USOO8975245B2
(12) United States Patent (10) Patent N0.: US 8,975,245 B2 Goodchild et a]. (45) Date of Patent: *hlar.10,2015
(54) ANAESTHETIC FORMULATION (56) References Cited (71) Applicant: DraWbridge Pharmaceuticals Pty Ltd, U.S. PATENT DOCUMENTS Southbank (AU) 2003/0055023 A1 3/2003 Rajewski et a1. 2003/0073665 A1 4/2003 Thompson et al. (72) Inventors: Juliet Marguerite Goodchild, Malvem (AU); Colin Stanley Goodchild, FOREIGN PATENT DOCUMENTS Malvern (AU); Benjamin James Boyd, Warrandyte (AU) EP 0399716 Bl 1/1994 W0 WO 93/17711 Al 9/1993 (73) Assignee: DraWbridge Pharmaceuticals Pty Ltd, W0 WO 01/70234 Al 9/2001 Southbank, Victoria (AU) W0 WO 2004/039426 A2 5/2004 OTHER PUBLICATIONS (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 Brewster, M.E. et a1. 1989 “Development of a Non-Surfactant For U.S.C. 154(b) by 0 days. mulation for Alfaxalone Through the Use of Chemically-Modi?ed This patent is subject to a terminal dis Cyclodextrins” Journal ofParenteral Science & Technology 43: 262 claimer. 265. Brewster, M.E. et al. 1995 “Preparation, Characterization, and Anes (21) Appl. N0.: 14/069,751 thetic Properties of 2-hydroxypr0pyl-[5-cyclodextrin Complexes of Pregnenolone in Rat and Mouse” Journal of Pharmaceutical Sci (22) Filed: Nov. 1, 2013 ences 84: 1154-1159. Chisari, M. et al. 2009 “The In?uence of Neuroactive Steroid (65) Prior Publication Data Lipophilicity 0n GABAA Receptor Modulation: Evidence for a Low Af?nity Interaction” J Neurophysiol 102: 1254-1264. US 2014/0066417 A1 Mar. 6, 2014 Egan, T.D. et al. 2003 “The Pharmacokinetics and Pharmacodynam ics of Propofol in a Modi?ed Cyclodextrin Formulation (Captisol®) Related US. Application Data Versus Propofol in a Lipid Formulation (Diprivan®): An (63) Continuation of application No. 13/574,201, ?led as Electroencephalographic and Hemodynamic Study in a Porcine application No. PCT/AU2011/000050 on Jan. 19, Model” Anesth Analg 97:72-79. Ramsay, et al. 1974 “Controlled Sedation with Alphaxalone 2011, now Pat. No. 8,697,678. Alphadolone” British Medical Journal 2: 656-659. (60) Provisional application No. 61/297,249, ?led on Jan. Sneyd, J .R. et al. 1997 “Computer-controlled infusion of ORG 21, 2010, provisional application No. 61/385,318, 21465, a water soluble steroid i.v. anaesthetic agent, into human ?led on Sep. 22, 2010. volunteers” British J Anaethesia 79: 433-439. Supplemental European Search Report, in corresponding European (51) Int. Cl. Application No. EP 11 73 4245, dated Jan. 17,2014. A61K 31/56 (2006.01) A61K 47/40 (2006.01) Primary Examiner * Paul Zarek A61K 31/57 (2006.01) (74) Attorney, Agent, or Firm * Knobbe, Martens Olson & A61K 47/48 (2006.01) Bear, LLP B82Y5/00 (2011.01) A61K 31/5 73 (2006.01) (57) ABSTRACT (52) US. Cl. The present invention relates generally to the ?eld of drug CPC ...... A61K47/40 (2013.01); A61K31/56 delivery systems for neuroactive steroid anaesthetic agents. (2013.01); A61K31/57 (2013.01); A61K More particularly, anaesthetic and sedative formulations are 47/48969 (2013.01); B82Y5/00 (2013.01); provided in the form of host/guest preparations comprising A61K31/573 (2013.01) one or more neuroactive steroid anaesthetics and a cyclodex USPC ...... 514/178 trin. Particular cyclodextrins contemplated include sulfoalkyl (58) Field of Classi?cation Search ether cyclodextrins and modi?ed forms thereof. CPC ...... A61K47/48969; A61K 31/573 See application ?le for complete search history. 9 Claims, 9 Drawing Sheets US. Patent Mar. 10, 2015 Sheet 1 0f9 US 8,975,245 B2
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time after iv in§ecti ermmioi m5 w?-Mfakhesin changeinpressure --- F‘i'm'x anCD Figure 11 US. Patent Mar. 10, 2015 Sheet 9 0f9 US 8,975,245 B2 % Qhange in mean diastotilc bicmd pressure time after iv inietrtiien {minut?s} £3 5 eihangeinpressure $6} Figure 12 US 8,975,245 B2 1 2 ANAESTHETIC FORMULATION Currently, a lipid formulation of di-isopropyl phenol (pro pofol) is the most highly used anaesthetic agent. Propofol, This application is a continuation of US. application Ser. however, can be contraindicated in certain at risk patients due No. 13/574,201, ?ledAug. 30, 2012, now US. Pat. No. 8,697, to its lowering effect on blood pressure, the effect it has on 678, which is the US. national phase of International Appli reducing cardiac output and it can adversely affect respiratory cation No. PCT/AU11/00050, ?led Jan. 19, 2011, which control. In particular, propofol is formulated in a lipid emul claims priority from US. Provisional Patent Application No. sion which can support microbial growth if contaminated. 61/297,249, ?led on 21 Jan. 2010, entitled “Anaesthetic for The formulation cannot, in fact, be sterilized. There have been mulation” AND U.S. Provisional Patent Application No. instances where microbially contaminated propofol formula 61/385,318 ?led on 22 Sep. 2010, entitled “Anaesthetic for tions have resulted in patients becoming infected. One other mulation,” the entire contents of which, are incorporated issue with the current propofol formulation is the pain herein by reference. induced following or during intravenous injection. Attempts to re-formulate in a water-based preparation have led to FIELD increased injection pain. Propofol also can lead to cardiovas The present invention relates generally to the ?eld of drug cular and respiratory depression and has a low therapeutic delivery systems for neuroactive steroid anaesthetic agents. index of 5, i.e. only 5 times the normal anaesthetic dose can More particularly, anaesthetic and sedative compositions are lead to death. Furthermore, the anaesthetic is incompatible provided in the form of host/ guest preparations comprising with plastic storage containers and plastic syringes which one or more neuroactive steroid anaesthetics and a cyclodex 20 complicates syringe delivery equipment which is frequently trin or a modi?ed form thereof. in standard use for intravenous anaesthesia and sedation. The drug can also cause hyperlipidaemia and can induce toxicity BACKGROUND when used in a larger dose by infusion. This is particularly problematic in the intensive care setting. Bibliographic details of references provided in the subject 25 A neuroactive steroid anaesthetic has the potential for speci?cation are listed at the end of the speci?cation. being more ef?cacious with fewer side effects than propofol. Reference to any prior art is not, and should not be taken as There is a need, therefore, to develop a suitable formulation an acknowledgment or any form of suggestion that this prior to enable the use of a neuroactive steroid anaesthetic agent in art forms part of the common general knowledge in any subjects. country. 30 Drug delivery systems aim to provide the required amount SUMMARY of drug systemically or to a targeted site for a time and under conditions suf?cient to have the desired effect. Some drug Throughout this speci?cation, unless the context requires delivery systems require carrier materials to mitigate particu otherwise, the word “comprise”, or variations such as “com lar undesirable properties of the drugs. One such type of 35 prises” or “comprising”, will be understood to imply the carrier molecule is a cyclodextrin which acts as a host for a selected guest molecule. inclusion of a stated element or integer or method step or Cyclodextrins are cyclic oligosaccharides with hydroxyl group of elements or integers or method steps but not the groups on their outer surface and a void central cavity which exclusion of any other element or integer or method step or has a lipophilic character. Cyclodextrins are capable of form 40 group of elements or integers or method steps. ing inclusion complexes with hydrophobic molecules. The The present invention provides a host/guest complex for stability of the resulting host/ guest complex depends on how mulation comprising a neuroactive steroid anaesthetic and a readily the guest molecule occupies the central cavity of the cyclodextrin or modi?ed form thereof for use in inducing host. anaesthesia or sedation in mammalian subjects. Generally, The most common cyclodextrins are (x-, [3- and y-cyclo 45 the neuroactive steroid anaesthetic is sparingly soluble. The dextrins consisting of 6, 7 and 8 0t-1,4-linked glucose units, host/ guest complex formulation is, therefore, a drug delivery respectively. Cyclodextrins have relatively low solubility in system for a neuroactive steroid anaesthetic. In an embodi water and organic solvents and this limits their use in phar ment, the cyclodextrin is a modi?ed polyanionic [3-cyclodex maceutical formulations. For a description of the general trin and the neuroactive steroid anaesthetic is selected from chemistry of cyclodextrin, reference can be made to From 50 alphaxalone, alphadolone, acebrochol, allopregnanolone, ming and Szejtlic (eds), Cyclodexlrins in Pharmacy, Kluwer: eltanolone(pregnanolone), ganaxolone, hydroxydione, Dordrecht, The Netherlands, 1994; Atwood, Davies, MacNi minaxolone, Org20599, Org21465 and tetrahydrodeoxycor col andVogtie (Eds), Comprehensive Supramolecular Chem ticosterone and a pharrnacologically acceptable derivative, istry Vol 4, Pergamon: Oxford UK, 1996; and Thomason, Cril salt or pro-drug form thereof. However, all cyclodextrins are Rev Ther Drug Carrier Sysl 14: 1, 1997. 