic agents including etomidate,  alfaxolone  and propoanidid. 
The aims of this study were to investigate an alternative formulation of TBE using HP-β-CD to improve solubility and stability of TBE, and to determine the anesthetic response to this new formulation following IP injection in mice.
| Materials and Methods|| |
TBE (2, 2, 2- tribomoethanol 97%, C 2 H 3 Br 3 O, MW 282.8), tert-amyl alcohol (99%) and methyl orange were purchased from Sigma Aldrich, (St. Louis, MO, USA). NaCl was purchased from BDH Chemicals (Palmerston North, New Zealand). HP-β-CD (Kleptose ® HPB, MW 1440) was obtained from a gift from Roquette Lestrem, France. CombiTitrant ® 5 and methanol (99.8%) were purchased from Merck (Auckland, New Zealand). Atropine was obtained as Phoenix Atropine injection from Provet NZ Ltd (Christchurch, New Zealand). Hematoxylin and eosin dyes for histological examination were obtained from Surgipath Medical (Illinois, USA).
TBE anesthetic solution
TBE is reported to have a solubility of 1 in 40 parts water at 40°C, soluble in alcohol, ether, bezene and chloroform and very soluble in tert-amyl alcohol  and is supplied as a white crystalline powder. A 1.25% w/v TBE anesthetic solution was produced following the method recommended by the Animal Welfare Office, University of Otago, by dissolving 0.5 g TBE in 0.25 g tert-amyl alcohol then diluting to 40 mL with 0.9% NaCl. Dilution of the tert-amyl alcohol solution produced a precipitate, which redissolved with stirring for 30-60 min on a magnetic stirrer. The final solution was filtered (0.2 μm) into an amber glass bottle, protected from light by covering the vial with foil and stored at 4°C.
The water content of HP-β-CD samples (0.05 g, n = 5) was determined by Karl Fischer titration (736 GP Titrino, Metrohm, USA). CombiTitrant ® 5 was used as a titrant, and methanol as a solvent. The mixture was calibrated with 10 μL of distilled water until a consistent value was obtained. The water content of the HP-β-CD solid was calculated to be 8.4% w/w. TBE has no appreciable ultra violet absorbance and so direct measurement of the binding constant for TBE and HP-β-CD could not be performed. An approximate phase solubility diagram was constructed by addition of small aliquots (5-30 mg) of TBE to HP-β-CD solutions (0-0.035 M) until no further TBE would dissolve. Solutions were protected from light to reduce the risk of chemical degradation during the observation period and stored at 20°C. Temperature was monitored using a Hobo data logger (Onset Computer Corp., Bourne, MA, USA).
Cyclodextrin formulations were prepared aseptically no more than 2 days prior to being used in the animal study. The cyclodextrin TBE formulation was prepared by dissolving 0.25 g TBE with 0.344 g HP-β-CD (weight corrected for water content) in 20 mL to give a concentration of 1.25% w/v in 0.012 M HP-β-CD. This mixture was dispersed using a magnetic stirrer (~1 h) and once fully dissolved, the solution was filtered (0.2 μm) and stored at 4°C and protected from light until administered.
Administration of formulations and assessment of anaesthesia
The procedures described had prior approval from the University of Otago Animal Ethics Committee, Dunedin and conformed to the University of Otago Code of Ethical Conduct for the Manipulation of Animals (1987) and the Animal Welfare Act (1999) in New Zealand.
Specific pathogen free, adult, female Balb/c mice of the same approximate weight (19-21 g) were maintained in group housing and acclimatized to the environment for at least 7 days prior to being used in the study. Animals were offered food and water ad libitum, and both were not withheld prior to, or following, anesthesia. Mice were randomly assigned to receive either the standard or cyclodextrin TBE anesthesia. Animals were removed from group housing, weighed and placed in individual cages on heating pads to maintain body temperature. Atropine (0.05 mg/kg) was administered subcutaneously 30 min prior to administration of the anesthetic formulations. The cyclodextrin TBE formulation and the standard TBE solution were compared using the dose regimen of 260 mg/kg TBE administered through the IP route (n = 6/group). Parameters observed in the animals were depth of anesthesia, time to righting reflex loss and gain.
The anesthetic efficacy of each formulation was assessed in the mice by monitoring several reflex responses. The time after administration of the anesthetic to loss of righting reflex, the animal's reflex to roll onto its front when placed on its back, was recorded. The intensity of the tail pinch reflex was assessed after pinching the tail of the mouse with plastic forceps. The intensity of the pedal withdrawal reflex was assessed when the interdigital skin on the fore and hind feet were pinched with plastic forceps. Tail and pedal withdrawal responses were scored on a scale from 0 to 3 (0 = no response through to 3 = strong response as in a conscious animal). The reflex scores for each time point were summed to give a combined reflex score (CRS) of 0-9 (0 = surgical anesthesia, 9 = fully responsive) for each animal.
