DELIVERY SYSTEMS FOR PROPOFOL

Abstract

Provided is a novel dilutable delivery systems and propofol microemulsions suitable for intravenous delivery of propofol.

Claims

1.-78. (canceled)

79. A propofol-microemulsion comprising an oil phase in the form of oil droplets dispersed in an aqueous diluent continuous phase, wherein the oil phase comprises propofol, at least one surfactant comprising polyethylene glycol 15-hydroxystearate (Solutol HS 15), at least one solvent comprising medium-chain triglycerides (MCT), at least one co-surfactant, and at least one co-solvent, the oil droplets having a size of at most 20 nm in the continuous phase, the propofol and the surfactant having diffusion coefficients having the same order of magnitude when in the microemulsion (as measured by SD-NMR), and the microemulsion being suitable for parenteral administration.

80. The propofol-microemulsion of claim 79, wherein the diffusion coefficients of propofol and the surfactant (when in the microemulsion) are at least of one order of magnitude smaller than the other components of the microemulsion.

81. The propofol-microemulsion of claim 79, wherein the diffusion coefficients of propofol and the surfactant (when in the microemulsion) are of an order of magnitude of 1?10.sup.?11 m.sup.2/sec, when in the microemulsion, as measured by SD-NMR.

82. The propofol-microemulsion of claim 79, wherein the polydispersity index (PDI) of the distribution of oil droplets is between about 0.03 and 0.08.

83. The propofol-microemulsion of claim 79, wherein the oil droplets size is between about 10 and 20 nm.

84. The propofol-microemulsion of claim 79, wherein said diluent is selected from water, water for injection, saline, dextrose solution, or a buffer having a pH between 3 and 9.

85. The propofol-microemulsion of claim 79, wherein the co-surfactant is different from said surfactant and is selected from polyols, diglycerides, polyoxyethylenes, lecithins and phospholipids, optionally wherein the co-surfactant is at least one polyol selected from ethylene glycol, glycerol, polyethylene glycol, polypropylene glycol, sorbitol, mannitol, lactitol and xylitol.

86. The propofol-microemulsion of claim 79, wherein the co-solvent is selected from ethanol, propanol, propylene glycol, and glycerol.

87. The propofol-microemulsion of claim 79, comprising propofol, Solutol HS 15, MCT, polyethylene glycol, propylene glycol, a co-solvent, and a diluent.

88. The propofol-microemulsion of claim 79, wherein the co-solvent is ethanol.

89. The propofol-microemulsion of claim 79, comprising between about 0.1 and 2 wt % propofol.

90. The propofol-microemulsion of claim 79, wherein (i) the weight ratio between propofol and the surfactant is between about 1:5 and 1:12, (ii) the weight ratio between said at least one solvent and the surfactant is between about 1:8 and 1:12, and/or (iii) the weight ratio between said at least one solvent and propofol is between about 1:2 and 1.25:1.

91. The propofol-microemulsion of claim 79, having one or more of the following characteristics: (i) being transparent, (ii) a turbidity value of between about 20 and 70 NTU, (iii) an osmolality value of between about 250 and 450 mOsm/Kg, (iv) a surface tension of between about 27 and 35 mN/m, and (v) being a Newtonian liquid.

92. A dilutable propofol-concentrate comprising propofol, at least one surfactant comprising polyethylene glycol 15-hydroxystearate (Solutol HS 15), at least one solvent comprising medium-chain triglycerides (MCT), at least one co-surfactant, and at least one co-solvent, the concentrate being substantially free of water.

93. A process for preparing a composition suitable for parenteral administration of propofol, comprising diluting a dilutable propofol-concentrate of claim 92 in a predetermined quantity of a pharmaceutically acceptable aqueous diluent, optionally wherein said predetermined quantity of diluent is between about 75-98 wt %.

94. A kit comprising means for holding a dilutable propofol-concentrate of claim 92 and at least one pharmaceutically acceptable aqueous diluent, and instructions of use.

95. A method for parenteral administration of propofol to a subject in need thereof, the method comprising diluting a dilutable propofol-concentrate of claim 92 to a predetermined effective amount in a pharmaceutically acceptable aqueous diluent, thereby obtaining a microemulsion suitable for parenteral administration, and administering said microemulsion parenterally to said subject.

