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`US 20100041769Al
`
`c19) United States
`c12) Patent Application Publication
`Pacheco et al.
`
`c10) Pub. No.: US 2010/0041769 Al
`Feb. 18, 2010
`(43) Pub. Date:
`
`(54) STABLE AND READY-TO-USE
`OIL-IN-WATER PROPOFOL
`MICRO EMULSION
`
`(76)
`
`Inventors:
`
`Ogari Pacheco, Itapira (BR);
`Roberto Moreira, Itapira (BR);
`Marisa Rizzi, Itapira (BR)
`
`Correspondence Address:
`BIRCH STEWART KOLASCH & BIRCH
`POBOX747
`FALLS CHURCH, VA 22040-0747 (US)
`
`(21)
`
`Appl. No.:
`
`12/447,247
`
`(22)
`
`PCT Filed:
`
`Oct. 23, 2007
`
`(86)
`
`PCT No.:
`
`PCT /BR2007 /000283
`
`§ 371 (c)(l),
`(2), ( 4) Date:
`
`Jul. 23, 2009
`
`(30)
`
`Foreign Application Priority Data
`
`Oct. 27, 2006
`
`(BR) ............................... PI0604377-1
`
`Publication Classification
`
`(51)
`
`Int. Cl.
`A61K 31105
`A61P 23100
`
`(2006.01)
`(2006.01)
`
`(52) U.S. Cl. ........................................................ 5141731
`
`(57)
`
`ABSTRACT
`
`The present invention describes a new propofol-containing
`anesthetic pharmaceutical composition for parenteral admin(cid:173)
`istration, in the form of an oil-in-water microemulsion in
`which the oily phase is constituted by propofol in the form of
`particles with size comprised between 1 and 100 nm using a
`single surfactant selected from the group consisting of poly(cid:173)
`ethylene glycol stearates with general formula C 17H35COO.
`(OCH2CH2)nH or C 1 7H35COO. (OCH2CH2 )n.COOC 1 7H35 .
`The anesthetic pharmaceutical composition of the present
`invention is more potent for induction of hypnosis and anes(cid:173)
`thesia, has a ready-to-use presentation and highly stable par(cid:173)
`ticle size, presenting improved physicochemical properties,
`and preventing the potential risks of undesirable effects
`encountered in the state-of-the-art propofol formulations.
`
`Petition for Inter Partes Review of US 8,338,470
`Amneal Pharmaceuticals LLC – Exhibit 1032 – Page 1
`
`

`

`US 2010/0041769 Al
`
`Feb. 18, 2010
`
`1
`
`STABLE AND READY-TO-USE
`OIL-IN-WATER PROPOFOL
`MICRO EMULSION
`
`FIELD OF THE INVENTION
`
`[0001] The present invention describes an injectable ready(cid:173)
`to-use anesthetic pharmaceutical composition for parenteral
`administration containing propofol as the active agent in the
`form of an oil-in-water microemulsion, whose disperse
`hydrophobic particles present much reduced and stable
`dimensions, which confer a transparent aspect to the micro(cid:173)
`emulsion.
`
`STATE OF THE ART
`
`[0002] The active agent Propofol, the active agent of the
`present invention, is also designated by the chemical name
`2,6-Bis-(1-methylethyl)-phenol. Processes for its preparation
`are described, for example, in the patents U.S. Pat. No. 2,831,
`898 and U.S. Pat. No. 4,447,657, whereas its anesthetic and
`sedative/hypnotic activity
`in mammals has been first
`described in the patent U.S. Pat. No. 4,056,635.
`[0003] An injectable anesthetic lipid emulsion containing
`propofol at 1 % and 2% concentrations (w/v) is currently
`available in the market under the brand name DRIPIVAN®.
`In Brazil, this medicine is also found under the brand name
`PROPOVAN®.
`[0004] Propofol has a short-term action, being adequate for
`induction and maintenance of general anesthesia; sedation
`during local surgical techniques; sedation for ventilated
`patients receiving intensive care; and conscious sedation for
`surgeries and diagnostic procedures conducted at intensive
`care units. It is usually administered by single or repeated
`intravenous injections by bolus dose or continuous infusion,
`being rapidly cleared from blood stream and metabolized. For
`this reason, deep anesthesia is easily controlled and patient
`recovery after drug withdrawal is usually fast.
