A Water-Soluble Intravenous Anesthetic Preparation in Sulfobutyl

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and the other 2 ) = 27.7 ALTH was withdrawn ALTH 2 50 Both reviews agreed that agreed Both reviews 3 = 43.6 mg/ 50 -Cyclodextrin This prompted a drug dis- a drug This prompted Ken B. Johnson Editor: Ken B. Section 1 β www.anesthesia-analgesia.org ≈ 20). Gyermek and Soyka suggested alphaxa-

50 (Anesth Analg 2015;120:1025–31) he sedating and anesthetic properties of steroid com- of steroid properties and anesthetic he sedating reported in 1942 by Hans Selye in first pounds were Anesthesia & Analgesia. ÷ ED

50 The use of Cremophor EL in veterinary products was also in veterinary products EL The use of Cremophor ALTH was characterized by many of the features of an ideal was characterized by many of the features ALTH of action; no irritat- IV anesthetic: rapid onset and offset and on blood vessels; and minor cardiovascular ing effects (TI) index therapeutic wide a with depression respiratory (LD - the market in 1984 because of hypersensitivity reac from excipient. EL tions caused by the Cremophor this address To species. canine in particularly problematic, a new veterinary formulation of alphaxalone was problem, lone should be reformulated in a water base to avoid the lone should be reformulated ALTH. excipient of EL Cremophor published in an editorial in the British Journal of Anaesthesia in 1980 by Prys-Roberts and Sear. covery program that led to the IV anesthetic formulation of covery program This was used in clini- Althesin (ALTH). alphaxalone called 1972 to 1984 in many countries. cal anesthetic practice from one by ALTH, of summarized the properties reviews Two Gyermek and Soyka in Anesthesiology in 1975, T Markus W. Hollmann PharmacologyPreclinical W. Markus Editor: Section Anesthetic Clinical Pharmacology Clinical Anesthetic International Society for AnaestheticInternational Pharmacology for Society

) values for loss of righting reflex being 2.8, 3.0, and 4.6 mg/kg, respec- and 4.6 mg/kg, 3.0, 2.8, ) values for loss of righting reflex being 50 †Drawbridge Pharmaceuticals Pty Ltd, Drug Delivery, Disposition and Dynamics, Dynamics, and Disposition Delivery, ‡Drug Number 5 Number • mg/kg). A dose of alphaxalone 53 mg/kg as ALTH caused 10 of 10 rats to die (LD as ALTH mg/kg). A dose of alphaxalone 53 mg/kg Alphaxalone is a neuroactive steroid anesthetic that is poorly water soluble. steroid anesthetic that is poorly Alphaxalone is a neuroactive water BACKGROUND: additive. surfactant nonionic a EL, Cremophor® using formulated Althesin® as 1972 in was It versatilea short-acting was product The coun- many in practice clinical in used anesthetic IV because of hypersensitivity from clinical practice withdrawn 1984. It was tries from 1972 to compared the properties reported we the investigations to Cremophor EL. In - of 3 anes here, β-cyclodextrin solution of alphaxalone dissolved in 7-sulfobutyl-ether- aqueous thetics: a new enables drug that solubilization in with a lipophilic cavity molecule a water-soluble (SBECD, of alphaxalone; and propofol. a Cremophor EL preparation water); solu- prepared: one using 13% w/v (10 mg/mL) were solutions of alphaxalone METHODS: Two and the other a solution of alphaxalone prepared as tion of SBECD in 0.9% saline (PHAX) mg/mL; (10 propofol of solution A (ALTH). EL Cremophor 20% using literature the in described - used as a comparator anesthetic. Jugular IV cath bean oil emulsion was in 10% v/v soya PROP) Separate anesthesia. under halothane g) (180–220 male Wistar rats implanted in eters were from 1.2 or PROP ALTH, injections of PHAX, given IV groups of 10 implanted rats each were mg/kg to lethal doses. Doses of each drug that caused anesthesia (loss of righting reflex and probit analysis. The drugs calculated by of rats were response to tail pinch) and lethality in 50% also compared for effects on arterialwere blood pressure and heart rate. with 50% sedation and anesthesia, caused dose-related and PROP ALTH, IV PHAX, RESULTS: effective dose (ED kg), whereas none died when given the same doses of alphaxalone formulated in SBECD. PHAX when given the same doses of alphaxalone formulated whereas none died in SBECD. kg), tested of 84 mg/kg.- caused 20% lethality at the maximal dose PHAX caused less cardiovascu the 3 drug-free no effects. Control experiments with vehicles showed lar depression than PROP. for both formula same dose at the anesthesia caused fast-onset Alphaxalone CONCLUSIONS: - SBECD as a drug-solubilizing The use of excipient did not alter the anes- tions (PHAX and ALTH). PHAX in alphaxalone of index therapeutic the increase did it but alphaxalone, of effect thetic higher safety margin than the propofol lipid formulation PHAX has a and compared with ALTH. also the alphaxalone formulation in Cremophor EL (ALTH). tively. PROP led to death in 10 of 10 rats at doses >30 mg/kg (50% lethal dose (LD led to death in 10 of 10 rats at doses >30 mg/kg (50% PROP tively. Volume 120 120 Volume •