55 contemplated herein including y and 0t cyclodextrins or their Alphaxalone [Alfaxalone or 3-0t-hydroxy-5-(x-,pregnan modi?ed forms as well as their salts. The term “derivative” 11,20-dione] is a potent neuroactive steroid anaesthetic cur rently used in veterinary medicine (Child et al., British Jour includes deuterated derivatives of the neuroactive steroid nal ofAnaethesia 4312-13, 1971). anaesthetic. Deuterated derivatives are contemplated for use as improved medicaments. One or more hydrogen atoms may Alphaxalone was widely used around the world as an intra 60 venous anaesthetic together with alphadolone [Althesin; be replaced by deuterium. Modi?ed forms of cyclodextrins Alfathesin] in human patients until 1983. Although these include methylated, hydroxyalkylated, branched, alkylated, anaesthetics have a high therapeutic index, they were never acylated and anionic cyclodextrins. By “alkylated” includes theless Withdrawn from clinical practice due to occasional, an alkyl ether derivative as well - unpredictable yet severe anaphylactoid reactions to a poly 65 cyclodextrin. The agent “alphadolone” includes its salt, ethoxylated castor oil excipient (Cremophor EL [Registered alphadolone acetate. Reference to a cyclodextrin or a modi Trademark]) . ?ed form thereof includes its salts (e.g. a sodium salt). US 8,975,245 B2 3 4 Accordingly, one aspect of the present invention is directed rahydrodeoxycorticosterone and a pharmacologically to an anaesthetic or sedative composition comprising a neu acceptable derivative, salt or pro-drug form thereof formu roactive steroid anaesthetic formulated with a cyclodextrin or lated with a sulfoalkyl ether cyclodextrin or modi?ed form modi?ed form thereof. thereof to generate a sterilizable composition with a thera The anaesthetic or sedative formulation of the present peutic index of greater than 5. invention exhibits features such as being sterilizable, causes In an embodiment, the composition is also storable in a reduced incidence of pain on injection, has a larger therapeu plastic container. tic index relative to propofol (including a therapeutic index The formulation may comprise a buffer to maintain pH greater than 5), is capable of storage in a plastic container and within a range of from about pH5 .5 to pH8. Alternatively, the induces a rapid induction of anaesthesia to surgical levels formulation may not be buffered wherein the pH of the for with similar or more rapid awakening time than propofol or mulation may be from about pH3 to about pH9.5. The formu Althesin (alphaxalone and alphadolone). lation may also comprise a preservative, anti-microbial agent Hence, another aspect of the present invention provides an and/or an agent which reduces toxicity. In addition, to anaesthetic or sedative composition comprising a neuroactive improve solubility and/or stability, a co-polymer may be steroid anaesthetic and a cyclodextrin or modi?ed form included. Examples of suitable co-polymers include thereof wherein the anaesthetic and cyclodextrin are formu hydroxyl propyl methyl cellulose (HPMC), polyvinyl pyrol lated to provide an anaesthetic composition which exhibits a lidone (PVP), and carboxymethyl cellulose (CMC). property selected from being sterilizable, exhibiting minimal The present invention further contemplates inducing or pain on intravenous injection, having a therapeutic index maintaining by infusion or intermittent bolus administration, greater than 5 and is storable in a plastic container. In an 20 anaesthesia or sedation in a subject, the method comprising embodiment, the formulation has one, two, three or all four of administering an anaesthetic-effective amount of a neuroac these properties. tive steroid anaesthetic formulated with a cyclodextrin, for a In a related embodiment, the present invention provides an time and under conditions to induce anaesthesia or sedation. anaesthetic or sedative delivery host/ guest composition com More particularly, the present invention provides a method prising a cyclodextrin host or a modi?ed form thereof with a 25 of inducing or maintaining by infusion or intermittent bolus neuroactive steroid anaesthetic drug guest, the host/ guest administration, anaesthesia or sedation in a subject, the composition formulated to be sterilizable, administrable by method comprising administering an anaesthetic-effective intravenous injection with minimal pain and to exhibit a amount of a neuroactive steroid anaesthetic selected from therapeutic index of greater than 5. The formulation may also alphaxalone, alphadolone, acebrochol, allopregnanolone, be storable in a plastic container. 30 eltanolone (pregnanolone), ganaxolone, hydroxydione, More particularly, the present invention provides an anaes minaxolone, Org20599, Org2l465 and tetrahydrodeoxycor thetic or sedative composition comprising a neuroactive ste ticosterone and pharmacologically acceptable derivatives, roid anaesthetic selected from alphaxalone, alphadolone, ace salts or pro-drug forms thereof formulated with a cyclodex brochol, allopregnanolone, eltanolone (pregnanolone), trin or modi?ed form thereof for a time and under conditions ganaxolone, hydroxydione, minaxolone, Org20599, 35 suf?cient to induce anaesthesia or sedation wherein the Org2l465 and tetrahydrodeoxycorticosterone and pharma anaesthetic or sedative formulation exhibits a property cologically acceptable derivatives, salts and pro-drug forms selected from being sterilizable, exhibiting minimal pain on thereof formulated with a cyclodextrin or modi?ed form intravenous injection and having a therapeutic index greater thereof. than 5. Even more particularly, the present invention is directed to 40 In an embodiment, the formulation is also storable in a an anaesthetic or sedative composition comprising a neuro plastic container. active steroid anaesthetic selected from alphaxalone, Generally, the molar ratio of neuroactive steroid anaes alphadolone, acebrochol, allopregnanolone, eltanolone thetic to cyclodextrin is from about 1:1 to about 1:6, more (pregnanolone), ganaxolone, hydroxydione, minaxolone, particularly about 1:1 to about 1:4, even more particularly Org20599, Org2l465 and tetrahydrodeoxycorticosterone 45 about 1:1 to 1:3 and still more particularly about 1:2. and pharmacologically acceptable derivatives, salts and pro These aspects of the present invention extend to inducing drug forms thereof formulated with a cyclodextrin or a modi or maintaining by infusion or intermittent bolus administra ?ed form thereof wherein the composition exhibits a property tion, anaesthesia or sedation or both in subjects. selected from being sterilizable, exhibiting minimal pain on The formulation may be packaged for sale with a set of intravenous injection, having a therapeutic index greater than 50 instructions. The instructions may include a patient manage 5 and is storable in a plastic container. ment protocol comprising administering to the patient an A particular cyclodextrin useful in the practice of the effective amount of neuroactive steroid anaesthetic such as present invention is a sulfoalkyl ether cyclodextrin such as (7) selected from alphaxalone, alphadolone, acebrochol, allo sulfobutyl ether [3-cyclodextrin. This compound can be pre pregnanolone, eltanolone (pregnanolone), ganaxolone, pared as described in Us. Pat. No. 5,376,645. Another useful 55 hydroxydione, minaxolone, Org20599, Org2l465 and tet cyclodextrin is an alkyl ether derivative including a sulfoalkyl rahydrodeoxycorticosterone and pharmacologically accept ether-alkyl ether cyclodextrin. However, the present inven able derivatives, salts and pro-drug forms thereof formulated tion extends to other cyclodextrin derivatives such as methy with a cyclodextrin for a time and under conditions suf?cient lated, hydroxyalkylated, branched, acylated and anionic to induce anaesthesia. As indicated above, a suitable cyclo forms. The anaesthetic formulation of the present invention 