`[0005] Propofol's characteristic of having a rapid start of
`action is attributed, in great part, to its considerable liposolu(cid:173)
`bility, as this characteristic provides a fast crossing of the
`hematoencephalic barrier. In order to ensure the adequate
`velocity for induction of anesthesia, administration of propo(cid:173)
`fol directly to the blood stream is of great interest.
`[0006] Nevertheless, the low solubility of propofol in water
`to
`the development of formulations suitable
`hinders
`parenteral administration, as the bodily fluids are basically
`constituted of water.
`[0007] A usual technique to increase the hydrophilicity of a
`certain drug is to obtain ionizable salts derived from this drug
`in order to tum it more soluble in water, thus allowing the
`release of the active free base in vivo. However, salt ionization
`depends on its pKa and, occasionally, the relation between the
`physiologic pH and the resulting salt pKa may be incompat(cid:173)
`ible with the adequate degree of drug ionization that is nec(cid:173)
`essary for its effective distribution and absorption.
`[0008]
`In spite of the significant number of studies using
`the above-mentioned approach, its application to propofol
`was not possible. In this case, the propofol-carrier pharma(cid:173)
`ceutical composition plays a key role on the increase of the
`hydrosoluble characteristics of the final product in an attempt
`to provide an adequate drug delivery via blood stream.
`[0009] The most common technique for development of
`more adequate formulations for intravenous administration
`has been the incorporation of propofol to pharmaceutical
`
`compositions in the form of oil-in-water emulsions in which
`the drug is solubilized in the disperse phase, which is usually
`constituted of fatty acids, vegetable oils and/or triglycerides.
`[0010] The state of the art presents propofol in oil-in-water
`emulsions constituted by vegetal oil, preferably soy oil,
`which incorporate a phospholipid, such as egg lecithin, as a
`tensoactive agent. As an example, propofol is supplied in an
`Intralipid® emulsion, a lipid emulsion usually administered
`intravenously for hypercaloric parenteral nutrition.
`[0011] Nevertheless, unsaturated fatty acids that constitute
`the Intralipid® emulsion are highly susceptible to peroxida(cid:173)
`tion, producing hydroperoxides, which may explain the tox(cid:173)
`icity and carcinogenic risk of such lipid emulsions [Helblock
`HJ; et al "Toxic hydroperoxides in intravenous lipid emul(cid:173)
`sions used in preterm infants", Pedaytrics 91, 83 (1993)]. In
`addition, such emulsions are supportive to microbial growth,
`leading to the occurrence of postoperative infectious events
`[Heidmann E et al "The association of propofol usage with
`postoperative wound infection rate in clean wounds: a retro(cid:173)
`spective study" Vet Surg 28(4), 1999; Seeberger MD et al
`"Efficacy of specific aseptic precaution for preventing propo(cid:173)
`fol-related infections: analysis by a quality-assurance pro(cid:173)
`grammer using the explicit outcome method". J Hosp Infect
`39(1), 1998; Sklar GE "Propofol and postoperative infec(cid:173)
`tions" Ann Pharmacother 31(12). 1997].
`[0012] Consequently, the oil-in-water emulsions depended
`on the incorporation of preservatives to prevent the oxidation
`of the lipid components. The use of preservatives is also
`necessary to prevent microbial growth or the use of extremely
`aseptic techniques to avoid the contamination and develop(cid:173)
`ment of microorganisms in the formulations.
`[0013] Several documents on the state of the art have shown
`attempts to prevent or eliminate this problem.
`[0014] Patents EP 814787 and U.S. Pat. No. 5,714,520
`describe a composition containing disodium EDTA in a suf(cid:173)
`ficient amount to prevent microbial growth. Nevertheless, the
`incorporation of di sodium EDTA to a formulation is not rec(cid:173)
`ognized by the USP (United States Pharmacopeia) standards
`as a preventive action against microbial growth [Sklar GE
`"Propofol and postoperative infections" Ann Pharmacother
`31 (12);1521-1523. 1997; and WO 99/39696)].
`[0015] Patent application WO 99/39696 describes the use
`of a sulfite, preferably sodium metabisulfite, in a non-toxic
`amount to delay or suppress the growth of microbial contami(cid:173)
`nants. However, such substances may cause allergic reac(cid:173)
`tions.