DOI: 10.1213/ANE.0000000000000559 2015 May Copyright © 2014 International Anesthesia Research Society Anesthesia Research Copyright © 2014 International Monash Institute of Medical Research, Monash University, Monash University, *Monash Institute of Medical Research, the From Australia; Melbourne, Victoria, Malvern, Victoria, Australia; and Australia; Victoria, Malvern, (Parkville University Monash Sciences, Pharmaceutical of Institute Monash Australia. Campus), Parkville, Victoria, is FFPMANZCA, FANZCA, FRCA, PhD, BChir, MB MA, Goodchild, S. Colin with the Drawbridge Pharmaceuticals Pty Ltd, Malvern, affiliated currently Australia. Victoria, with the GlaxoSmithKline affiliated MSc, PhD, is currently Anton Kolosov, Singapore. Pte Ltd, Gateway West, Accepted for publication October 7, 2014. Monash from by an internal grant Funding: This work was funded entirely University. at the end of the article. See Disclosures Conflict of Interest: Anaes of Congress in part at the 15th World was presented This report Annual Meeting 2012; and the March Argentina, Aires, thesiologists, Buenos Boston, MA, May 2012. Society, Anesthesia Research of the International Drawbridge Pharmaceuticals Pty Ltd is the startup company set up by 2 of the inventors (C.S. Goodchild and J.M. Serrao) and that has subsumed the intellec- of Phaxan. and the task of development and commercialization tual property the authors. Reprints will not be available from FRCA, PhD, BChir, MB, MA, Goodchild, S. Colin to correspondence Address FFPMANZCA, Drawbridge Pharmaceuticals Pty Ltd, 23 Milton FANZCA, e-mail to colin@draw- Address Australia 3144. Parade, Malvern, Victoria, bridgepharmaceuticals.com.au. * ‡ * † Anesthetic Preparation in Sulfobutyl-Ether- Preparation Anesthetic FFPMANZCA, FANZCA, FRCA, PhD, BChir, MB, MA, Colin S. Goodchild, PhD and Ben J. Boyd, PhD, MSc, FRCA, Anton Kolosov, PhD, BS, MB, Juliet M. Serrao, Alphaxalone Reformulated: A Water-Soluble Intravenous Intravenous Reformulated:Alphaxalone A Water-Soluble

