Original Article

A cyclodextrin formulation to improve use of the anesthetic tribromoethanol (Avertin®)

Arlene McDowell, Jessica A. Fothergill, Azeem Khan1, Natalie J. Medlicott

New Zealand’s National ABSTRACT School of Pharmacy, Objective: Efficacy and safety concerns have been raised in the literature with the use of University of Otago, tribromoethanol (TBE) (Avertin®) for anesthesia in rats and mice when administered by intraperitoneal (IP) Dunedin, New Zealand, 1Deparment of Pharmacy injection. Despite the controversy, it remains in common usage as an anesthetic agent in laboratory rodents for and Pharmacology, short‑term surgical procedures. Cyclodextrins have been shown to improve drug solubility and were investigated University of Bath, here as an improved anesthetic formulation for mice. Materials and Methods: The phase solubility of TBE with Claverton Down, Bath, hydroxypropyl‑b‑cyclodextrin (HP‑β‑CD) was estimated. The efficacy of two anesthetic regimens was compared United Kingdom 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 Address for correspondence: parameters of time to induction of anesthesia, duration of anesthesia and recovery time were measured using Dr. Arlene McDowell, E‑mail: arlene.mcdowell@ a combined reflex score (CRS). Results and Discussion: Phase solubility studies showed a linear increase otago.ac.nz 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. Received : 20-11-13 Review completed : 20-11-13 Accepted : 20-11-13 KEY WORDS: Anesthetic, cyclodextrin, mice, solubility, tribromoethanol

ribromoethanol (TBE) is an injectable anesthetic mice due to the modest cardiodepressive effects compared to T used in biomedical research for short‑term surgical other injectable anesthetics.[3] TBE is generally administered procedures (5‑30 min) involving laboratory rodents. TBE by the intraperitoneal (IP) route and is reported to produce provides anesthetic effect through its action as a general good surgical anesthesia and muscle relaxation with rapid central nervous system depressant and is metabolized in the induction and recovery within 80 min.[4] The recommended liver by conjugation with glucuronic acid prior to excretion dose for surgical anesthesia in mice is 240 mg/kg IP,[2] although in the urine as TBE glucuronate.[1] It is commonly used in the range used by researchers is between 125 mg/kg and embryo transfer procedures to produce transgenic mice and has 500 mg/kg,[5] TBE has been marketed under the trade names minimal effects on embryonic development and reproductive Avertin, Bromethol, Ethobrom, Narcolan and Narkolan (a 66.7% performance[2] and in studies using echocardiography in solution of pharmaceutical‑grade TBE in tert‑amyl alcohol), however these products are no longer manufactured,[5] so Access this article online researchers must now formulate their own solution for use from Quick Response Code: non‑pharmaceutical‑grade TBE. Adverse effects reported with Website: the use of the extemporaneous preparation of TBE include www.jpbsonline.org intestinal ileus and fibrous abdominal adhesions as well as increased morbidity, particularly if given subsequent doses.[6‑8] DOI: TBE undergoes photodegradation to dibromoacetaldehyde 10.4103/0975-7406.124303 and hydrobromic acid,[9] both irritants and some authors have attributed adverse effects to these degradation products.

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.

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However, a study carried out by Lieggi et al.[10] found no of Otago, by dissolving 0.5 g TBE in 0.25 g tert‑amyl alcohol then correlation between dibromoacetaldehyde concentration in diluting to 40 mL with 0.9% NaCl. Dilution of the tert‑amyl the injection preparations and occurrence of adverse effects. alcohol solution produced a precipitate, which redissolved with The current formulation practice, which involves heating solid stirring for 30‑60 min on a magnetic stirrer. The final solution was TBE in an organic co‑solvent (tert‑amyl alcohol), may be less filtered (0.2 µm) into an amber glass bottle, protected from light than optimal with respect to drug solubility and stability and by covering the vial with foil and stored at 4°C. our research will address these limitations by investigating a new approach that may improve the utility of this anesthetic agent. Cyclodextrin formulation