`[0016]
`It is important to emphasize that propofol is the drug
`of choice for use as a long-term sedative medication in bed(cid:173)
`ridden patients. Therefore, the great amount of oil present in
`these oil-in-wateremulsions and the prolonged exposure may
`cause problems associated to lipid overload in the blood
`stream, which produces an elevation in lipidemia. This lipid
`hyperalimentation may exceed the patient capacity to elimi(cid:173)
`nate fats from blood stream resulting in the so-called Fat
`Overload Syndrome [Lindholm M "Critically ill patients and
`fat emulsions", Minerva Anestesiol 58(10), 1992], which
`causes sudden elevation of triglycerides serum levels,
`increase of bilirrubin levels in blood stream, "fatty liver",
`fever, hepatosplenomegaly, coagulopathy and other dysfunc(cid:173)
`tions in different organs [Haber et al. "Fat overload syndrome.
`An autopsy study with evaluation of the coagulopathy". Am J
`Clin Pathol 90(2):223-227. 1988]. Furthermore, tolerance to
`fat in some ill patients may decrease, leading to secondary
`metabolic alterations.
`
`Petition for Inter Partes Review of US 8,338,470
`Amneal Pharmaceuticals LLC – Exhibit 1032 – Page 2
`
`

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`US 2010/0041769 Al
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`Feb. 18, 2010
`
`2
`
`[0017] An additional problem related to the use of intrave(cid:173)
`nous emulsions consists in the voluminous dimensions of the
`particles in the disperse phase (103 to 104 nm). Very dense
`particles confer greater instability between the phases and
`considerable turbidity to formulation. This fact hinders the
`visual control of sterility of the emulsion and may cause
`embolism.
`[0018] The risk posed by the particle size of the emulsions
`may be overcome by the development of microemulsions.
`The microemulsions are prepared with the use of surfactants
`that are adequate for solubilization of the drug under consid(cid:173)
`eration and are able to decrease the interfacial tension.
`[0019] Patent U.S. Pat. No. 5,637,625 refers to a formula(cid:173)
`tion containing propofol microparticles wrapped by a fat-free
`and triglyceride-free phospholipid layer. Such microparticles
`have dimensions between 100 nm and 200 nm. The aqueous
`phase of the referred formulation is constituted by glucose/
`phosphate buffer with pH adjusted to 7 .0. Although the patent
`reports that the formulation does not propitiate the develop(cid:173)
`ment of microorganisms for not containing oils as a microbial
`nutrient, the inclusion of glucose or another carbohydrate in
`the formulation accounts to favors microbial growth.
`[0020] Patent U.S. Pat. No. 6,071,974 incorporates propo(cid:173)
`fol in the solvent 2,5-dimethylisosorbide that is miscible with
`water. According to the inventors, it constitutes a formulation
`of comparable efficacy to that of the currently marketed for(cid:173)
`mulation.
`[0021] Patents U.S. Pat. No. 6,623,765 and GB 2359747
`describe injectable microemulsions containing propofol and,
`additionally, a surfactant constituted by long-chain polymers,
`specifically poloxamers, for the formation of micelles.
`[0022] Patent U.S. Pat. No. 6,743,436 describes an inject(cid:173)
`able composition in the form of oil-in-water microemulsion
`for propofol incorporating poloxamer as surfactant. The
`patent refers to poloxamer as an adequate surfactant to form
`microemulsions with a particle size of 100 nm or below.
`Additionally, it mentions the use of co-surfactants selected
`from the group consisting ofSOLUTOL HS 15 (Macrogol 15
`Hydroxystearate), egg
`lecithin, LABRASOL (polyoxy
`caprylic glyceride), polyoxyl 10 oleyl ether, TWEEN, ethanol
`and polyethylene glycol. According to the patent, poloxamer
`407 (0.1to5%) is the preferred surfactant, butpoloxamer 188
`is also mentioned among its examples.