Downloaded from https://journals.lww.com/anesthesia-analgesia by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD3bhnalqTQiPv0Qe2oObD6Borpr8ynxk/m7SI60vZRsDj7xUAQhxAE7Q== on 07/29/2020 Downloaded from https://journals.lww.com/anesthesia-analgesia by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD3bhnalqTQiPv0Qe2oObD6Borpr8ynxk/m7SI60vZRsDj7xUAQhxAE7Q== on 07/29/2020 Water-Soluble Formulation of Alphaxalone developed using hydoxypropyl-β-cyclodextrin (HPBCD) as 1. The presence of a normal righting reflex, scored as the drug-solubilizing excipient. This is marketed as Alfaxan present or absent, CD-RTU (ALF).4–6 Dissolution of alphaxalone in an aque- 2. Any response to tail pinch by a 25-cm artery clamp ous vehicle suitable for human use has proved to be dif- applied to the base of the tail to a maximal force of ficult. However, 7-sulfobutyl-ether-β-cyclodextrin (SBECD) touching the first ratchet, scored present or absent, and has been used to dissolve hydrophobic drugs in water for 3. Time (seconds) that the rat was able to walk on a IV injections suitable for human use,7–11 but has not been rotarod (a rotating cylinder; Ugo Basile Srl, Comerio, investigated previously as an excipient to enable a human Italy): the normal run time is 120 seconds in nonse- product of alphaxalone free from Cremophor EL. dated rats. Time from drug injection to attaining this The studies reported here compare the anesthetic prop- value was a measure of time taken to recover from the erties and TIs of 3 anesthetic preparations: propofol in the sedating effects of the anesthetics. form of an injectable soya bean oil emulsion, and 2 preparations of alphaxalone, one using Cremophor EL and the Cardiovascular Effects other using SBECD as excipients. Male Wistar rats (180–220 g weight) were implanted with internal jugular vein catheters as above. Twenty-four hours later, 3 groups of 5 rats each received IV injections of PROP METHODS (6.6 mg/kg), ALTH (3.3 mg/kg), or PHAX (3.3 mg/kg) being This research was approved by Monash Medical Centre Animal the ED dose of each drug for loss of righting reflex shown in Ethics Committee B (approval number MMC-B 2010/04). The 95 Figure 1. Systolic and diastolic blood pressure and heart rate present study was in compliance with the guidelines and rec- were measured using a noninvasive tail cuff blood pressure ommendations from the Code of Practice for the Use of Animals recorder with piezo-ceramic pulse detection (BP Recorders From Municipal Pounds in Scientific Procedures established by series 58000; Ugo Basile Srl). These variables were measured the Bureau of Animal Welfare of Victoria, Australia. All rats every 5 minutes for 15 minutes before and at 0.5, 1, 2, 3, 4, 5, were housed in plastic boxes at room temperature with food 7.5, 10, 15, and 20 minutes after the IV drug treatments. and water available ad libitum. A 12:12 light/dark cycle with lights on at 8:00 am was maintained, and the tests were per- Lethal Dose formed between 9:00 am and 7:00 pm. Male Wistar rats (180–220 g weight) were implanted with internal jugular vein catheters as above. Twenty-four hours Drug Preparations later, groups of 10 rats received an IV injection from a range The propofol used in these studies was Diprivan® (propofol 10 mg/mL in 10% soya bean oil emulsion; AstraZeneca, North A Ryde, NSW, Australia). Alphaxalone and alphadolone were purchased from Steraloids Inc., Newport, RI. SBECD was obtained as Captisol® from Ligand Pharmaceuticals (formerly Cydex Pharmaceuticals), Shawnee Mission, KS. Alphaxalone was prepared as ALTH as described previously: alphaxalone 9 mg/mL, plus alphadolone 3 mg/mL in 20% w/v Cremophor EL.12 Because alphadolone is reported to have the same anesthetic properties as alphaxalone but with 30% to 50% of the potency,12 for comparison with the other drugs, ALTH will be B considered alphaxalone 10 mg/mL. The third anesthetic preparation, PHAX, was also alphaxalone 10 mg/mL, prepared as follows: alphaxalone 300 mg (0.9 mmol), SBECD 3889 mg (1.8 mmol) dissolved in 30 mL 0.9% sodium chloride solution. The method used was first dissolution of the SBECD in the final volume of saline by stirring and then adding the alphaxalone while continuing to stir. No heating or ultrasonication was nec- essary to dissolve the alphaxalone in this solution. C