Cyclodextrins are cyclic oligosaccharides that possess a The water content of HP‑β‑CD samples (0.05 g, n = 5) was hydrophobic core and hydrophilic exterior and are used as determined by Karl Fischer titration (736 GP Titrino, Metrohm, water‑soluble drug carriers for hydrophobic injectable drugs.[11] USA). CombiTitrant®5 was used as a titrant, and methanol as On mixing with suitably sized drugs in an aqueous solution, a solvent. The mixture was calibrated with 10 μL of distilled inclusion complexes are formed where hydrophilic water water until a consistent value was obtained. The water content molecules drive inclusion of hydrophobic drug molecules within of the HP‑β‑CD solid was calculated to be 8.4% w/w. TBE has no the cyclodextrin cavity. A dynamic equilibrium between the appreciable ultra violet absorbance and so direct measurement free drug, free cyclodextrin and drug: Cyclodextrin complex is of the binding constant for TBE and HP‑β‑CD could not established in solution.[12] These reversible inclusion complexes be performed. An approximate phase solubility diagram was improve the drug’s aqueous solubility, chemical stability and constructed by addition of small aliquots (5‑30 mg) of TBE to potentially reduces irritancy at the injection site.[13,14] Medlicott HP‑β‑CD solutions (0‑0.035 M) until no further TBE would et al.[15] demonstrated that encapsulation of melphalan with a dissolve. Solutions were protected from light to reduce the risk hydroxypropyl-β-cyclodextrin (HP‑β‑CD) formulation resulted of chemical degradation during the observation period and in less damage to venous endothelial cells in vitro compared with stored at 20°C. Temperature was monitored using a Hobo data co‑solvents that are typically used to improve solubility. Of the logger (Onset Computer Corp., Bourne, MA, USA). cyclodextrins available HP‑β‑CD is popular because it has high solubility and low toxicity when administered parenterally[16] Cyclodextrin formulations were prepared aseptically no more than and has been used previously to increase the aqueous solubility 2 days prior to being used in the animal study. The cyclodextrin of anesthetic agents including etomidate,[17] alfaxolone[18] and TBE formulation was prepared by dissolving 0.25 g TBE with propoanidid.[14] 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 The aims of this study were to investigate an alternative mixture was dispersed using a magnetic stirrer (~1 h) and once formulation of TBE using HP‑β‑CD to improve solubility and fully dissolved, the solution was filtered (0.2 µm) and stored at stability of TBE, and to determine the anesthetic response to 4°C and protected from light until administered. this new formulation following IP injection in mice. Administration of formulations and assessment of Materials and Methods anaesthesia

Chemicals The procedures described had prior approval from the University of Otago Animal Ethics Committee, Dunedin and conformed

TBE (2,2,2- tribomoethanol 97%, C2H3Br3O, MW 282.8), to the University of Otago Code of Ethical Conduct for the tert‑amyl alcohol (99%) and methyl orange were purchased Manipulation of Animals (1987) and the Animal Welfare from Sigma Aldrich, (St. Louis, MO, USA). NaCl was purchased Act (1999) in New Zealand. from BDH Chemicals (Palmerston North, New Zealand). HP‑β‑CD (Kleptose® HPB, MW 1440) was obtained from a Specific pathogen free, adult, female Balb/c mice of the same gift from Roquette Lestrem, France. CombiTitrant® 5 and approximate weight (19‑21 g) were maintained in group housing methanol (99.8%) were purchased from Merck (Auckland, and acclimatized to the environment for at least 7 days prior to New Zealand). Atropine was obtained as Phoenix Atropine being used in the study. Animals were offered food and water injection from Provet NZ Ltd (Christchurch, New Zealand). ad libitum, and both were not withheld prior to, or following, Hematoxylin and eosin dyes for histological examination were anesthesia. Mice were randomly assigned to receive either obtained from Surgipath Medical (Illinois, USA). the standard or cyclodextrin TBE anesthesia. Animals were removed from group housing, weighed and placed in individual TBE anesthetic solution cages on heating pads to maintain body temperature. Atropine (0.05 mg/kg) was administered subcutaneously 30 min prior to TBE is reported to have a solubility of 1 in 40 parts water at 40°C, administration of the anesthetic formulations. The cyclodextrin soluble in alcohol, ether, bezene and chloroform and very soluble TBE formulation and the standard TBE solution were compared in tert‑amyl alcohol[5] and is supplied as a white crystalline powder. using the dose regimen of 260 mg/kg TBE administered through A 1.25% w/v TBE anesthetic solution was produced following the the IP route (n = 6/group). Parameters observed in the animals method recommended by the Animal Welfare Office, University were depth of anesthesia, time to righting reflex loss and gain.