96. The method of claim 94, wherein said diluent is water, saline, dextrose solution, or a buffer having a pH between 3 and 9.

97. A method of inducing an anesthetic effect to a subject in need thereof, comprising administering to the subject a propofol-microemulsion of claim 79.

98. A method for preventing irritancy or reducing pain during administration of propofol in a site of administration, the method comprising providing a propofol-microemulsion of claim 79 and administering said propofol-microemulsion to a patient in need thereof at a site of administration, the propofol being maintained within the oil droplets of the microemulsion during administration.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0112] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0113] FIGS. 1A-1C shows a propofol microemulsion of the invention (FIG. 1A: B9A formulation; FIG. 1B: B6A formulation), compared to a propofol lipid emulsion prepared by high-shear mixing (or cavitation) according to Example 1 of reference [1] (FIG. 1C).

[0114] FIG. 2A shows droplet size distribution, as measured by DLS (Dynamic Light Scattering) analysis for a microemulsion prepared by diluting a 6 wt % propofol concentrate by water for injection; water concentration in the microemulsion ranging from 83.33 to 95 wt %.

[0115] FIG. 2B shows droplets size distribution, as measured by DLS for a microemulsion prepared by diluting a 9 wt % propofol concentrate by water for injection; water concentration in the microemulsion ranging from 88.88 to 95 wt %.

[0116] FIG. 2C shows droplets size distribution, as measured by DLS for CLE (Commercial Lipid Emulsion, Propofol-Lipuro?).

[0117] FIGS. 3A-3B show pH changes in accelerated stability studies of B6- (FIG. 3A) and B9-based (FIG. 3B) diluted formulations.

[0118] FIGS. 4A-4B show droplet size changes in accelerated stability studies of B6-(FIG. 4A) and B9-based (FIG. 4B) diluted formulations.

[0119] FIGS. 5A-5D shows microemulsions loaded with 1 wt % of propofol (FIG. 5A), 2,4-isomer of propofol (FIG. 5B), BHA (FIG. 5C), and TBHQ (FIG. 5D).

[0120] FIGS. 6A-6B shows hemolysis test results for B6A (FIG. 6A) and B9A (FIG. 6B) diluted propofol microemulsion placed on blood agar plates, compared with positive control causing hemolysis of Triton X100 and 0.9% saline, a negative control that do not cause hemolysis.

[0121] FIG. 7A shows the mean plasma concentration of propofol in study 1 following single intravenous Bolus administration, with formulations at a concentration of 10 mg/ml Propofol, to three non-naive male beagle dogs of prototype commercial lipid emulsion (Propofol-? Lipuro), B6A and B9A microemulsions at 6 mg/kg.

[0122] FIG. 7B shows the mean plasma concentration of propofol in study 2 following single intravenous Bolus administration of three formulations with a concentration of 10 mg/ml, to six naive male beagle dogs of: prototype commercial lipid emulsion (Propofol-? Lipuro), B6A and B9A formulation at 6 mg/kg.

DETAILED DESCRIPTION OF EMBODIMENTS

Example I: Propofol-Microemulsions Compared to Commercial Emulsions

[0123] 1 wt % propofol commercial liquid emulsion (CLE) Propofol-Lipuro? 1% was used as reference for comparison with the propofol-microemulsions of the invention.

[0124] Two dilutable propofol-concentrates were prepared, containing 6 wt % and 9 wt % of propofol (B6 and B9, accordingly), according to the following preparation protocol.

[0125] B6 Concentrate

[0126] Solutol HS-15 (also known as Kolliphor HS-15) was heated to about 40-60? C. Following heating, the Solutol was introduced into a vessel, together with propylene glycol (PG), MCT, polyethylene glycol 400 (PEG 400) and ethanol, and mixed for 10 minutes at 50-400 rpm. Egg Lecithin E80 (egg phospholipids with 80% phosphatidylcholin) was added, and mixed for 60 minutes, at 40-50? C. and mixed at 50-400 rpm. The mixture was then left to cool down to room temperature. Once the mixture has cooled, propofol was added and mixed form 5-30 minutes at 50-400 rpm, to thereby obtain a concentrate containing 6 wt % propofol.