Rat Experiments Anesthesia Male Wistar rats (180–220 g weight; Monash University Animal Services) were implanted with internal jugular IV catheters under halothane anesthesia. The catheters were tunneled under the skin and brought out to the dorsum Figure 1. Probit plots (A, PROP; B, ALTH; C, PHAX) for loss of righting of the neck, where they were additionally anchored with reflex and response to tail pinch. These plots were used to calculate nylon sutures. Twenty-four hours later, each rat was placed the ED50 values to compare potencies of the anesthetic preparations briefly in a Plexiglass restrainer to facilitate an IV injec- shown in Table 1. Data for loss of righting reflex are shown as blue symbols (squares) and the probit regression by blue lines (solid for tion over 15 seconds of a range of doses of PROP, ALTH, mean and dotted for ±95% confidence interval [CI]). Data for loss of or PHAX (1.2–15 mg/kg). The following were assessed at tail pinch response are shown as red symbols (circles) and the probit regular time intervals after the IV injection: regression by red lines (solid for mean and dotted for ± 95% CI).

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of doses of PHAX, ALTH, or PROP, with each member of with Tukey post hoc test (GraphPad Prism ver. 6.05, GraphPad a group of 10 receiving the same formulation and dose of Software, Inc., La Jolla, CA). drug. The dose of anesthetic drug injected was increased in The time from induction to full recovery to a normal subsequent groups of 10 until all rats in the group died. The rotarod run time (120 seconds) was noted for all the indi- number of rats that died was recorded for each group. viduals in the group of 10 rats that received the minimal anesthetic dose of each anesthetic preparation. These were Effect of SBECD on Lethality combined as means (95% CI) for each drug treatment To test whether the low toxicity of alphaxalone as PHAX and compared using ANOVA with Tukey post hoc test was attributable to the SBECD excipient, 20 rats with (GraphPad Prism ver. 6.05). indwelling jugular IV catheters were divided into 2 groups The area under the curve was calculated for systolic and of 10 rats each. They were all given IV injections of ALTH diastolic blood pressures and heart rate for each individual at a dose that had, in previous experiments reported above, rat. This was achieved by the calculation: (measurement at caused all rats to die (alphaxalone dose 52.5 mg/kg IV). time point x − measurement at time point y before x) x the Sixty seconds before the ALTH injection, a premedication time in minutes between time points x and y. All values for IV injection was given: each rat experiment from time 0 (drug injection) to 20 min- 1. Group 1 (10 rats) received 5.3 mL/kg 0.9% sodium utes later were added together to calculate a single value for chloride solution. area under the curve for that rat and that parameter. These 2. Group 2 (10 rats) received 5.3 mL/kg 13% solution of values were combined for drug treatment, plotted as box SBECD in 0.9% sodium chloride solution. This dose and whisker, and compared using Kruskal-Wallis tests with of SBECD was chosen because this was the amount of Dunn post hoc correction for multiple comparisons. SBECD administered when 52.5 mg/kg alphaxalone For the lethality experiments, the number of rats that was given as PHAX. died in each group of 10 treated with the same drug and The number of rats that died in each group of 10 after the dose was subjected to probit regression analysis using SPSS subsequent ALTH injection was recorded. Statistics 18 to produce a probit plot to calculate the esti-