60 dextrin includes a sulfoalkyl ether dextrin, such as (7) sul enables injectable administration to mammalian subjects and fobutyl ether [3-cyclodextrin as well as alkyl ether derivatives in particular human patients. such as sulfoalkyl-alkyl ether cyclodextrins. Other deriva Another aspect of the present invention provides an anaes tives include methylated, hydroxyalkylated, branched, alky thetic or sedative composition comprising a neuroactive ste lated, acylated and anionic cyclodextrins. roid selected from alphaxalone, alphadolone, acebrochol, 65 The present invention further contemplates the use of a allopregnanolone, eltanolone (pregnanolone), ganaxolone, neuroactive steroid anaesthetic and a cyclodextrin or modi hydroxydione, minaxolone, Org20599, Org2l465 and tet ?ed form thereof, in the manufacture of a medicament to US 8,975,245 B2 5 6 induce anaesthesia in a subject. In a particular embodiment, steroid anaesthetic is sparingly soluble in water. The drug the neuroactive steroid anaesthetic is selected from alphaxa delivery system comprises a host carrier in the form of a lone, alphadolone, acebrochol, allopregnanolone, eltanolone cyclodextrin or modi?ed form thereof. Reference to “cyclo (pregnanolone), ganaxolone, hydroxydione, minaxolone, dextrin” includes in one embodiment an (x-, [3- or y-cyclodex Org20599, Org21465 and tetrahydrodeoxycorticosterone trin or a modi?ed or derivatized form thereof. Reference to and pharmacologically acceptable derivatives, salts and pro “cyclodextrin” in another embodiment includes a sulfoalkyl drug forms thereof. ether dextrin such as (7) sulfobutyl ether [3-cyclodextrin or an Kits comprising in compartmental form a neuroactive ste alkyl ether derivative thereof such as a sulfobutyl ether-alkyl roid anaesthetic in a ?rst compartment and a cyclodextrin, ether cyclodextrin. Derivatives of cyclodextrins include such as a sulfoalkyl ether cyclodextrin, in a second compart methylated, hydroxyalkylated, branched, alkylated, acylated ment and optionally excipients and/or co-polymers in a third and anionic cyclodextrins. By “alkylated” includes an alkyl or other compartment are also contemplated herein. The kit ether derivative such as an alkyl ether-alkyl ether cyclodex may be in the form of a modi?ed syringe. trin. Particular cyclodextrins contemplated herein are shown Labeled forms of the neuroactive steroid anaesthetic are in Table 7 [Uekama et al., Chem. Rev. 98: 2045-2076, 1998] also useful in monitoring and tracking the anaesthetic during and include [3-cyclodextrin sulfobutyl ethers, ethyl ethers, sedation or anaesthesia. Kits and apparatus are therefore pro [3-cyclodextrin sulfobutyl ethers (?at), y-cyclodextrin sul vided herein to assist in the monitoring of labelled neuroac fobutyl ethers and (x-cyclodextrin sulfobutyl ethers and their tive steroid anaesthetics. Labeled derivatives include deuter salts (e.g. sodium salts). ated, tritiated and other labeled agents. The drug delivery system of the present invention enables 20 a neuroactive steroid anaesthetic to be administered to a sub BRIEF DESCRIPTION OF THE FIGURES ject in a sterilized form. Furthermore, the delivery itself is with less pain compared to the intravenous administration of FIGS. 1a through If are graphical representations of propofol. The formulation of the present invention addition experiments on male Wistar rats implanted with indwelling ally has a therapeutic index greater than 5 (meaning that internal jugular vein intravenous catheters under halothane 25 administration of greater than 5 times the anaesthetic dose anaesthesia and then provided with propofol (a,b), Althesin can lead to death in a test animal). By “greater than 5” means [alphaxalone and alphadolone acetate] (c,d) or PhaxanCD a therapeutic index of between 5 and 200 including 10, 20, 30, [alphaxalone in a 1:2 molar complexation ratio with (7) sul 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, fobutyl ether [3-cyclodextrin] (e,f). 180, 190 and 200 as well as integers or fractions in between. FIG. 2 is a graphical representation of lethal dosing of two 30 The formulation of the present invention is also storable in a alphaxalone preparations [PhaxanCD and Althesin in rats]. plastic container and is compatible for use in plastic delivery FIG. 3 is a graphical representation of a probit plot for apparatus. lethality dosing of an Althesin preparation in rats. Accordingly, an aspect of the present invention provides an FIG. 4 is a graphical representation of sleep time in rats anaesthetic or sedative delivery host/ guest composition com using repeated doses of PhaxancD (alphaxalone in a 1:2 molar 35 prising a cyclodextrin host or modi?ed form thereof with a complexation ratio with (7) sulfobutyl ether [3-cyclodextrin). neuroactive steroid anaesthetic drug guest, the host/ guest FIG. 5 is a graphical representation of righting re?ex time composition formulated to be sterilizable, administrable by response curves in rats using pregnanolone in a (7) sulfobutyl intravenous injection with minimal pain and to exhibit a ether [3-cyclodextrin. therapeutic index of greater than 5. In an embodiment, the FIG. 6 is a graphical representation of tail pinch time 40 formulation may also be storable in a plastic container. The response curve in rats using pregnanolone in a (7) sulfobutyl formulation may exhibit one, two, three or all four of these ether [3-cyclodextrin. properties. FIG. 7 is a graphical representation of rotarod test time By “reduced pain” means compared to a formulation com response curve in rats using pregnanolone in a (7) sulfobutyl prising propofol as a reference. ether [3-cyclodextrin. 45 The formulation is useful for inducing anaesthesia or seda FIG. 8 is a graphical representation of righting re?ex time tion in mammalian subjects and in particular human subjects. response curves in rats using alphadolone in a (7) sulfobutyl In an embodiment, the neuroactive steroid is selected from ether [3-cyclodextrin. alphaxalone, alphadolone, acebrochol, allopregnanolone, FIG. 9 is a graphical representation of tail pinch time eltanolone (pregnanolone), ganaxolone, hydroxydione, response curves in rats using alphadolone in a (7) sulfobutyl 50 minaxolone, Org 20599, Org 21465 and tetrahydrodeoxycor ether [3-cyclodextrin. ticosterone and a pharmacologically acceptable derivative, FIG. 10 is a graphical representation of rotarod test time salt or pro-drug form thereof. response curves in rats using alphadolone in a (7) sulfobutyl An example of a pharmacologically acceptable salt is ether [3-cyclodextrin. alphadolone acetate, which is encompassed by the present FIG. 11 is a graphical representation of percentage change 55 invention. An example of a derivative of a neuroactive steroid in mean systolic blood pressure in rats after injection with anaesthetic is a deuterated derivative. A “modi?ed” cyclodex propofol, Althesin or PhaxanCD. trin includes a derivative of a cyclodextrin. FIG. 12 is a graphical representation of percentage change Accordingly, another aspect of the present invention is in mean diastolic blood pressure in rats after injection with directed to a drug delivery host/guest composition compris propofol, Althesin or PhaxanCD. 60 ing a cyclodextrin host or modi?ed form thereof with a neu Reference to “PhaxanCD” means an alphaxalone prepara roactive steroid anaesthetic drug guest selected from alphaxa tion with (7) sulfobutyl ether [3-cyclodextrin. lone, alphadolone, acebrochol, allopregnanolone, eltanolone (pregnanolone), ganaxolone, hydroxydione, minaxolone, DETAILED DESCRIPTION Org 20599, Org 21465 and tetrahydrodeoxycorticosterone 65 and a pharmacologically acceptable derivative, salt or pro The present invention provides a drug delivery system for drug form thereof, the host/ guest composition being steriliZ a neuroactive steroid anaesthetic. Generally, the neuroactive able, administrable by intravenous injection with minimal US 8,975,245 B2 7 8 pain, exhibiting a therapeutic index of greater than 5 and/or Another aspect of the present invention is directed to a drug storable in a plastic container. The formulation can also ini delivery host/guest composition comprising a sulfoalkyl tiate rapid induction of anaesthesia to surgical levels with ether dextrin ho st or modi?ed form thereof with a neuroactive similar or more rapid wakening time compared to propofol. steroid anaesthetic drug guest selected from alphaxalone, As indicated above, the formulation may exhibit one, two, alphadolone, acebrochol, allopregnanolone, eltanolone three or all of these properties. (pregnanolone), ganaxolone, hydroxydione, minaxolone, The