Animals were observed for 2 h following recovery and then daily for 14 days. 14 days following administration of the anesthetic formulations, a necropsy was performed and tissue samples of the liver and gut were collected for histological examination. The tissue samples were fixed with 10% formalin, embedded in paraffin and sectioned with a Leica RM2025 rotary microtome (Nussloch, Germany) to 3 μm thick sections. Tissue sections were stained with hematoxylin and eosin for visualization using light microscopy.
Data are averaged and reported as mean ± SD, unless otherwise stated. Statistical analysis was performed using statistical package for the social sciences software (IBM, version 19). P < 0.05 was considered to be significant. The comparison between respiratory rate with standard and cyclodextrin TBE formulations was undertaken by repeated measures analysis of variance model. The time to loss and gain, of righting reflex was compared between the two treatment groups using an independent samples t-test.
| Results and Discussion|| |
The solubility of TBE in water is reported to be 1 in 40 (2.5% w/v) at 40°C.  In the present study, we observed the solubility of TBE to increase linearly with increasing HP-β-CD concentration [Figure 1]. The solubility in the absence of HP-β-CD was 28 mg/mL (2.8% w/v) and temperature recorded over the study period was 21 ± 1.5°C. The A L shaped diagram with a slope of greater than 1 suggested a higher order interaction with respect to the drug (>1:1) exists between TBE and HP-β-CD.  Since TBE is a small molecule it is possible that 2:1 or greater molar ratio TBE: HP-β-CD complex was formed. Using Equation 1, a 2:1 binding constant of 380 M−2 was estimated.
|Figure 1: Phase-solubility profile of tribromoethanol as a function of hydroxypropyl-β-cyclodextrin concentration at 20°C. Data points represent mean of duplicates. Least squares linear regression produced a slope = 1.608 (Standard error = 0.065); intercept = 0.1037 (SE = 0.0037); R2 = 0.993|
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Where S 0 = solubility (M) of TBE in the absence of HP-β-CD and the slope is from the TBE solubility versus HP-β-CD concentration graph [Figure 1].
A notable difference between the preparations of the formulations was that TBE could be readily dissolved directly in the cyclodextrin solution prior to use, whereas for the standard formulation TBE required dissolution in an organic solvent followed by dilution with normal saline. When the organic solution was diluted, TBE precipitation was observed and time was required to allow this to re-dissolve prior to use.
The use of cyclodextrin formulations can reduce precipitation in vivo as discussed by Brewster and Loftsson.  The relationship between the solubility of a drug that forms a complexation with a cyclodextrin is commonly linear. Consequently, upon administration, dilution of the drug-cyclodextrin complex causing dissociation should also occur in a linear fashion, and without precipitation. This is in contrast to preparations made using organic solvents where the solubilization of the drug is based on a non-linear relationship and so upon dilution following in vivo administration, the ability of organic solvents to solubilize the drug is rapidly reduced leading to precipitation. 
Owing to the disparity in doses reported in the literature (125-500 mg/kg), a preliminary study was undertaken to determine an appropriate dose of TBE to achieve anesthesia in mice. Consistent with other studies, at a dose of 260 mg/kg there was good surgical anesthesia, however there was also some evidence of respiratory depression (i.e. vocalizations, labored breathing). Consequently, atropine was administered 30 min prior to anesthesia (0.05 mg/kg) by subcutaneous injection. The inclusion of atropine improved the anesthesia and reduced respiratory depression.
A comparison of anesthesia between the standard TBE solution and the cyclodextrin TBE formulation [Figure 2] shows the trend for the standard formulation to produce a deeper level of anesthesia, although the difference was not statistically significant. The lowest CRS attained was 0 for the standard formulation and 2 for the cyclodextrin formulation [Figure 2]. Short and variable anesthetic times with TBE (dose 300 mg/kg) have been reported by Avila et al.  and these authors also comment that the duration of anesthesia was sometimes too short to conduct their intended measurements. The effectiveness of the standard TBE formulation compared to the cyclodextrin formulation may be attributed to the presence of organic solvent (concentration = 0.625% in the injected solution), which is known to cause central nervous system depression and ataxia on its own,  and which is not present in the cyclodextrin TBE formulation. Complexation of the TBE with the cyclodextrin would also result in reduced free drug concentrations compared to the standard formulation. It may be that rather than a comparison of the same dose (260 mg/kg) between the two formulations, a higher dose of the cyclodextrin formulation may be required to have the same effect as the standard formulation. Binding of TBE by HP-β-CD supports the observation of reduced pharmacodynamic effect in the present study. However, Stella and Rajewski reported that release of drugs from cyclodextrin complexes occurs very rapidly once administered to the body.  Recovery from the TBE anesthetic was more rapid for the cyclodextrin group as all mice had regained a CRS of 9 by 30 min following administration compared to up to 1 h for the standard TBE formulation. The time to loss and gain of righting reflex reflects the level of anesthesia reached with the two formulations. Results from observation of the righting reflex [Table 1] show that this reflex is lost approximately 30 s faster with the standard formulation (P = 0.006) and it appears to take longer to return, although this result was not significant. At a relatively high TBE dose of 400 mg/kg, the time interval between loss and regain of righting reflex has reported to be between 0 min and 120 min, with a mean of 21.5 min. 