mated dose that caused death in 50% of subjects (LD50). The Controls TI was then calculated for each drug: TI = LD ÷ ED . Indwelling jugular IV catheters were implanted for con- 50 50 The results from the experiments investigating the effect trol experiments in 15 rats. Five rats were given a 20% solu- on lethality of previous administration of SBECD were tion of Cremophor EL, another 5, 13% SBECD, and 5 more, entered into a contingency table and compared statistically 10% soya bean oil emulsion, with all 3 groups being given a using Fisher exact test. dose and volume equal to that administered in the experiments above at the highest doses of alphaxalone (for ALTH, PHAX) and propofol. RESULTS Anesthesia Statistics All 3 anesthetic preparations caused dose-related effects The number of rats in each group for the experiments on loss of righting reflex and tail pinch response that above was chosen before the experiments were performed, occurred within 15 seconds of drug injection. There were and no changes to those planned numbers of experiments no involuntary muscle movements observed with any drug. were made once the series of experiments was started. For Figure 1 shows the probit plots for righting reflex and tail most experiments, it was decided to use 10 rats in each pinch responses of rats treated with PROP (Fig. 1A), ALTH comparator group because this is the number most used (Fig. 1B), and PHAX (Fig. 1C). The alphaxalone formula- in experiments of this type reported in the literature using tions, PHAX (SBECD-enabled), and ALTH (Cremophor probit regression analysis to compare anesthetic potencies.13 EL-enabled) were equipotent for the dose required for loss For the experiments on cardiovascular effects of the 3 anes- of righting reflex (ED50 = 2.8, 2.2–4.3 mg/kg, and 3, 2.4–4.5 thetic preparations, n = 5 per group was chosen because this mg/kg; mean, 95% CI for PHAX and ALTH, respectively), is the number used successfully in recent studies comparing and furthermore, both were more potent than PROP for this 14 cardiovascular effects of different anesthetics in rats. end point (ED50 for PROP = 4.6, 3.8–5.0 mg/kg; mean, 95% The number of rats in each group of 10 similarly treated CI) (Table 1). animals that lost righting reflex or did not respond to tail Figure 2 shows the mean walking time on the rotarod for pinch was subjected to probit regression analysis using SPSS groups of 10 rats at each dose of the 3 anesthetic prepara- Statistics 18 (SPSS Inc., Chicago, IL) to produce graphs of tions: PROP (Fig. 2A), ALTH (Fig. 2B), and PHAX (Fig. 2C). probit versus log dose (probit plot) to calculate the estimated It can be seen from these that recovery from sedation to a dose that caused anesthesia in 50% of subjects (ED50) for normal 120-second score in this test occurred similarly for righting reflex and tail pinch. all 3 drugs at the minimal anesthetic dose (5 mg/kg of The minimal anesthetic dose was also recorded for each IV alphaxalone as ALTH and PHAX and 10 mg/kg PROP; preparation, that is, the dose of drug that led to all rats in that Table 1). group of 10 losing righting reflex. The duration of the loss of righting reflex was noted for each individual in the group of Cardiovascular Effects 10 that received the minimal anesthetic dose. These were com- Figure 3 shows the cardiovascular effects of the ED95 bined as means (95% confidence interval [CI]) for each drug dose for loss of righting reflex for each anesthetic: PROP treatment and compared using analysis of variance (ANOVA) (Fig. 3A), ALTH (Fig. 3B), and PHAX (Fig. 3C). These are

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Table 1. Doses Causing Loss of Righting Reflex and Tail Pinch Responses and Lethality Plus Recovery Rates for 2 Alphaxalone Preparations (PHAX and ALTH) and Propofol (PROP) ALTH PHAX PROP Minimal anesthetic dose causing all rats in a group of 10 to lose righting reflex, mg/kg 5 5 10

ED50 dose for loss of righting reflex, mg/kg mean (95% CI) 3.0 (2.4–4.5) 2.8 (2.2–4.3) 4.6 (3.8–5)

ED50 dose for loss of tail pinch response, mg/kg mean (95% CI) 6.5 (4.7–8.4) 6.6 (5–8.3) 8.4 (6.7–10.4)

LD50, mg/kg mean (95% CI) 43.6 (40.7–46.6) >84 27.7 (26.3–29) Duration of loss of righting reflex after minimal anesthetic dose that caused all 10 rats to 2.8 (1.8–3.8)a 2.2 (1.6–2.8)a 2.8 (2.1–3.6)a lose righting reflex, minutes mean (95% CI) Time (minutes) to complete recovery of rotarod Performance after minimal anesthetic dose 19.9 (17.1–22.7)b 17 (15.4–18.5)b 16 (14.6–17.3)b (all 10 rats lost righting reflex), mean (95% CI)

The doses of alphaxalone given as ALTH and PHAX for loss of righting reflex and tail pinch response are not statistically different, but the LD50 dose for alphaxalone as PHAX was greater than alphaxalone as ALTH. The duration of loss of righting reflex was not statistically significantly different between treatments at the minimal doses of each anesthetic that caused 10 of 10 rats to lose the righting reflex. The time to complete recovery to normal rotarod running times was statistically significantly less after PROP compared with ALTH but no different for PROP versus PHAX or ALTH versus PHAX.