Journal of Pharmacy and Bioallied Sciences January-March 2014 Vol 6 Issue 1 17  McDowell, et al.: A cyclodextrin formulation for Avertin

The anesthetic efficacy of each formulation was assessed in A notable difference between the preparations of the the mice by monitoring several reflex responses. The time after formulations was that TBE could be readily dissolved directly in administration of the anesthetic to loss of righting reflex, the the cyclodextrin solution prior to use, whereas for the standard animal’s reflex to roll onto its front when placed on its back, formulation TBE required dissolution in an organic solvent was recorded. The intensity of the tail pinch reflex was assessed followed by dilution with normal saline. When the organic after pinching the tail of the mouse with plastic forceps. The solution was diluted, TBE precipitation was observed and time intensity of the pedal withdrawal reflex was assessed when the was required to allow this to re‑dissolve prior to use. interdigital skin on the fore and hind feet were pinched with plastic forceps. Tail and pedal withdrawal responses were scored The use of cyclodextrin formulations can reduce precipitation on a scale from 0 to 3 (0 = no response through to 3 = strong in vivo as discussed by Brewster and Loftsson.[11] The relationship response as in a conscious animal). The reflex scores for each between the solubility of a drug that forms a complexation time point were summed to give a combined reflex score (CRS) with a cyclodextrin is commonly linear. Consequently, upon of 0‑9 (0 = surgical anesthesia, 9 = fully responsive) for each administration, dilution of the drug‑cyclodextrin complex animal. causing dissociation should also occur in a linear fashion, and without precipitation. This is in contrast to preparations made Animals were observed for 2 h following recovery and then using organic solvents where the solubilization of the drug daily for 14 days. 14 days following administration of the is based on a non‑linear relationship and so upon dilution anesthetic formulations, a necropsy was performed and tissue following in vivo administration, the ability of organic solvents to samples of the liver and gut were collected for histological solubilize the drug is rapidly reduced leading to precipitation.[11] examination. The tissue samples were fixed with 10% formalin, embedded in paraffin and sectioned with a Leica RM2025 Owing to the disparity in doses reported in the literature rotary microtome (Nussloch, Germany) to 3 μm thick sections. (125‑500 mg/kg), a preliminary study was undertaken to Tissue sections were stained with hematoxylin and eosin for determine an appropriate dose of TBE to achieve anesthesia visualization using light microscopy. in mice. Consistent with other studies, at a dose of 260 mg/kg there was good surgical anesthesia, however there was also some Statistical analysis evidence of respiratory depression (i.e. vocalizations, labored breathing). Consequently, atropine was administered 30 min Data are averaged and reported as mean ± SD, unless otherwise prior to anesthesia (0.05 mg/kg) by subcutaneous injection. stated. Statistical analysis was performed using statistical The inclusion of atropine improved the anesthesia and reduced package for the social sciences software (IBM, version 19). respiratory depression. P < 0.05 was considered to be significant. The comparison between respiratory rate with standard and cyclodextrin TBE A comparison of anesthesia between the standard TBE solution formulations was undertaken by repeated measures analysis of and the cyclodextrin TBE formulation Figure 2 shows the variance model. The time to loss and gain, of righting reflex trend for the standard formulation to produce a deeper level was compared between the two treatment groups using an of anesthesia, although the difference was not statistically independent samples t‑test. significant. The lowest CRS attained was 0 for the standard formulation and 2 for the cyclodextrin formulation [Figure 2]. Results and Discussion  The solubility of TBE in water is reported to be 1 in 40 (2.5% w/v)  at 40°C.[19] 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 AL 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.[11] Since TBE is a small molecule it is possible  that 2:1 or greater molar ratio TBE: HP‑β‑CD complex was

(VWLPDWHG7%(VROXELOLW\ 0  formed. Using Equation 1, a 2:1 binding constant of 380 M−2 was estimated.        slope +3ȕ&'FRQF 0 K = (1) 2:1 s(2 2-slope) 0 Figure 1: Phase‑solubility profile of tribromoethanol as a function of hydroxypropyl‑β‑cyclodextrin concentration at 20°C. Data Where S0 = solubility (M) of TBE in the absence of HP‑β‑CD points represent mean of duplicates. Least squares linear and the slope is from the TBE solubility versus HP‑β‑CD regression produced a slope = 1.608 (Standard error = 0.065); concentration graph [Figure 1]. intercept = 0.1037 (SE = 0.0037); R2 = 0.993