composition of the present invention may be referred Org 20599, Org 21465 and tetrahydrodeoxycorticosterone to as a formulation, host/guest composition, drug delivery and a pharmacologically acceptable derivative, salt or pro system, medicament, anaesthetic or sedative as well as more drug form thereof, the host/guest composition to be steriliZ able, administrable by intravenous injection with minimal descriptively such as an anaesthetic formulation or sedative pain and exhibiting a therapeutic index of greater than 5. formulation. In an embodiment, the composition is also storable in a Another aspect of the present invention provides an anaes plastic container. thetic or sedative formulation comprising a neuroactive ste Another aspect of the present invention provides an anaes roid anaesthetic and a cyclodextrin or modi?ed form thereof, thetic or sedative formulation comprising a neuroactive ste the formulation exhibiting properties including being steril roid anaesthetic and a sulfoalkyl ether dextrin or modi?ed izable, inducing reduced pain on intravenous administration, form thereof, the formulation exhibiting properties including having a therapeutic index of greater than 5 and/or being being sterilizable, inducing reduced pain on intravenous storable in a plastic container. administration, having a therapeutic index of greater than 5 More particularly, the present invention relates to an anaes 20 and/or being storable in a plastic container. thetic or sedative formulation comprising a neuroactive ste As indicated above one particularly useful sulfoalkyl ether roid anaesthetic selected from alphaxalone, alphadolone, ace dextrin is (7) sulfobutyl ether [3-cyclodextrin. Of the proper brochol, allopregnanolone, eltanolone (pregnanolone), ties exhibited, in a particular embodiment, the formulation ganaxolone, hydroxydione, minaxolone, Org 20599, Org exhibits two or more, three or more or all properties. These 21465 and tetrahydrodeoxycorticosterone and a pharmaco 25 properties include imitating rapid induction of anaesthesia to logically acceptable derivative, salt or pro-drug form thereof, surgical levels with similar or more rapid wakening time such the formulation exhibiting properties including being steril as compared to propofol. izable, inducing reduced pain on intravenous administration The formulation between the neuroactive steroid and and having a therapeutic index of greater than 5. cyclodextrin is generally in a molar ratio of from 1:1 to 1:6 In an embodiment, the formulation is also storable in a 30 (neuroactive steroid:cyclodextrin), more particularly about plastic container. 1:1 to 1:4, even more particularly about 1:1 to 1:3 and still The present invention extends to mixtures of two or more more particularly about 1:2. The range 1:1 to 1:6 includes 1:1, neuroactive steroid anaesthetic drugs such as a composition 1:1.1,1:1.2,1:1.3,1:1.4,1:1.5,1:1.6,1:1.7,1:1.8,1:1.9,1:2, comprising alphaxalone and alphadolone and/ or alphadolone 1:2.1,1:2.2,1:2.3,1:2.4,1:2.5,1:2.6,1:2.7,1:2.8,1:2.9,1:3, acetate or their pharmacologically acceptable derivatives, 35 1:3.1,1:3.2,1:3.3,1:3.4,1:3.5,1:3.6,1:3.7,1:3.8,1:3.9,1:4, salts or pro-drug forms. 1:4.1,1:4.2,1:4.3;1:4.4,1:4.5,1:4.6,1:4.7,1:4.8,1:4.9,1:5, A “pharmacologically acceptable derivative” is a deriva 1:5.1,1:5.2,1:5.3,1:5.4,1:5.5,1:5.6,1:5.7,1:5.8,1:5.9 and tive that still induces anaesthesia whilst not increasing 1:6. adverse side effects. The term “derivative” includes deuter Accordingly, the present invention provides a drug deliv ated derivatives where one or more hydrogen atoms are 40 ery host/ guest composition comprising a cyclodextrin host or replaced with deuterium. This can lead to improved ef?cacy. modi?ed form thereof with a neuroactive steroid drug guest, Furthermore, the anaesthetic agents may be subject to alky wherein the molar ratio of neuroactive steroid to cyclodextrin lation, alkoxylation, acetylation and/or phosphorylation to is from about 1:1 to about 1:6 and wherein the composition is generate other derivatives. Other types of derivatives include formulated to be sterilizable, administrable by intravenous deuterated or tritiated or other labeled forms useful for moni 45 injection with minimal pain and exhibiting a therapeutic toring and tracking the anaesthetic in the body. The terms index of greater than 5. In an embodiment, the formulation “derivative” and “modi?ed form” are used interchangeably may also be storable in a plastic container. herein. Salts of alphadolone include alphadolone acetate. More particularly, the present invention is directed to a Reference to pro-drugs include transported pro-drugs. drug delivery host/ guest composition comprising a cyclodex In an embodiment, the cyclodextrin is a [3-cyclodextrin or 50 trin selected from an (x-, [3- or y-cyclodextrin or a modi?ed a modi?ed form thereof such as but not limited to a sulfoalkyl form thereof including a sulfoalkyl ether dextrin or sulfoalkyl ether dextrin. A particularly useful sulfoalkyl ether dextrin is ether-alkyl ether derivative or other derivatives listed in Table (7) sulfobutyl ether [3-cyclodextrin. Alkyl ether derivatives 7 and a neuroactive steroid drug guest selected from alphaxa are also contemplated such as a sulfoalkyl ether-alkyl ether lone, alphadolone, acebrochol, allopregnanolone, eltanolone cyclodextrin. An example of an alkyl ether derivative is a 55 (pregnanolone), ganaxolone, hydroxydione, minaxolone, sulfobutyl ether-alkyl ether cyclodextrin. Other cyclodextrins Org 20599, Org 21465 and tetrahydrodeoxycorticosterone contemplated herein are listed in Table 7 and include methy and a pharmacologically acceptable derivative, salt or pro lated, hydroxyalkylated, alkylated, branched, acylated and drug form thereof, wherein the molar ratio of a neuroactive anionic derivatives. steroid to cyclodextrin is from about 1:1 to about 1:6 and Accordingly, an aspect of the present invention provides an 60 wherein the composition is formulated to be sterilizable, anaesthetic or sedative delivery host/ guest composition com admini strable by intravenous injection with minimal pain and prising a sulfoalkyl ether dextrin host or modi?ed form to exhibit a therapeutic index of greater than 5. In one embodi thereof with a neuroactive steroid anaesthetic drug guest, the ment, the (7) sulfobutyl ether [3-cyclodextrin comprises less host/ guest composition formulated to be sterilizable, admin than 100 ppm of a phosphate and has an absorption of less istrable by intravenous injection with minimal pain, exhibit 65 than 0.5 AU due to a drug-degrading enzyme, as determined ing a therapeutic index of greater than 5. In an embodiment, by UV/v is spectrophotometry at a wave length of 245 nm to the formulation may also be storable in a plastic container. 270 nm for an aqueous solution containing 300 mg of the US 8,975,245 B2 9 10 dextrin per ml of solution in a cell having a 1 cm path length. The neuroactive steroid anaesthetic may be used alone or in In an embodiment, the formulation may also be storable in a combination With another anaesthetic or sedative or other plastic container. active agent. In one embodiment, alphaxalone is used With The anaesthetic composition of the present invention may alphadolone or its salt, alphadolone acetate. Hence, reference in one embodiment comprise a buffer such as a phosphate or to “alphadolone” includes alphadolone acetate. The compo tris or citrate phosphate buffer to maintain the pH from about sition may comprise, therefore alphaxalone or alphadolone 5.5 to about pH8. This includes pH values of5.5, 6, 6.5, 7, 7.5 alone or a combination of alphaxalone and alphadolone or and 8. Alternatively, the composition does not comprise a any of their derivatives, salts or pro-drug forms. buffer and the pH being from about pH3 to about pH 9.5 such Hence, in a particular embodiment, the present invention as pH3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 or 9.5. further provides a composition comprising alphaxalone or a In a further aspect, the formulations of the present inven pharmacologically acceptable derivative, salt or pro-drug tion also include one or more agents such as excipients and/or thereof and/or alphadolone or a pharmacologically accept preservatives, microbial retardants. Other agents may also be included to reduce toxicity. Agents include, for example, able derivative, salt or pro-drug thereof formulated With a EDTA, benzyl alcohol, bisulphites, monoglyceryl ester of sulfoalkyl ether dextrin, such as (7) sulfobutyl ether [3-cyclo lauric acid (Monolaurin), capric acid and/or its soluble alka dextrin Wherein the molar ratio of alphaxalone and/or line salts or its monoglyceryl ester (Monocaprin), edetate, alphadolone to dextrin is from about 1:1 to about 1:6. Refer ence can conveniently be made to Remington’s Pharmaceu and capric acid and/or its soluble alkaline salts or its 20 monoglyceryl ester (Monocaprin) and edentate. The formu tical Sciences, Mack Publishing Company, Eaton, USA, 1990 lation may also contain one or more co-polymers to assist in and Rowe’s Handbook of Pharmaceutical Excipients, 2009 solubility or stability of the anaesthetic agent. Examples for formulation methods and reagents. include hydroxy propyl methyl cellulose (HPMC), polyvinyl The present invention contemplates inducing or maintain pyrollidone (PVP) and/or carboxymethyl cellulose (CMC). 