CI = confidence interval; ED50 = 50% effective dose; LD50 = 50% lethal dose. aP = 0.3594 1-way analysis of variance (ANOVA); analysis of residuals revealed they were normally distributed with equal variances between treatment groups (Bartlett statistic [corrected] = 2.658; P = 0.2648). bP = 0.0123 1-way ANOVA. Tukey corrected CIs: ALTH versus PHAX, −0.2137 to 6.014; ALTH versus PROP, −7.014 to −0.7863; PHAX versus PROP, −4.114 to 2.114. Analysis of residuals revealed they were normally distributed with equal variances between treatment groups (Bartlett statistic [corrected] = 5.570; P = 0.0617).

A Lethal Dose The probit plots for lethality are shown in Figure 4. The probit plots for ALTH and PROP were used to calculate the dose of drug that caused death in 50% of the cohort (the

LD50) (Table 1). Dose escalation of PROP to doses >30 mg/ kg caused death in all 10 rats in the group. ALTH caused dose-related mortality. A dose of 52 mg/kg alphaxalone as ALTH caused death in all 10 rats in the group. However, 52 B mg/kg alphaxalone as PHAX did not cause any rats to die in that group of 10 rats. Alphaxalone as PHAX at 71, 78, and then 84 mg/kg caused 20% mortality. The dose escalation was stopped at that point because the volume of injectate

exceeded 10% blood volume. Thus, the LD50 of alphaxa-

lone as PHAX is >84 mg/kg. The TI (LD50 ÷ ED50) is 14.8 for ALTH and >30 for PHAX, both being much higher than the C TI for PROP (6.5; Table 1). Effect of SBECD on Lethality All 20 rats were anesthetized by injection of 52.5 mg/ kg alphaxalone as ALTH. The lethality results are shown in Table 3. In Group 1, which had received saline only as a premedication before ALTH injection, 8 of the 10 rats died. Figure 2. Rotarod run times. Graph showing the mean running times However, in Group 2, which received SBECD premedica- on the rotarod (A, PROP; B, ALTH; C, PHAX) for all rats in each group tion, 2 of the 10 rats died. This reduction in mortality associ- that received anesthetic drugs at doses of 1.2 to 15 mg/kg; lines show ated with SBECD premedication was statistically significant mean run times for each dose against time of testing, n = 10/ group. (P = 0.0230; Fisher exact test). The groups of rats that received the minimal anesthetic doses for loss of righting reflex recovered similarly fast (PROP = 10 mg/kg, red line and diamond marker; ALTH and PHAX both = 5 mg/kg, blue line and Controls triangle marker). Statistical analysis of this recovery timing is shown None of the 3 vehicles caused sedation, anesthesia, or in Table 1. death in any rat when given alone at the highest doses and volumes used in the experiments above. shown as box and whisker plots of median, interquartile range, total range, and individual points for the area under DISCUSSION the curve of systolic and diastolic blood pressures and heart The main conclusions from this preclinical study in rats are: rate. Analyses of these data (Kruskal-Wallis test with Dunn post hoc correction for repeated measures; GraphPad Prism • The new water-soluble preparation of alphaxalone, ver. 6.05) are shown in Table 2. There were no significant PHAX, causes anesthesia with fast onset and offset differences in heart rate among the anesthetic treatments. timing equivalent to PROP. However, the depression of systolic and diastolic blood pres- • The 2 alphaxalone solutions, PHAX and ALTH, are sures was significantly greater after PROP compared with equipotent with the same ED50 values for loss of right- PHAX (Table 2). The effects on systolic and diastolic blood ing reflex and for the dose needed to abolish the tail pressures were the same for both alphaxalone preparations. pinch response.