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Short and variable anesthetic times with TBE (dose 300 mg/kg) righting reflex has reported to be between 0 min and 120 min, have been reported by Avila et al.[20] and these authors also with a mean of 21.5 min.[23] comment that the duration of anesthesia was sometimes too short to conduct their intended measurements. The Variable anesthetic effect is reported in the literature for TBE effectiveness of the standard TBE formulation compared to and comparisons may be confounded by the variety of doses the cyclodextrin formulation may be attributed to the presence used, animal characteristics (age, sex and species) and method of organic solvent (concentration = 0.625% in the injected of assessment of anesthesia. For example, a gender difference solution), which is known to cause central nervous system is reported for TBE with female mice having longer durations depression and ataxia on its own,[21] and which is not present of anesthesia.[23] The current experiments were performed to in the cyclodextrin TBE formulation. Complexation of the reduce sources of variability by using an inbred strain of mice, TBE with the cyclodextrin would also result in reduced free one gender (females) of similar age and weight and performing drug concentrations compared to the standard formulation. the experiments at the same time of day to reduce variation due It may be that rather than a comparison of the same dose to circadian rhythm. It is also noted in the present study that (260 mg/kg) between the two formulations, a higher dose of there was considerable individual variation in response to the the cyclodextrin formulation may be required to have the same anesthetic, with the group of mice that received the cyclodextrin effect as the standard formulation. Binding of TBE by HP‑β‑CD formulation showing less variation [Figure 2]. The observed supports the observation of reduced pharmacodynamic effect differences in response following the injection of TBE could be in the present study. However, Stella and Rajewski reported that in part due to the variable placement of the IP injection within release of drugs from cyclodextrin complexes occurs very rapidly the peritoneum. Injection of the formulations adjacent to gut once administered to the body.[22] Recovery from the TBE vasculature may lead to a more rapid and higher absorption, anesthetic was more rapid for the cyclodextrin group as all mice as diffusional distances for TBE are less. Such variability in had regained a CRS of 9 by 30 min following administration absorption of substances from the peritoneal cavity is known compared to up to 1 h for the standard TBE formulation. to vary between subjects, as has been seen with the peritoneal The time to loss and gain of righting reflex reflects the level absorption of insulin in humans.[24] There has also been criticism of anesthesia reached with the two formulations. Results of the use of the IP route as it is easy to cause damage to the from observation of the righting reflex [Table 1] show that intestine and visceral organs during administration of the this reflex is lost approximately 30 s faster with the standard injection. The use of a cyclodextrin formulation instead of formulation (P = 0.006) and it appears to take longer to return, one that includes an organic solvent may make it feasible for although this result was not significant. At a relatively high TBE administration to be performed subcutaneously rather than the dose of 400 mg/kg, the time interval between loss and regain of IP route; however the suitability of the subcutaneous site for TBE remains to be investigated. Table 1: Comparison of times to loss and gain of righting reflex between the standard tribromoethanol formulation and Some researchers report high rates of post‑anesthesia mortality, hydroxypropyl‑β‑cyclodextrin tribromoethanol formulation peritonitis, intestinal ileus and abdominal adhesions,[7] whereas Time to (min±SD) Standard Cyclodextrin Significance others have used TBE extensively with few adverse effects.[4] formulation formulation The mice in the current study were monitored daily for 14 days Loss of righting reflex 1.01±0.12 1.52±0.28 P=0.006 after administration of the anesthetic for clinical signs of illness, Regain righting reflex 34.2±12.8 22.5±9.5 P=0.104 i.e. weight loss, reduction in water intake, abnormal behavior. SD: Standard deviation At day 14, the mice were euthanized by cervical dislocation

 

 

 

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      7LPH PLQ 7LPH PLQ a b 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