`[0023] Nevertheless,
`injectable poloxamer-containing
`compositions have shown limitations with respect to the use
`of greater volumes or prolonged administration. These for(cid:173)
`mulations are associated to the Lipid Overload Syndrome,
`which causes damages to the patient, specifically for causing
`hypertriglyceridemia and hypercholesterolemia, with conse(cid:173)
`quent induction of atherosclerosis [ Jonhston T Pet al "Poten(cid:173)
`tial downregulation ofHMG-CoA reductase after prolonged
`administration of P-407 in C57BL/6 mice". J Cardiovasc
`Pharmacol. 34(6). 1999; Johnston T Pet al "Poloxamer 407-
`induced atherosclerosis in mice appears to be due to lipid
`derangements and not to its direct effects on endothelial cells
`and macrophages". Medaytors Inflamm. 12(3). 2003; Jonh(cid:173)
`ston T P "The P-407-induced murine model of dose-con(cid:173)
`trolled hyperlipidemia and atherosclerosis: a review of finds
`to date". J Cardiovasc Pharmacol. 43(4). 2004].
`[0024] Another propofol microemulsion constituted by a
`surfactant mixture has been described by Ryoo et al. [Ryoo H
`Ket al "Development of propofol-loadedmicroemulsion sys(cid:173)
`tem for parenteral delivery". Arch Pharm Res. 2005 28(12):
`1400-1404]. In their study, Ryoo et al. presented an acceler-
`
`ated stability assay (at 40° C.) conducted within only 8 weeks
`involving three different 1 % propofol microemulsions con(cid:173)
`taining a surfactant/ co-surfactant mixture comprised by Solu(cid:173)
`tol/Ethanol in a 5:1 ratio. The studied microemulsions were
`prepared containing 4, 6 and 8% by weight of the 5:1 Solutol/
`Ethanol mixture. The graph presented in that study shows that
`two out of three formulations demonstrated an increase in
`particle diameter to a value close to 100 nm in one case
`(mixture at 6%) and close to 200 nm in the other case (mixture
`at 4%) within only 8 weeks of follow-up of the stability assay.
`In addition of containing a surfactant mixture, this formula(cid:173)
`tion is not able to maintain the desired stability regarding
`particle size.
`[0025] Patent application W02005/0797 58 describes bases
`for a self-microemulsifiable composition comprising two or
`four components. The base that comprehends two compo(cid:173)
`nents is constituted by a first component formed by a surfac(cid:173)
`tant containing polyethylene glycol and a liquid propofol
`containing vitamin E; and by a second component that is a
`saline isotonic aqueous carrier, or dextrose. The carrier is
`mixed to the first component at the moment of use. On the
`other hand, the base comprising four components is more
`complex, being constituted essentially by a surfactant con(cid:173)
`taining polyethylene glycol, a vitamin E-containing liquid
`propofol, a co-solvent immiscible in water, and ethanol. The
`document affirms that the base is stable for undetermined
`time; however, no stability assay is presented to confirm this
`statement. One of the advantages attributed to this invention
`would be the possibility of preparing a formulation contain(cid:173)
`ing propofol at high concentrations. Nevertheless, the author
`does not justify the importance of increasing propofol con(cid:173)
`centration. On the contrary, it is known that high concentra(cid:173)
`tions of propofol may cause side effects characterized, for
`example, by myocardial failure, metabolic acidosis and rab(cid:173)
`domyolisis [De Cosmo, Get al. "Sedation in PACU: The Role
`of Propofol" Current Drug Targets. 2005. 6 (7): 741]
`[0026]
`In view of the deficiencies found in the state of the
`art referring to the excipients employed in injectable propofol
`compositions, there is the need for development of ready-to(cid:173)
`use microemulsions that present smaller particle size and
`high stability and that minimize the side effects inherent to
`propofol or those related to Overload Syndromes.
`
`DESCRIPTION OF THE INVENTION
`
`In this context, the present invention describes an
`[0027]
`injectable anesthetic pharmaceutical composition, contain(cid:173)
`ing propofol as the active agent, in the form of a highly stable
`ready-to-use oil-in-water microemulsion, whose disperse
`hydrophobic particles present much reduced dimensions.