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A systolic blood pressure Table 2. Results of Statistical Analysis of 0 Cardiovascular Measurements After ED95 Doses of PHAX, ALTH, and PROP )

in -100 Dunn multiple comparisons

.m Kruskal-Wallis test test adjusted P g PROP PROP ALTH

mH -200

m * Cardiovascular versus versus versus (

C parameters P ALTH PHAX PHAX -300 AU Systolic blood pressure 0.0105 0.1687 0.0175 >0.9999 Diastolic blood pressure 0.0092 0.0710 0.0267 >0.9999 -400 Heart rate 0.5824 >0.9999 0.9666 >0.9999 H X OP R LT HA P A P PROP treatment caused greater decreases in systolic and diastolic blood anesthetic drug pressures than PHAX. There were no statistically significant differences between treatments for effects on heart rate. There were no statistically significant differences between PHAX and ALTH for effects on systolic or B diastolic blood pressure diastolic blood pressure. P values shown are exact. 50

) # in m . 0 mmHg (

C -50 AU

-100 Figure 4. Probit plots for lethality. Dose escalation of anesthetic H OP AX LT H led to death of some rats in each group of 10, eventually leading to R A P P death of all 10 subjects treated with PROP and ALTH. The dose that anesthetic drug caused death in 50% of rats (LD50) is 27.7 mg/kg for PROP and 43.6 mg/kg for ALTH. Much higher doses of alphaxalone as PHAX were C heart rate needed to cause death compared with either ALTH or PROP, and the 1000 lethality reached a ceiling of death in 2 of the 10 rats per group at 84.0 mg/kg. Dose escalation beyond this was not possible because

of the large volume of IV injection; therefore, the LD50 for PHAX is n) i 500 >84 mg/kg. Data for each anesthetic are shown as individual points m . for the proportion of rats out of 10 that died at each dose (PROP as pm 0 circles; ALTH as triangles; and PHAX as squares). Probit regressions (b are shown as means (solid lines) ± 95% confidence interval; green UC

A -500 for PROP, red for ALTH, and blue for PHAX.

-1000 P H X T A RO L H Table 3. Effect of Previous Administration of P A P SBECD on the Lethality of ALTH anesthetic drug Premedication Number of rats Number of rats Figure 3. Cardiovascular effects—box and whisker plots. The car- (5.25 mL/kg IV) that died that lived diovascular effects of ED95 doses for loss of righting reflex of the Group 1, saline 8 2 3 anesthetics are shown: PROP (green box and circles); ALTH (red Group 2, 13% SBECD 2 8 box and triangles); and PHAX (blue box and squares). Medians are shown by horizontal lines inside the boxes that indicate the inter- P = 0.0230; Fisher exact test. * quartile range. The short horizontal lines joined to the boxes by ver- tical lines indicate total range, and the symbols indicate individual * values for area under the curve for that particular cardiovascular and that it causes less cardiovascular depression. It is interest- parameter; n = 5 rats per treatment group. The results from all ing to note that PHAX is also less toxic than the alphaxalone 5 rats appear as point symbols on all plots with none overlaying formulation in Cremophor EL (ALTH), with TI being >30 for others. Significantly less depression of systolic blood pressure by PHAX, and 14.8 and 6.0 for ALTH and PROP, respectively PHAX compared with PROP, P = 0.0175 (see Table 2). Significantly (Table 1). None of the 3 drug-free vehicles caused sedation, less depression* of diastolic blood pressure by PHAX compared with PROP, P = 0.0267 (see Table 2). † anesthesia, or death, indicating that neither the differences between the sedating and anesthetic properties nor the safety or lethality of the formulations of alphaxalone or propofol • The 2 alphaxalone solutions, PHAX and ALTH, have were attributable to dose-related toxicity of the excipients. the same recovery times for sedation after the same In the experiments testing lethality, escalating doses of doses of alphaxalone, as revealed by the rotarod test. the drugs were given IV at the same rate (15 seconds) into • PHAX has a higher TI than PROP or ALTH. the jugular vein. No cardiovascular or respiratory support was given, so the deaths could have been a result of car- Neuroactive steroid anesthetics are characterized by high diovascular or respiratory embarrassment or a combination TIs, with that for alphaxalone commonly quoted at 20 for of both. Clinical sedating and anesthetic doses of propofol rodents.3 The experiments in rats reported here show that cause depression of arterial blood pressure and respira- PHAX is less toxic than the propofol lipid formulation PROP, tion. Furthermore, it has been reported that alphaxalone as