25 ing by infusion or intermittent bolus administration, anaes Conveniently, the neuroactive steroid anaesthetic is pro thesia in a human subject, the method comprising adminis vided at a concentration of from about 0.5 to 100 mg/ml in a tering an anaesthetic-effective amount of a neuroactive saline suspension comprising the cyclodextrin. Such a con steroid anaesthetic formulated With a cyclodextrin, such as centration includes 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 30 sulfoalkyl ether dextrin, for a time and under conditions to 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, induce anaesthesia. 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, More particularly, the present invention provides a method 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, of inducing or maintaining by infusion or intermittent bolus 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, administration, anaesthesia in a human subject, the method 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 35 comprising administering an anaesthetic-effective amount of 94, 95, 96, 97, 98, 99 and 100 mg/ml of drug. As indicated a neuroactive steroid anaesthetic selected from alphaxalone, above, the composition is generally formulated so that the alphadolone, acebrochol, allopregnanolone, eltanolone molar ratio of neuroactive steroid to cyclodextrin is from (pregnanolone), ganaxolone, hydroxydione, minaxolone, about 1:1 to about 1:6, particularly from about 1:1 to 1:4, even Org20599, Org21465 and tetrahydrodeoxycorticosterone 40 more particularly from about 1:1 to 1:3 and most particularly and a pharmacologically acceptable derivative, salt or pro about 1:2. drug form thereof formulated With a cyclodextrin, such as (7) Reference to any particular neuroactive steroid or their sulfobutyl ether [3-cyclodextrin, for a time and under condi salts includes a racemic mixture of enantiomers of each tions suf?cient to induce anaesthesia, Wherein the anaesthetic anaesthetic as well as a single enantiomer of the agent. 45 formulation is sterilizable, administrateable by intravenous In a particular embodiment, the neuroactive steroid is injection With minimal pain and exhibits a therapeutic index alphaxalone, alphadolone and/ or alphadolone acetate. In one of greater than 5. Additionally, the formulation can initiate embodiment, alphaxalone is in the formulation at a concen rapid anaesthesia With similar or more rapid wakening time tration of from 1 to 100 mg/ml such as 10 mg/ml. In another compared With propofol or Althesin (Registered Trademark). embodiment, alphadolone or alphadolone acetate is present at The formulation may also be storable in a plastic container. 0.5 to 50 mg/ml such as 3 mg/ml. The present invention extends to inducing or maintaining The formulations herein are for in vivo delivery meaning by infusion or intermittent bolus administration, sedation. that the neuroactive steroid anaesthetic is delivered by intra Hence, another aspect of the present invention provides a venous, sub-cutaneous, intraperitoneal, intrathecal, intra 55 method of inducing or maintaining by infusion or intermittent muscular, intravitreal, transdermal, suppository (rectal), pes bolus administration, sedation in a subject, the method com sary (vaginal), inhalation, intranasal and the like. Most prising administering a sedation-effective amount of a neu effectively, the formulation is an intravenous (iv) formula roactive steroid anaesthetic formulated With a cyclodextrin tion. such as sulfoalkyl ether dextrin, for example, (7) sulfobutyl Accordingly, another aspect of the present invention pro 60 ether [3-cyclodextrin, for a time and under conditions suf? vides an injectable formulation of a neuroactive steroid cient to induce sedation. anaesthetic selected to be sterilizable, administrable by intra Reference to “(7) sulfobutyl ether [3-cyclodextrin” includes venous injection With minimal pain, exhibiting a therapeutic methylated, hydroxyalkylated, branched, alkylated, acylated index of greater than 5 and storable in a plastic container 65 and anionic derivatives thereof such as a sulfobutyl ether formulated With cyclodextrin, such as (7) sulfobutyl ether alkyl ether [3-cyclodextrin. Other derives include [3-cyclodex [3-cyclodextrin or an alkyl ether derivative. trin sulfobutyl ethers, ethyl ethers, [3-cyclodextrin sulfobutyl US 8,975,245 B2 11 12 ethers (?at), y-cyclodextrin sulfobutyl ethers and (x-cyclodex (pregnanolone), ganaxolone, hydroxydione, minaxolone, trin sulfobutyl ethers and their salts (e.g. sodium salts). Org20599, Org21465 and tetrahydrodeoxycorticosterone As indicated above, a particular subject is a human subject. and a pharmacologically acceptable derivative, salt or pro The anaesthetic formulation may be packaged for sale with drug form thereof formulated; 5 instructions for use. The use includes a patient management (iii) the cyclodextrin is selected from an (x-, [3-, and y-cyclo protocol comprising administering to the patient an effective dextrin or a modi?ed form thereof; amount of neuroactive steroid anaesthetic such as selected (iv) a buffer is optionally present and when present the pH of the formulation is from about pH5.5 to about pH8.0 and in from alphaxalone, alphadolone and pharmacologically the absence of buffer, the pH is from about pH3 to about acceptable derivatives, salts and pro-drug forms thereof for pH9.5; mulated with a cyclodextrin such as a sulfoalkyl ether dextrin, (v) the formulation is sterilizable; for example, (7) sulfobutyl ether [3-cyclodextrin, for a time (vi) intravenous injection of the formulation induces less pain and under conditions suf?cient to induce anaesthesia. than from a propofol formulation; The present invention further contemplates the use of a (vii) the therapeutic index of the formulation is greater than 5; neuroactive steroid anaesthetic and a cyclodextrin, such as a sulfoalkyl ether dextrin, for example, (7) sulfobutyl ether (viii) the formulation can be stored in a plastic container; and [3-cyclodextrin, in the manufacture of a medicament to induce (ix) the formulation can initiate rapid induction of anaesthesia anaesthesia in a subject such as a human subject. In a particu to surgical levels with similar or more rapid wakening time lar embodiment, the neuroactive steroid anaesthetic is 20 compared to propofol. selected from alphaxalone, alphadolone and pharmacologi In a particular embodiment, an anaesthetic or sedative for cally acceptable derivatives, salts and pro-drug forms thereof. mulation is provided comprising a sulfoalkyl ether or sul In another embodiment, the anaesthetic is selected from ace foalkyl ether-alkyl ether dextrin, a neuroactive steroid anaes brochol, allopregnanolone, eltanolone (pregnanolone), thetic such as alphaxalone or alphadolone and one or more ganaxolone, hydroxydione, minaxolone, Org20599, 25 co-polymers such as HPMC, PVP and/or CMC. Org21465 and tetrahydrodeoxycorticosterone and a pharma In a particular embodiment, the neuroactive steroid anaes cologically acceptable derivative, salt or pro-drug form thetic is formulated with a sulfoalkyl ether dextrin such as (7) thereof. sulfobutyl ether [3-cyclodextrin. In terms of an anaesthetic-effective amount, this is gener The present invention further contemplates a method for ally around 0.25 mg/kg to about 100 mg/kg body weight. A 30 formulating an anaesthetic or sedative composition, the sedative-effective amount is provided in similar or lower method generating a host/guest composition comprising a amounts and includes from about 0.05 mg/kg to about 10 cyclodextrin and a neuroactive steroid. In an embodiment, the mg/kg body weight. cyclodextrin is a sulfoalkyl ether or sulfoalkyl ether-alkyl The present invention further provides a kit. The kit may be ether dextrin