`[0028] Surprisingly, the propofol microemulsion of the
`present invention was more potent for induction and mainte(cid:173)
`nance of hypnosis and anesthesia compared to the propofol
`compositions existing in the prior art. It was noticed that the
`ready-to-use microemulsion of the present invention pro(cid:173)
`duces anesthetic and hypnotic effects equivalent to those
`obtained with the conventional formulations containing 1 %
`(w/v) propofol, though with half the concentration (0.5%
`w/v). The use of lower propofol doses to reach the desired
`anesthetic and hypnotic effect minimizes the risks of side
`effects and potential adverse reactions inherent to propofol.
`[0029] Therefore, the microemulsion of the present inven(cid:173)
`tion is characterized by the propofol being comprised in the
`range between 0.1 and 5% (w/v) of the final composition.
`Preferably, propofol is comprised in the 0.1to2% (w/v) range
`
`Petition for Inter Partes Review of US 8,338,470
`Amneal Pharmaceuticals LLC – Exhibit 1032 – Page 3
`
`

`

`US 2010/0041769 Al
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`Feb. 18, 2010
`
`3
`
`and, even more preferably, propofol is comprised in the range
`between 0.5% and 1 % (w/v) of the final composition.
`[0030] An important aspect of the ready-to-use microemul(cid:173)
`sion of the present invention, resides in the fact that its dis(cid:173)
`perse particles present much reduced dimensions, compre(cid:173)
`hended between 1 and 100 nm, more specifically, between 1
`and 50 nm, which remained stable for at least 12 months. Due
`to this characteristic, the microemulsion of the present inven(cid:173)
`tion has a transparent aspect and viscosity comparable to that
`of an aqueous solution.
`[0031] The microemulsion of the present invention is also
`characterized by containing a single surfactant selected from
`the group consisting of polyethylene glycol stearates, prefer(cid:173)
`is C 17H35COO.
`ably non-ionic.
`Its general
`formula
`(OCH2CH2 )nH or C 17H35COO.(OCH2CH2 )n.COOC 1 7H35
`and it is comprised within the range between 1 and 50% (w/v)
`of final composition, or preferably between 5 and 20% (w/v)
`of the final composition.
`[0032] Preferably, the microemulsion of the present inven(cid:173)
`tion uses SOLUTOL HS 15 (Macrogol 15 Hydroxystearate)
`as surfactant, but it is not restrictive to the scope of the present
`invention. Other surfactants adequate for the development of
`the present invention are those comprised in the group con(cid:173)
`sisting ofpolyoxyethylene (4) monostearate, polyoxyethyl(cid:173)
`ene (6) monostearate, polyoxyethylene (8) monostearate,
`polyoxyethylene (12) monostearate, polyoxyethylene (20)
`monostearate, polyoxyethylene (30) stearate, polyoxyethyl(cid:173)
`ene (40) monostearate, polyoxyethylene (50) monostearate,
`polyoxyethylene (100) monostearate, polyoxyethylene (150)
`stearate, polyoxyethylene ( 4) distearate, polyoxyethylene (8)
`distearate, polyoxyethylene (12) distearate, polyoxyethylene
`(32) distearate, polyoxyethylene (150) distearate.
`[0033] Optionally, the microemulsion of the present inven(cid:173)
`tion may contain agents for adjustment of pH and agents for
`adjustment of osmolarity that are pharmaceutically accept(cid:173)
`able for the intravenous environment. The preferred agents
`for such purposes, but not limiting the scope, are sodium
`hydroxide and glycerol, respectively.
`[0034] A microemulsion of the present invention may be
`sterilized by filtration through a sterilizing membrane with
`0.22 µm-diameter pores.
`[0035] The administration route of the pharmaceutical
`composition of the present invention is, preferably, the intra(cid:173)
`venous route. Nevertheless, the formulation is also adequate
`for intramuscular, subcutaneous, intradermal and spinal
`administration.
`[0036] One aspect of the present invention is that the phar(cid:173)
`maceutical composition obtained by the ingredients and pro(cid:173)
`cesses described herein provides a thermodynamically stable
`product with a single homogeneous phase and transparent
`appearance.
`[0037] Another aspect of the present invention is that the
`microemulsion described herein is highly stable and ready(cid:173)
`to-use, with particle size maintained below 50 nm, even after
`the 12-month duration of the stability assay, unlike the most
`similar microemulsions known in the state of the art.