|Figure 2: Influence of anesthetic formulation on combined reflex score for mice treated with (a) standard tribromoethanol or (b) hydroxypropyl-β-cyclodextrin tribromoethanol (n = 6/group). Dose of tribromoethanol was 260 mg/kg + atropine 0.05 mg/kg. CRS score 0 = surgical anesthesia and 9 = fully responsive|
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|Table 1: Comparison of times to loss and gain of righting reflex between the standard tribromoethanol formulation and hydroxypropyl-β-cyclodextrin tribromoethanol formulation|
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Variable anesthetic effect is reported in the literature for TBE and comparisons may be confounded by the variety of doses used, animal characteristics (age, sex and species) and method of assessment of anesthesia. For example, a gender difference is reported for TBE with female mice having longer durations of anesthesia.  The current experiments were performed to reduce sources of variability by using an inbred strain of mice, one gender (females) of similar age and weight and performing the experiments at the same time of day to reduce variation due to circadian rhythm. It is also noted in the present study that there was considerable individual variation in response to the anesthetic, with the group of mice that received the cyclodextrin formulation showing less variation [Figure 2]. The observed differences in response following the injection of TBE could be in part due to the variable placement of the IP injection within the peritoneum. Injection of the formulations adjacent to gut vasculature may lead to a more rapid and higher absorption, as diffusional distances for TBE are less. Such variability in absorption of substances from the peritoneal cavity is known to vary between subjects, as has been seen with the peritoneal absorption of insulin in humans.  There has also been criticism of the use of the IP route as it is easy to cause damage to the intestine and visceral organs during administration of the injection. The use of a cyclodextrin formulation instead of one that includes an organic solvent may make it feasible for administration to be performed subcutaneously rather than the IP route; however the suitability of the subcutaneous site for TBE remains to be investigated.
Some researchers report high rates of post-anesthesia mortality, peritonitis, intestinal ileus and abdominal adhesions,  whereas others have used TBE extensively with few adverse effects.  The mice in the current study were monitored daily for 14 days after administration of the anesthetic for clinical signs of illness, i.e. weight loss, reduction in water intake, abnormal behavior. At day 14, the mice were euthanized by cervical dislocation and a necropsy was undertaken. No evidence of inflammation or gross lesions was identified in the abdominal cavity of the animals. Tissue samples were taken from the spleen, kidney, bowel and liver for histological examination. There was no evidence of inflammation or fibrosis found in any of the tissues examined and there was no difference between the two TBE formulations investigated [Figure 3]. Norris and Turner  investigated the use of TBE in Mongolian gerbils at varying concentrations (1.25-2.25%) and doses (225-450 mg/kg) and found that mortality rate increased with the use of the 2.25% solution and at higher doses for the 1.25% solution. The use of the lower concentration (1.25%) solution and the relatively low dose rate in the present study may be a contributing factor why no inflammation was observed in the tissues. In addition, the absence of pathological findings may be because of the period of time between the animals receiving the formulation and the euthanasia. The post mortem conducted 14 days after anesthetic administration may have provided time for any inflammation to resolved,  however Thompson et al.  investigated histopathological changes to the liver, spleen, heart and kidney 3 and 6 h after the onset of anesthesia following an IP injection of TBE in mice (dose 160 mg/kg) and also report no detectable pathologic changes in the tissue.
|Figure 3: Photomicrographs of bowel section from mice administered (a) standard tribromoethanol formulation and (b) cyclodextrin tribromoethanol formulation. m = Mucosa, vi = Villus and goblet cells (arrow). Sections of liver parenchyma 14 days after administration of (c) standard tribromoethanol formulation and (d) cyclodextrin tribromoethanol formulation. V = Vein, hepatocyte nuclei are stained a blue/black colour (arrow) and red blood cells visible in the sinusoids|
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| Conclusion|| |
TBE could be formulated as a 1.25% w/v solution 0.035 M HP-β-CD in a single step without the need for an organic solvent. This study has also identified the value in using atropine in addition to TBE for anesthesia. The reduced anesthetic effect of the cyclodextrin formulation compared to the standard formulation may have been due to TBE complexation with the cyclodextrin complex. No evidence of peritonitis or abdominal adhesions were observed with either formulation, suggesting that freshly prepared TBE solutions (standard or HP-β-CD) at a concentration of 1.25% were not significantly irritant.
| Acknowledgments|| |
We are grateful to Dr. John Schofield (Director of Animal Welfare, University of Otago) and Dr Gail Williams (Department of Pathology, University of Otago) for performing the autopsies and pathology of the mouse tissue, respectively. We thank Brian Niven (Department of Maths and Statistics, University of Otago) for advice on statistical analysis and Natalie Baker (Department of Microbiology and Immunology) for information on the CRS pain scale in mice.
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[Figure 1], [Figure 2], [Figure 3]
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