such as (7) sulfobutyl either [3-cyclodextrin or in any form including a syringe or modi?ed syringe. A kit may 35 sulfobutyl ether-alkyl ether [3-cyclodextrin. Other cyclodex comprise alphaxalone and/or alphadolone or other neuroac trins include [3-cyclodextrin sulfobutyl ether-ethyl ether, tive steroid anaesthetic or their derivatives, salts or pro-drug [3-cyclodextrin sulfobutyl ether (?at), y-cyclodextrin sulfobu forms in one or more compartments and a sulfoalkyl ether tyl ether, (x-cyclodextrin sulfobutyl ether and their sodium dextrin in a further compartment as well as excipients in salts. subsequent compartments. The contents of the compartments 40 may be admixed prior to use. The present invention is further described by the following In a particular embodiment, the present invention provides non-limiting Examples. When a neuroactive steroid anaes a formulation comprising alphaxalone and/or alphadolone thetic is formulated with a cyclodextrin, it is referred to as and/ or pharmacologically acceptable derivatives, salts or pro “neuroactive steroid anaestheticCD”. An example is Phax drug forms thereof complexed with the sulfobutyl ether anCD, which comprises alphaxalone formulated with a cyclo cyclodextrin, for use in inducing or maintaining by infusion dextrin which in this case is (7) sulfobutyl ether [3-cyclodex or intermittent bolus administration, anaesthesia or sedation trin. Other examples include pregnanoloneCD and in human subject. alphadoloneCD. The anaesthetic forms may be labeled such as deuterated or tritiated forms or by other labels to facilitate monitoring and 50 EXAMPLE 1 tracking of the anaesthetics in the body. Kits and apparatus are provided, therefore, to monitor labeled neuroactive ste roid anaesthetics. Anaesthetic Effects of Alphaxalone in 30% w/v (7) Whilst the present invention is particularly directed to Sulfobutyl Ether [3-Cyclodextrin anaesthetic formulations for use in humans, the formulations 55 may also be used in animals such as for clinical trials or Alphaxalone was formulated as 6 ml clear colorless liquid veterinary use. Non-human animals contemplated herein containing: include rats, mice, guinea pigs, hamsters, sheep, pigs, dogs, Alphaxalone 60 mg (10 mg/ml); cats, horses, cows, goats, camels and non-human primates. (7) sulfobutyl ether [3-cyclodextrin 1800 mg; Hence, the present invention provides an anaesthetic or sedative composition comprising a neuroactive steroid anaes Saline (0.9% w/v) 6 ml. thetic and a cyclodextrin or modi?ed form thereof wherein This is a molar complexation ratio of alphaxalone to (7) the composition has the following properties: sulfobutyl ether [3-cyclodextrin of 1:4.6. Male Wistar rats (i) the neuroactive steroid and cyclodextrin are formulated in (weight [wt] 270-315 g) with indwelling jugular intravenous a molar ratio of from about 1:1 to about 1:6; 65 catheters were put in a Perspex restrainer and given the inj ec (ii) the neuroactive steroid is selected from alphaxalone, tions with the attached observations upon release from the alphadolone, acebrochol, allopregnanolone, eltanolone restrainer shown in Table 1. US 8,975,245 B2 13 14 TABLE 1 Effects of alphaxalone formulation on Wistar rats TIME PRC PRC PRC PRC MINUTES PRC447 446 468 461 448 PRC 469 PRC 455 C5 C10 C15 C20 C20 C25 0 s S l s S 2 s s S R 3 s s S R 4 s R ' RR 5 R RR R 6 RR R 7 RR R 8 s RR R 9 s RR R 10 s RR RR 11 s R 12 s R ; l3 s S I 14 ss S no sedation C : cyclodextin formulation [5, 10, IS, 20, 25 and 100 rug/kg] 30 The rats given 25 and 100 mg/kg body weight had recov ether [3-cyclodextrin formulation and the Althesin (Regis ered by 60 minutes; they did not die or suffer any adverse tered Trademark) formulation. This would indicate that the effects at these doses. In these experiments, it can be seen that new formulation is expected to behave in a similar fashion to intravenous injection of alphaxalone 10 mg/ml dissolved in the way Althesin (Registered Trademark) has behaved in the (7) sulfobutyl ether [3-cyclodextrin caused loss of conscious 35 past, particularly with respect to doses needed for anaesthesia ness at doses above 10 mg/kg and there was a dose related and sedation and also the speed of recovery. A sample table of anaesthetic effect with a wide safety margin; rats that received blood levels that can be expected from these experiments 25 and 100 mg/kg body weight (two times and ten times the appears in Table 2. anaesthetic dose) did not die indicating a wide safety margin for the preparation. This is clearly different from a formula 40 TABLE 2 tion of alphaxalone in hydroxypropyl [3-cyclodextrin. Such a preparation (AlfaxanCD-RTU) made by Jurox Pty, New Variable Units castle NSW Australia has a published LDSO, the dose of Duration ofAnaesthesia Min 35.0 [9.0] alphaxalone that causes death in 50% of rats when given AUC Min * rug/L 58.6 [11.7] 45 tl/z (eh-m) Min 38.2 [5.6] intravenously, of 19 mg/kg body weight, a ?gure very signi? CIT mL/rnin/kg 38.0 [8.2] cantly below that shown here for alphaxalone formulated in VC L/kg 0.5 [0.3] sulfobutyl ether [3-cyclodextrin [Alfaxan CD-RTU Material Vdss L/kg 1.8 [0.6] safety Data Sheet; Jurox Pty, Newcastle NSW Australia]. EXAMPLE 2 50 EXAMPLE 3 Phannacokinetics in the Rat Anaesthetic Effects of Alphaxalone in (7) Sulfobutyl Ether [3-Cyclodextrin Compared with Alphaxalone as Two groups of ten rats with implanted internal jugular Althesin (Registered Trademark) and Propofol intravenous and carotid intraarterial catheters receive bolus 55 intravenous injections via the jugular vein of 10 mg/kg body Male Wistar rats (wt 150-220 g weight) were implanted weight of (7) sulfobutyl ether [3-cyclodextrin formulation of with indwelling internal jugular vein intravenous catheters alphaxalone (n:10 rats) or a mixture of alphaxalone and under halothane anaesthesia. Twenty four hours later each rat alphadolone in CremophorEL (a polyethoxylated Caster oil), received an intravenous injection from a range of doses of 1.1 ml/kg (n:10 rats). Blood taken from the carotid artery or 60 either: propofol (10 mg/ml in 10% w/v Intralipid emulsion; tail at a number of time intervals after this injection is ana Diprivan [Registered Trademark]); Althesin [Registered lyzed for alphaxalone blood levels. These are ?tted to a three Trademark) (alphaxalone 9 mg/ ml plus alphadolone acetate 3 compartment pharmacokinetic model and meanzsem for key mg/ml in 20% w/v CremophorEL); or PhaxanCD (alphaxa parameters are calculated for both preparations of steroid lone 10 mg/ml in a 1:2 molar complexation ratio with Capti anaesthetic. 65 sol (Registered Trademark)i(7) sulfobutyl ether [3-cyclo It is expected that there will be no signi?cant difference dextrin). The following were assessed at regular time between the PK parameters calculated for the (7) sulfobutyl intervals after the intravenous injection: US 8,975,245 B2 15 16 .righting re?ex: scored as: 1 normal; 2 slow; 3 some attempt; EXAMPLE 4 4 noneithis was a measure of onset and duration of unconsciousness; Lethal Dose Finding for Alphaxalone Anaesthetic . tail pinch response: scored as: 1 normal; 2 weak; 3 just Preparations present; 4 noneithis was a measure of onset and duration of surgical anaesthesia; and This series of experiments was undertaken to determine the . time to when the rat was able to walk on the rotarod (a LD50 and LD95 doses for alphaxalone formulated in Cremo rotating cylinder) measured in seconds: the maximum nor phorEL [Althesin (Registered Trademark)] and (7) sulfobutyl mal run time is 120 s in non sedated ratsiattaining this ether [3-cyclodextrin (PhaxanCD); i.e., the doses of alphaxa value was a measure of time taken to attain full recovery 10 lone in Captisol (Registered Trademark) that caused 50% and from the sedating effects of the anaesthetic injections. 95% Of SllbjeCtS to die. _Male Wistar rats (Wt 150-220 ~g Results from groups often rats treated with the same anaes- Welght) Were Implanted Wlth IHdWelllng Internal Jugular veln thetic and dose were combined for statistical purposes. Rats 11m avenous catheters under h910than9ana95th951a; Twenty that attained a score of 4 for loss of righting re?ex were four hours later each rat received an intravenous injection deemed to have lost consciousness and those that scored a 4 15 fronli a railglf Ofldosesgof elthelr: ‘i‘ltheslmh(%e1515tered Trade; for loss of tail pinch response were deemed to be surgically mar ) .