`[0038] The microemulsion of the present invention may be
`prepared by conventional techniques for preparing micro(cid:173)
`emulsions described in the state of the art. Preferably, the
`microemulsion of the present invention may be prepared
`according to the following steps:
`(a) Providing a first receptacle containing an amount
`[0039]
`between 1 to 50% (w/v) of a non-ionic polyethylene glycol
`
`stearate surfactant, maintaining the system under constant
`stirring, preferably under heating around 50° C., until fusion
`of the surfactant;
`(b) Adding approximately 5-10% of the total water
`[0040]
`of the final composition and an amount between 0.1and5%
`(w/v) ofpropofol to the mixture of the first receptacle, main(cid:173)
`taining the system under constant stirring;
`(c) Providing a second receptacle with a stirring
`[0041]
`system containing 50-85% of total water for the final compo(cid:173)
`sition;
`( d) Adding the mixture of the first receptacle to the
`[0042]
`second receptacle, under constant agitation, until homogeni(cid:173)
`zation;
`( e) Completing the final volume of the composition
`[0043]
`with water, under constant agitation, until homogenization;
`(f) Sterilizing the final composition using a 0.22 µm
`[0044]
`filtrating membrane.
`[0045] Optionally, a pharmaceutically acceptable osmolar(cid:173)
`ity agent can be added to the mixture together with the accom(cid:173)
`plishment of the step (d).
`[0046] Yet, a pharmaceutically acceptable pH adjustment
`agent can be added during the step ( e) to provide a final pH
`between 5.0 and 8.5.
`In addition to the physicochemical improvements
`[0047]
`and the surprising verification of the increase of the power for
`the anesthetic and hypnotic effect obtained with the above(cid:173)
`mentioned pharmaceutical composition of the present inven(cid:173)
`tion, the microemulsion of the present invention has also
`other advantages over the compositions known in the state of
`the art, such as: (i) the fact that the composition of the present
`invention does not present lipid- or lecithin-derived compo(cid:173)
`nents eliminates the risks of formation of toxic sub-products
`from degradation/oxidation of these components; (ii) the
`composition of the present invention does not present com(cid:173)
`ponents derived from oils and sugars, which are factors that
`favor the growth of microorganisms and, as result, the poten(cid:173)
`tial contamination of the product and patients is minimal; (iii)
`when administered during prolonged periods, the composi(cid:173)
`tion of the present invention eliminates the damage associated
`with the lipid Overload Syndrome; (iv) because the compo(cid:173)
`sition of the present invention has surfactant of another
`nature, it also eliminates the damages associated with the
`Lipid Overload Syndrome that can occur with poloxamer(cid:173)
`containing formulations administered for a prolonged period;
`(v) because the composition of the present invention presents
`particles of much reduced dimensions, it eliminates the risks
`associated with embolism; (vi) because it requires half the
`dose to reach the desired anesthetic and hypnotic effects with
`propofol, the composition of the present invention minimizes
`potential side effects and adverse reactions associated with
`higher doses of propofol used until the present moment; (vii)
`the highly stable and ready-to-use microemulsion of the
`present invention can be stored for long periods without
`undergoing alterations in its physicochemical aspect and pre(cid:173)
`senting easiness of handling and administration to patient.
`[0048] Therefore, the pharmaceutical composition of the
`present invention permits either short-term or long-term
`parenteral use of propofol, with increased safety and reliabil(cid:173)
`ity.
`[0049] The following Examples aim to demonstrate, in a
`non-restrictive manner, the best form for performing the
`
`Petition for Inter Partes Review of US 8,338,470
`Amneal Pharmaceuticals LLC – Exhibit 1032 – Page 4
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`US 2010/0041769 Al
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`Feb. 18, 2010
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`4
`
`present invention, as well as its advantages in comparison to
`the state-of-the-art formulations.
`
`Example 1
`
`Preparation of 1 % and 0.5% Propofol-Containing
`Microemulsions
`
`[0050] One accomplishment of the present invention may
`be illustrated by preparation of MICROEMULSION 1 and
`MICRO EMULSION 2, whose preferred propofol and excipi(cid:173)
`ent ratios are described in Table 1 below:
`
`TABLE 1
`
`Propofol microemulsions.