Journal of Pharmacy and Bioallied Sciences January-March 2014 Vol 6 Issue 1 19  McDowell, et al.: A cyclodextrin formulation for Avertin and a necropsy was undertaken. No evidence of inflammation either formulation, suggesting that freshly prepared TBE or gross lesions was identified in the abdominal cavity of the solutions (standard or HP‑β‑CD) at a concentration of 1.25% animals. Tissue samples were taken from the spleen, kidney, were not significantly irritant. bowel and liver for histological examination. There was no evidence of inflammation or fibrosis found in any of the tissues Acknowledgments examined and there was no difference between the two TBE formulations investigated [Figure 3]. Norris and Turner[25] We are grateful to Dr. John Schofield (Director of Animal Welfare, investigated the use of TBE in Mongolian gerbils at varying University of Otago) and Dr Gail Williams (Department of Pathology, concentrations (1.25‑2.25%) and doses (225‑450 mg/kg) and University of Otago) for performing the autopsies and pathology of found that mortality rate increased with the use of the 2.25% the mouse tissue, respectively. We thank Brian Niven (Department solution and at higher doses for the 1.25% solution. The use of Maths and Statistics, University of Otago) for advice on statistical analysis and Natalie Baker (Department of Microbiology and of the lower concentration (1.25%) solution and the relatively Immunology) for information on the CRS pain scale in mice. low dose rate in the present study may be a contributing factor why no inflammation was observed in the tissues. 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Frijlink HW, Franssen EJ, Eissens AC, Oosting R, Lerk CF, Meijer DK. a b The effects of cyclodextrins on the disposition of intravenously injected drugs in the rat. Pharm Res 1991;8:380‑4. 14. MacKenzie CR, Fawcett JP, Boulton DW, Tucker IG. Formulation and evaluation of a propanidid hydroxypropyl‑beta‑cyclodextrin solution for intravenous anesthesia. Int J Pharm 1997;159:191‑6. 15. Medlicott NJ, Foster KA, Audus KL, Gupta S, Stella VJ. Comparison of the effects of potential parenteral vehicles for poorly water soluble anticancer drugs (organic cosolvents and cyclodextrin solutions) on cultured endothelial cells (HUV‑EC). J Pharm Sci 1998;87:1138‑43. 16. Brewster M, Estes K, Bodor N. An intravenous toxicity study of c d 2‑hydroxyproply‑β‑cyclodextrin, a useful drug solubilizer, in rats and Figure 3: Photomicrographs of bowel section from mice monkeys. Int J Pharm 1990;59:231‑43. administered (a) standard tribromoethanol formulation and 17. Doenicke A, Roizen MF, Nebauer AE, Kugler A, Hoernecke R, Beger‑Hintzen H. A comparison of two formulations for etomidate, (b) cyclodextrin tribromoethanol formulation. m = Mucosa, vi = Villus 2‑hydroxypropyl‑beta‑cyclodextrin (HPCD) and propylene glycol. and goblet cells (arrow). Sections of liver parenchyma 14 days Anesth Analg 1994;79:933‑9. after administration of (c) standard tribromoethanol formulation and 18. Brewster ME, Estes KS, Bodor N. Development of a non‑surfactant (d) cyclodextrin tribromoethanol formulation. V = Vein, hepatocyte formulation for alfaxalone through the use of chemically‑modified nuclei are stained a blue/black colour (arrow) and red blood cells cyclodextrins. J Parenter Sci Technol 1989;43:262‑5. visible in the sinusoids 19. The Merck Index: An Encyclopeadia of Chemicals,

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Drugs and Biologicals. 14th ed.. Whitehouse Station, NJ: Merck the hypnotic drugs tribromoethanol, urethane, pentobarbital, and and Co.; 2006. propofol within outbred ICR mice. Exp Anim 2002;51:119‑24. 20. Avila MY, Carré DA, Stone RA, Civan MM. Reliable measurement 24. Fine A, Parry D, Ariano R, Dent W. Marked variation in peritoneal of mouse intraocular pressure by a servo‑null micropipette system. insulin absorption in peritoneal dialysis. Perit Dial Int 2000;20:652‑5. Invest Ophthalmol Vis Sci 2001;42:1841‑6. 25. Norris ML, Turner WD. An evaluation of tribromoethanol (TBE) as an 21. Lindamood C 3rd, Farnell DR, Giles HD, Prejean JD, Collins JJ, anaesthetic agent in the Mongolian gerbil (Meriones unguiculatus). Takahashi K, et al. Subchronic toxicity studies of t‑butyl alcohol in Lab Anim 1983;17:324‑9. rats and mice. Fundam Appl Toxicol 1992;19:91‑100. 22. Stella VJ, Rajewski RA. Cyclodextrins: Their future in drug formulation and delivery. Pharm Res 1997;14:556-7. Source of Support: Nil, Conflict of Interest: None declared. 23. Koizumi T, Maeda H, Hioki K. Sleep‑time variation for ethanol and

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