(a p Oaxa one mg/m p us a p a 0 one acetate . . . . mg/ml 1n 20 A) CremophorEL); or PhaxanCD (alphaxalone 10 anaesthetlzed. The number of rats 1n each group of 10 s1m1- / 1 - 1_2 1 1 - - - h C - 1 R larl treated animals that scored 4 were sub'ected to robit mg m In a ' m0 ar comp exatlon rano Wlt apnso ( Sg y _ _ _ _ _ J p 1stered Trademark)i(7)sulfobutyl ether [3-cyclodextr1n). regressmn analySIS usmg SPSS Stalls?“ 18 to prOduce The number of rats that died was recorded for each group of graphs Of PrObll value V 10% dose (PrOblt P190 9119 also to 20 10 rats given the same dose of drug. Results from groups of calculate the est1mated dose that caused anaesthes1a 1n 50 and ten rats treated With the same anaesthetic and dose were 95% Of subjects (ADSO and A1395, respectively) for 11110011- combined for statistical purposes. The graph of the raw data is sciousness (righting re?ex measurements) and surgical ana- shown in FIG. 2 [% rats died in each dosage group v dose]. esthesia (tail pinch responses). The rotarod walking times At doses of alphaxalone between 50 and 60 mg/kg all the were also plotted for each dose and treatment. This was used 25 rats in the Althesin (Registered Trademark) groups died as a measurement of complete recovery. The results are whereas none died in the PhaxanCD groups that received the shown in FIGS. 1a through If; rotarod performance for each same doses of alphaxalone. The lethality values for PhaxanCD anaesthetic, n:10 rats per dose. showed a ceiling at 20%; no more than 20% rats died even if Table 3 below summarises the results of this series of the dose of alphaxalone administered as PhaxanCD were experiments. It can be seen that PhaxanCD is equipotent with 30 increased. The percentages of rats that died in all the different Althesin (Registered Trademark) in causing unconsciousness dosage treatment groups were subjected to a probit regression and surgical anaesthesia and both are more potent than pro- analysis using SPSS Statistics 18 and the probit values were pofol in this respect. Recovery from unconsciousness caused plotted on a graph against the log dose of anaesthetic; this is by PhaxanCD is just as fast as with propofol. However; recov- called a probit plot. This is shown in FIG. 3. ery from PhaxanCD is slightly slower than propofol but faster 35 The probit plot for Althesin (Registered Trademark) was than Althesin (Registered Trademark) if the depth of anaes- used to calculate the dose of alphaxalone in this formulation thesia is taken to a surgical level. Control experiments that caused death in 50% and 95% rats; the LD50 and LD95 revealed that the vehicles given alone intravenously, 20% respectively). These values were 43.6 mg/kg [LDSO] and 51.5 CremophorEL, 10% lntralipid and 13% Captisol had no mg/kg [LD95]. As the dose of alphaxalone was increased the sedating or anaesthetic effects. number of rats that died increased in proportion when the TABLE 3 Althesin (Registered Trademark) PhaxanCD propofol ANOVA with Tukey post hoc minimum dose causing all 10 rats to lose righting re?ex mgkg 5 5 10 ED5O dose for loss ofrighting re?ex mgkg 2.95 2.79 4.63 ED95 dose for loss ofrighting re?ex mgkg 4.39 4.26 8.4 minimum dose causing all 10 rats to lose tail pinch response 15 15 15 Ina/kg ED5O dose for loss oftail pinch response re?ex mgkg 6.46 6.56 8.4 ED95 dose for loss oftail pinch response mg/kg 14.09 8.56 14.46 duration ofloss ofrighting re?ex at dose causing all 10 rats mean = 3.6 1.9 2.5 not signi?cant ANOVA p = 0.0527 to lose righting re?ex (minutes) SD = 2.18 0.84 1.15 duration ofloss oftail pinch response at dose causing all mean = 13.45 4.65 4.05 Althesin vs PhaxanCD *** P < 0.001; 10 rats to lose tail pinch response (minutes) SD = 3.9 2.55 1.28 Althesin vs propofol *** P < 0.001; PhaxanCD vs propofol ns P > 0.05 time (minutes) to overall (complete) recovery of rotarod mean = 19.9 17 16 Althesin vs PhaxanCD ns P > 0.05; performance after minimum anaesthetic dose (lost RR) SD = 3.93 2.16 1.89 Althesin vs propofol * P < 0.05; PhaxanCD vs propofol ns P > 0.05 time (minutes) to overall (complete) recovery ofrotarod mean = 39 32.5 23.1 Althesin vs PhaxanCD *** P < 0.001; performance after minimum surgical anaesthetic dose SD = 3.16 3.54 3.25 Althesin vs propofol *** P < 0.001; (lost tail pinch) PhaxanCD vs propofol *** P < 0.001 It can be concluded from this set of experiments that: alphaxalone was given as Althesin (Registered Trademark). Phaxan is an effective intravenous anaesthetic causin fast By contra“ there was a ceiling effeCt Of the lethality Of the CD g 65 alphaxalone in (7) sulfobutyl ether [3-cyclodextrin (Phax onset of general anaesthesia after intravenous injection. anCD) formulation. The alphaxalone was much less assessed It is equipotent with Althesin and twice as potent as propofol. by lethality compared with alphaxalone formulated toxic as US 8,975,245 B2 1 7 18 with CremophorEL (Althesin [Registered Trademark]). A 10 rats each. They were all given intravenous injections of dose of 52 mg/kg alphaxalone as Althesin (Registered Trade alphaxalone formulated in 20% Cremophor at a dose that had, mark) caused all 10 rats in that group to die but 64 mg/kg in previous experiments reported in example 4, caused all rats alphaxalone caused no deaths in the 10 rats which received to die (Althesin [Registered Trademark]; alphaxalone dose that dose of alphaxalone formulated with (7) sulfobutyl ether 52.5 mg/kg ivithis equals 16 times the AD95 for Althesin [3-cyclodextrin (PhaxanCD). Furthermore, unlike the probit (Registered Trademark) at which it is expected a very high plot for Althesin (Registered Trademark), which showed a proportion or all rats will die). Sixty seconds before the Alth direct proportional relationship of increasing lethality with esin (Registered Trademark) injection a premedication inj ec increasing dose, alphaxalone formulated in (7) sulfobutyl tion was given: ether [3-cyclodextrin (PhaxanCD) showed a ceiling effect for group 1 (10 rats) received 5.3 mls/kg 0.9% sodium chloride lethality; when the dose of alphaxalone in this preparation solution 60 seconds before 52.5 mg/kg alphaxalone as was increased to 71, 78 and then 84 mg/kg only 20% of rats Althesin (Registered Trademark); died in each group. Thus, it was not possible to ?nd the dose group 2 (10 rats) received 5.3 mls/kg 13% solution of (7) of alphaxalone in this formulation that caused death in 50% sulfobutyl ether [3-cyclodextrin in 0.9% sodium chloride and 95% rats (the LD50 and LD95, respectively). In any event, solution 60 seconds before 52.5 mg/kg alphaxalone as both of these values are greater than 84 mg/kg which is more Althesin (Registered Trademark). than double the equivalent values for Althesin (Registered The number of rats that died in each group was recorded Trademark) and four times the value of the LD50 for alphaxa shown in Table 5. All 20 rats were anaesthetized by the injection of 52.5 mg/kg alphaxalone as Althesin (Registered lone formulated in hydroxypropyl [3-cyclodextrin manufac 20 tured by Jurox [Alfaxan CD-RTU Material Safety Data Sheet. Trademark). However, the presence of (7) sulfobutyl ether Jurox Pty, Newcastle NSW Australia]. [3-cyclodextrin caused a statistically and clinically signi?cant These results make the therapeutic index (ratio of dose that reduction in the mortality caused by the alphaxalone. causes death in 50% subjects (LDSO) divided by the dose that TABLE 5 causes anaesthesia in 50% subjects (the ADSO) to be 14.8 for 25 Althesin (Registered Trademark) and >30.2 for alphaxalone ALL RATS THAT SURVIVED WERE formulated in (7) sulfobutyl ether [3-cyclodextrin (Captisol ANAESTHETISED >1 HOUR [Registered Trademark] PhaxanCD). This difference is not premed given 60 s before Althesin @ 16 xAD95 due to differences in toxicity of the excipients. Table 4 below premed volume = 5.25 mls/kg 11 died 11 did not die shows the results of experiments in 10 rats with indwelling 30 jugular intravenous catheters. Five rats were given a 20% SALINE PREMED 8 2 solution of Cremophor EL intravenously and another ?ve rats SBECD7 PREMED 2 8 were given a solution of (7) sulfobutyl ether [3-cyclodextrin, Study Of The Effect ofCaptisol on the Death Rate ofAlthesin both being administered at a dose and volume equal to that Fisher’s Exact Test The two-sided P value is 0.0230, considered signi?cant. administered in the experiments above at the highest dose of 35 alphaxalone. Neither excipient caused death in any rat indi The row/column association is statistically signi?cant. cating that the difference in the safety/lethality of the two This is a much unexpected result. formulations of alphaxalone was not due to dose related tox icity of the excipients. EXAMPLE 6 40 TABLE 4 The Effect on Sleep Times of Repeated Dosing with n/lO died vehicle dose = ml/kg PhaxanCD 0 