`
`Components
`
`Propofol
`Macrogol 15 Hydroxy(cid:173)
`stearate (SOLUTOL
`HS 15)
`Glycerol (Glycerin BCA
`FCTA)
`Sodium Hydroxide PA
`Water for Injection
`
`MICROEMULSION 1 MICROEMULSION 2
`o/ow/v
`o/ow/v
`
`10
`
`2.5
`
`0.5
`10
`
`2.5
`
`q.s.t. pH 5.0 to 8.5
`q.s.t 100
`
`q.s.t. pH 5.0 to 8.5
`q.s.t. 100
`
`[0051] According to the present invention, MICRO EMUL(cid:173)
`SIONS 1 and 2 were prepared as follows:
`[0052] Seventy percent of the total water for injection was
`added to a stainless steel reactor presenting a stirring system.
`Separately, the macrogol 15 hydroxystearate (SOLUTOL HS
`15) was added to a stainless steel receptacle and heated up to
`50° C., under constant stirring, for complete fusion of the
`product. Next, 6% of the total water for injection and propofol
`were added to the fused surfactant, under constant agitation.
`
`[0053] The content of the stainless steel receptacle was
`added to the reactor containing 70% of the total water for
`injection, together with glycerol, under constant stirring, until
`total homogenization was obtained. The pH was adjusted to
`the 5.0 to 8.5 range, using a 1 N sodium hydroxide solution,
`previously prepared with water for injection. The final vol(cid:173)
`ume of the composition was completed with water for injec(cid:173)
`tion, checking again the final pH.
`[0054] The final composition was filtered inAP-15 257-25
`Pre-Filter and GVWP 293-25 sterilizing membrane with 0.22
`µm-diameter pores.
`[0055] At the end of this process, the product was enclosed
`in appropriate sterile flasks.
`[0056] The resulting formulation presents as a transparent
`microemulsion free of foreign particles.
`
`Example 2
`Stability of the Propofol Microemulsion of the
`Present Invention
`[0057] Freshly prepared MICROEMULSIONS 1 and 2
`according to Example 1 were submitted to tests to evaluate
`their characteristics. The evaluation of particle size was the
`most important issue for the present invention because the
`stabilization ofthis parameter is one of the deficiencies of the
`propofol microemulsions described in the art.
`[0058] Particle size was monitored during the 12-month
`stability assay under normal temperature conditions (Table 2)
`and during the 180-day accelerate stability assay at 40° C.
`(Table 3).
`[0059] The results displayed on Tables 2 and 3 demonstrate
`that there was no significant alteration in particle size during
`the monitoring time. Even the older samples presented sig(cid:173)
`nificantly smaller particle size than the maximum limit of 50
`nm established by the present invention as the preferential
`limit.
`
`TABLE2
`
`Effect of the storage time, under the usual conditions of long-term stability
`assays, on the particle size of the propofol microemulsions.
`
`Particle sizes (established limit: up to 100 nm)
`30° C. ± 2° C. and relative humidity of75% ± 5%
`
`Microemulsion
`
`12
`9
`INITIAL MONTH MONTHS MONTHS MONTHS MONTHS
`
`2
`
`16.2
`19.2
`
`17.5
`29.6
`
`17.1
`36.0
`
`19.2
`27.1
`
`17.3
`17
`
`16.6
`18.8
`
`TABLE3
`
`Effect of the storage time, Wlder the usual conditions of accelerate
`stability assays, on the particle size of the propofol microemulsions
`
`Particle sizes (established limit: up to 100 nm)
`40° C. ± 2° C. and relative humidity of75% ± 5%
`
`Microemulsion
`
`12
`9
`INITIAL MONTH MONTHS MONTHS MONTHS MONTHS
`
`2
`
`19.2
`19.2
`
`29.6
`29.6
`
`36.0
`32.1
`
`27.1
`37.6
`
`17
`28.9
`
`18.8
`19.2
`
`Petition for Inter Partes Review of US 8,338,470
`Amneal Pharmaceuticals LLC – Exhibit 1032 – Page 5
`
`

`

`US 2010/0041769 Al
`
`Feb. 18, 2010
`
`5
`
`Example 3
`
`Comparative Analysis of the Stability of State-of(cid:173)
`the-Art Propofol Microemulsions and the Micro(cid:173)
`emulsion of the Present Invention
`
`[0060] Formulations described in the reference Patent U.S.