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ALTH or ALF, caused minimal cardiovascular effects at clin- Contribution: This author helped design the study, conduct the ical and supraclinical doses, so the wider margin of safety study, analyze the data, and write the manuscript. between therapeutic and lethal doses of PHAX compared Attestation: Colin S. Goodchild has seen the original study data, with PROP was to be expected. reviewed the analysis of the data, approved the final manuscript, Alphaxalone has been successfully formulated in and is the author responsible for archiving the study files. HPBCD as a veterinary anesthetic (ALF), and it is used Conflicts of Interest: Colin S. Goodchild has equity inter- widely in veterinary practice, being reported to have a high est in Drawbridge Pharmaceuticals. He is cited as inventor on Phaxan patents and is also a director and owner of Drawbridge safety margin and low cardiovascular toxicity.6,15–20 The data Pharmaceuticals, to which the Phaxan patents have been in these papers indicate that the HPBCD formulation is assigned. well tolerated in many species, and complement activation Name: Juliet M. Serrao, MB, BS, PhD, FRCA. and histamine release are not features of this formulation, Contribution: This author helped analyze the data and write compared with the veterinary equivalent of ALTH, Saffan® the manuscript. which contained Cremophor EL. Clearly, one has to ask the Attestation: Juliet M. Serrao has seen the original study data, question “why not use this in humans?” The answer is one reviewed the analysis of the data, and approved the final of relative toxicity for HPBCD versus SBECD. manuscript. In early development, IV voriconazole was formulated Conflicts of Interest: Juliet M. Serrao has equity interest in with HPBCD. The clinical voriconazole formulation that Drawbridge Pharmaceuticals. She is cited on Phaxan patents was eventually released used SBECD in the formulation as inventor, and she is a director and owner of Drawbridge instead of HPBCD because of differences in toxicity seen in Pharmaceuticals, to which the Phaxan patents have been assigned. animal models.21 These include hemolysis, the occurrence of Name: Anton Kolosov, MSc, PhD. foamy macrophages, and pancreatic cancers with HPBCD Contribution: This author helped conduct the study and write not seen with SBECD.22,23 Many of these effects are attribut- the manuscript. Attestation: able to membrane destabilization through the removal of Anton Kolosov has seen the original study data, reviewed the analysis of the data, and approved the final lipids with HPBCD, which has not been seen with SBECD. manuscript. It was unclear why PHAX should have a TI >30, when Conflicts of Interest: The author has no conflicts of interest to 20 was reported in the literature for ALTH in rodents. declare. PHAX is clearly different from the veterinary formulation Name: Ben J. Boyd, PhD. of alphaxalone in HPBCD, ALF, for which the LD50 is stated Contribution: This author helped conduct the study and write in the product insert to be 19 mg/kg IV in rats (TI = 7.6). The the manuscript. explanation for these differences is the presence of SBECD Attestation: Ben J. Boyd has seen the original study data, in PHAX. The series of experiments in which SBECD and reviewed the analysis of the data, and approved the final saline were given as a premedication before a lethal dose of manuscript. ALTH showed clearly that the SBECD was responsible for Conflicts of Interest: Ben J. Boyd will receive royalties from the higher TI of PHAX compared with ALTH. Monash University if Phaxan is successfully commercialized by The use of SBECD as excipient to aid dissolution of alphax- Drawbridge Pharmaceuticals. He is cited on the Phaxan patents alone avoids the major impediment to reintroduction of this as inventor, and as an employee of Monash University, he will useful agent into human anesthetic practice, that is, hyper- receive a share of the royalties that Monash University receives sensitivity reactions to Cremophor EL; the excipient used to from any commercialization of Phaxan. This manuscript was handled by: Markus W. Hollmann, MD, dissolve alphaxalone as ALTH marketed as a human and PhD, DEAA. veterinary anesthetic from 1972 to 1984. ALTH was used in every area of anesthetic practice, including prolonged inten- REFERENCES sive care sedation and the emerging areas of day case anes- 1. Selye H. Studies concerning the correlation between anesthetic thesia and procedural sedation. 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