13% Captisol 9.0 The mechanism responsible for the (7) sulfobutyl ether 0 20% Cremophor EL 4.6 45 [3-cyclodextrin in causing the ceiling effect on alphaxalone toxicity is unknown. It is known that alphaxalone is very poorly soluble in water and thus the vast majority of alphaxa EXAMPLE 5 lone molecules are complexed with the cyclodextrin mol ecules in the ratio of 1 :2 (the complexation ratio). It is known Demonstration of the Limitation of Alphaxalone 50 that some of the alphaxalone will dissociate from the cyclo Toxicity by Captisol [Registered Trademark] dextrin complex when PhaxanCD is injected intravenously. (Sulfobutyl Ether [3-cyclodextrin) The question posed by the unique property of the alphaxalone sulfobutyl ether cyclodextrin complex exhibiting a ceiling Since the potency in causing anaesthesia is the same for a effect for toxicity is whether this is caused by limiting the bolus dose of alphaxalone whether given in 20% Cremo 55 amount of alphaxalone released from the complex or whether phorEL (Althesin [Registered Trademark]) or in (7) sulfobu there occurs a “mopping up” of alphaxalone molecules that tyl ether [3-cyclodextrin (PhaxanCD), then the ceiling effect might otherwise penetrate the brain to cause toxicity, by for toxicity must be related to the (7) sulfobutyl ether [3-cy excess uncomplexed cyclodextrin molecules freed up by clodextrin when higher doses of alphaxalone in the (7) sul alphaxalone metabolism by the liver. The effect of the latter fobutyl ether [3-cyclodextrin formulation are administered. 60 would be predicted to cause a progressive decrease in the Such a property has not been described for intravenous anaes level of free alphaxalone as the concentration of uncom thetics before. Furthermore, this property has not been plexed sulfobutyl ether cyclodextrin increased as a result of: described before for alphaxalone formulated in other cyclo liver metabolism of the alphaxalone; dextrins. more doses of PhaxanCD being administered thereby making In order to test whether the low toxicity of PhaxanCD was 65 more uncomplexed cyclodextrin available. due to the Captisol excipient, twenty rats with indwelling If this were the case one would predict tolerance to jugular intravenous catheters were divided into two groups of repeated doses of alphaxalone formulated in (7) sulfobutyl US 8,975,245 B2 19 20 ether [3-cyclodextrin i.e., repeated injections of PhaxanCD time with each successive dose. On the contrary the sleep time would cause progressively less sedation and anaesthetic increased with each of the ?rst four doses and then remained effect. constant thereafter with each subsequent dose. In order to test this ?ve rats with indwelling jugular intra venous catheters were given repeated injections of the mini EXAMPLE 7 mum dose of alphaxalone in (7) sulfobutyl ether [3-cyclodex trin (5 mg/kg PhaxanCD) that had caused 10/10 rats to be Ceiling Toxicity anaesthetized as judged by complete loss of righting re?ex. The time was measured for each rat to begin recovery of The following assumption is made: righting re?ex (progress from a score of 4 to a score of 3 in the A. that intravenous injection of Althesin (Registered Trade righting re?ex as described in example 2 above) and then mark) leads to an instantaneous dispersal of alphaxalone in another dose of 5 mg/kg PhaxanCD was given. The time to the the plasma but, since anaesthesia is caused by drug injec beginning of recovery of the righting re?ex after the second tion in one circulation and also alphaxalone is cleared from dose of anaesthetic was measured and then another dose of 5 the blood by the liver on ?rst pass, the level achieved by the mg/kg PhaxanCD was administered iv and the process was mixing in the plasma will only reach 30% of the theoretical repeated eight more times. The progressive recovery times for maximum assuming instantaneous mixing. the ?rst to the tenth doses are shown in the histogram (FIG. 4) The following is considered: and table 6 below as means (sem) for those ?ve rats. It can be . alphaxalone is soluble in water to 0.03 mg/ml; seen that there was a signi?cant progressive increase in recov 20 2. alphaxalone is 35% protein bound in plasma; ery times after each of the ?rst four doses. Further doses 5-10 3. plasma volume is 31 ml/kg in rat (Davies and Morris, caused no further signi?cant increase and more importantly, Pharmaceutical Research, 10(7):1093-95, 1993); no decrease, in sleeping time. These results indicate that the 4. both Althesin (Registered Trademark) and PhaxanCD have alphaxalone toxicity ceiling effect caused by the (7)sulfobu alphaxalone concentrations of 10 mg/ml; tyl ether [3-cyclodextrin is probably due to the controlled 25 5. for induction of anaesthesia Althesin (Registered Trade release of the alphaxalone from the complex at a rate su?i mark) and PhaxanCD are equipotent; a minimum of 4.3 cient to cause anaesthesia but no greater than that, no matter mg/kg alphaxalone by either preparation cause sleep in how much of the complex is given. This is a unique balance most (95%) rats; between the chemical relationship of the alphaxalone and the 6. after an initial bolus injection a drug preparation will (7) sulfobutyl ether [3-cyclodextrin, the low solubility of the 30 equilibrate with the plasma volume during the ?rst sleep alphaxalone in water environments, including biological ?u cycle but later the drug will disperse into the extracellular ids, the amount of alphaxalone needed to penetrate the brain ?uid [ECF] which is:297 ml/kg (Davies and Morris, 1993 to cause anaesthesia and the pharmacokinetics of alphaxa supra); lone. The results are shown in FIG. 4 and Table 6. None of this 7. Captisol is distributed to ECF and restricted to this space; was expected nor could have been predicted from prior art. 35 and 8. Alphaxalone is only released from the Captisol into an TABLE 6 aqueous environment if the level of free alphaxalone in the dose sleep time mins aqueous environment is less than saturation i.e., <0.03 mg/ml [fact 1]. number mean sem 40 It is proposed herein that: ?rst 0.8 0.14 I. From A and 5 above, the plasma level of drug needed to second 2.5 0.35 cause sleep:dose administered as Althesin/plasma vol third 2.9 0.41 ume:4.3/31:0.14 mg/ml. fourth 3.2 0.45 II. Applying A, the plasma concentration is expected to be ??h 3.7 0.38 45 30% of this when the blood has circulated to mix the drug sixth 4.1 0.21 seventh 4.1 0.21 effectively:0.046 mg/ml. eighth 4.2 0.14 III. From this, the level of free unbound alphaxalone in the ninth 4.3 0.34 plasma associated with anaesthesia induction after a single tenth 4.2 0.14 iv bolus injection:65% of total (from fact 50 2):0.045><0.65:0.03 mg/ml. One way ANOVA [Tukey Kramer post hoc] applied to the IV. Proposition III above is exactly the known solubility of data of sleep times after X 10 repeated dosing of 5 rats with alphaxalone in water. PhaxanCD 5 mg/kg revealed that there was a progressive sta V. Combining points 5 and 8 with proposition IV, it is pro tistically signi?cant increase in sleeping time from the ?rst to posed herein that the ?rst induction dose of PhaxanCD the second, second to the third and third to the fourth but no 55 caused anaesthesia by releasing all of the alphaxalone from increase in sleeping times thereafter. Furthermore it is impor the complex just achieving the anaesthetic level and free tant to note that there was not a progressive decrease in alphaxalone saturation level. sleeping times that would be predicted if the mechanism for VI. When the second dose of anaesthetic was administered in low toxicity and the ceiling effect of PhaxanCD was due to Example 9 the rat was starting to recover from the anaes uncomplexed “free” sulfobutyl ether cyclodextrin [Captisol 60 thetic because some free alphaxalone had been metabo (Registered Trademark)] mopping up alphaxalone from the lized by the liver, some free alphaxalone had been redis blood. If that were the case then the amount of free uncom tributed to the ECF and some of the Captisol containing plexed Captisol would increase as more doses were given and alphaxalone had also redistributed to the ECF. Thus, the as alphaxalone was metabolized by the liver so leading to a free alphaxalone level fell and alphaxalone left the brain so progressive increase in free uncomplexed Captisol as the 65 causing awakening. Thus, a further dose was given. Unlike repeated exposure experiment progressed. If the alphaxalone the ?rst dose, there was still alphaxalone in the blood so were mopped up by this then one would expect less sleeping only some of the alphaxalone was released from the com