`Pat. No. 6,743,436 (Examples 1, 5 and 6) were elected for
`comparative purposes as well as to demonstrate the physico(cid:173)
`chemical improvements reached with the pharmaceutical
`composition of the present invention, considering the teach(cid:173)
`ings of the closest state-of-the-art to this new composition.
`[0061] When the compositions were reproduced according
`to the teachings of the Patent used as a reference it was
`observed that, in general, the turbidity did not meet the micro(cid:173)
`emulsion parameters and the analysis demonstrated particle
`size much above the limit of 100 mn (as shown in Table 4).
`
`TABLE4
`
`Determination of the particle sizes obtained in accelerated
`stability assays (40° C. ± 2° C.).
`
`60 DAYS
`30 DAYS
`INITIAL
`Specification: Particle Size (up to 100 nm)
`
`90 DAYS
`
`Example 1 *
`Example 5 *
`Example 6 **
`
`571.4
`1755.8
`33.2
`
`814.2
`1543.6
`30.5
`
`877.1
`2218.8
`36.6
`
`669.6
`1873.4
`38.4
`
`* Poloxarner as surfactant in combination with Solutol HS 15 as co-surfac(cid:173)
`~a;'~oloxamer in combination witb co-surfactants otber tban Saluto! HS 15.
`
`In contrast with these results, the composition of the
`[0062]
`present invention, as observed in the Example 2 of the present
`invention, was completely transparent and its particle size,
`even after 12 months of stability monitoring, remained stable,
`with dimensions significantly below 50mn. This clearly dem(cid:173)
`onstrates the physicochemical improvements reached with
`the pharmaceutical composition of the present invention.
`
`Example 4
`
`Assessment of Sterility of the Present Invention
`Microemulsion (Microemulsion 1)
`
`[0063] The microemulsion prepared according to the
`Example 1 of the present invention was submitted to a sterility
`assay by incubation in three different culture media, under
`specific temperature conditions that are adequate for micro(cid:173)
`biological growth in each culture medium, observing whether
`or not development of colony forming units (c.f.u.) and tur(cid:173)
`bidity of the medium occurred within 14 days of incubation.
`The culture media used are presented in the following table:
`
`TABLES
`
`Culture media used for tbe sterility assay with
`MICROEMULSION 1 oftbe present invention.
`
`CULTURE MEDIUM
`
`Tryptic Soy Brotb (TSB)
`Fluid Thyoglicollate
`Medium (THIO)
`Peptone Water
`
`INCUBATION
`TEMPERATURE
`
`INCUBATION
`TIME
`
`25° c.
`35° c.
`
`25° c.
`
`14 days
`14 days
`
`14 days
`
`[0064] After the incubation period, no signs of bacterial
`growth were observed in any of the culture media, confirming
`the safety of the formulation under the perspective of sterility.
`
`Example 5
`
`Comparison of Pharmacological Parameters
`Observed with 1 % Microemulsion, 0.5% Micro(cid:173)
`emulsion and 1 % Commercial Emulsion
`
`In vivo assays were performed to compare the phar(cid:173)
`[0065]
`macological parameters of the present invention formula(cid:173)
`tions, at 1%and0.5% concentrations, to the pharmacological
`parameters presented by the 1 % commercially available
`emulsion (Propovan®). The hypnotic and anesthetic activi(cid:173)
`ties were investigated by means of intravenous infusion in 6
`rats weighing 260 to 350 g. The animals were anesthetized by
`ethylic ether inhalation and, subsequently, placed in ventral
`decubitus. An incision was made at the anterior cervical
`region and the jugular vein was dissected. A catheter filled
`with heparin was tunneled through the subcutaneous cell
`tissue up to the posterior cervical region where it was fixed to
`the skin. The assay was performed only when the animal
`presented complete recovery from ether anesthesia (±60
`min). The propofol formulations were infused during 1 hour
`through an infusion pump (B. Braun) at a volume of 40
`µUmin. The hypnotic and anesthetic latencies were deter(cid:173)
`mined by the analysis of the time elapsed between the begin(cid:173)
`ning of drug infusion and the loss of postural reflex and
`absence ofreaction to painful stimulus (pressure of the skin
`on the planar region of the hindfoot), respectively. The
`obtained res