[0127] B9 Concentrate

[0128] Solutol HS-15 (also known as Kolliphor HS-15) was heated to about 40-60? C. Following heating, the Solutol was introduced into a vessel, together with propylene glycol, MCT, PEG 400 and ethanol, and mixed for 10 minutes at 50-400 rpm. Propofol was then added and mixed form 5-30 minutes at 50-400 rpm, to thereby obtain a concentrate containing 9 wt % propofol.

[0129] B10 and B11 compositions, containing 10 and 11 wt % propofol, respectively, were prepared in a similar manner Compositions of the concentrates are detailed in Table 1.

[0130] In order to obtain the propofol-microemulsions, the concentrates were diluted with an appropriate amount of water for injection for 20-60 minutes at 200-400 rpm, to obtain microemulsions having a propofol concentration of 1 wt %. It is of note that preparation of larger batches of concentrate and/or microemulsion may be carried out under inert atmosphere (such as a flow of nitrogen) in order to prevent oxidation of propofol. The compositions of the microemulsions are detailed in Table 2.

TABLE-US-00001 TABLE 1 Dilutable propofol-concentrate compositions, 6-11 wt % propofol PEG Solutol PG MCT 400 Ethanol Lecithin Propofol B6 70.246 1.88 7.52 7.52 4.954 1.88 6 B9 68.203 3.688 6.08 6.949 6.08 0 9 B10 65.537 3.646 6.014 6.87 7.933 0 10 B11 60.113 6.955 5.945 6.797 9.190 0 11 * All quantities in wt %.

TABLE-US-00002 TABLE 2 Propofol-microemulsions, 1 wt % propofol PEG Pro- Solutol PG MCT 400 Ethanol Lecithin pofol Water B6A 11.707 0.313 1.253 1.253 0.825 0.313 1 83.333 B9A 7.578 0.409 0.675 0.772 0.675 0 1 88.88 B10A 6.554 0.365 0.601 0.687 0.793 0 1 90.00 B11A 5.465 0.632 0.540 0.618 0.835 0 1 90.91 * All quantities in wt %.

[0131] The physical properties of propofol-microemulsions prepared from B6, B9, B10 and B11 concentrates, in comparison to CLE, are provided in Tables 3-1 and 3-2.

TABLE-US-00003 TABLE 3-1 Properties of B6 and B9 based microemulsions compared to commercial lipid emulsion (CLE) Microemulsion prepared from propofol concentrate Parameter B6A B9A CLE Transparency Yes Yes No Color Clear yellowish Clear yellowish White opaque Microscopy.sup.a Uniform Uniform Uniform Turbidity (NTU).sup.b 26.2 38.18 NA pH.sup.c 7.27 7.24 7.50 Droplet size (nm).sup.d 15-16 16-17 300-400 Poly Dispersion Index 0.079 0.043 0.222 (PDI).sup.d Osmolality (mOsm/Kg).sup.e 389 302 333.5 Surface Tension (mM/m) 32.208 33.358 NA .sup.aMicroscopy analysis: Nikon Eclipse 80i .sup.bTurbidity evaluation: HI 83414 Turbidity and free/Total Chlorine Meter by HANNA instruments (using calibration curve samples and WFI of 0.13NTU as reference) .sup.cpH measurements: SevenEasy Metller Toledo .sup.dDrop size examination: Zeta sizer, nano sizer (nano-s), MALVERN instrument .sup.eFiske? Micro-Osmometer (model 210)

TABLE-US-00004 TABLE 3-2 Properties of B10 and B11 based microemulsions compared to CLE Microemulsion prepared from propofol concentrate Parameter B10A B11A CLE Transparency Yes Yes No Color Clear yellowish Clear yellowish White opaque Microscopy Uniform Uniform Uniform Turbidity (NTU) 45.3 61.7 NA Droplet size (nm) 18-19 19-20 300-400 Poly Dispersion Index 0.051 0.085 0.222 (PDI)

[0132] As clearly shown in Tables 3-1 and 3-2, B6A, B9A, B10A and B11A microemulsions have significantly different properties from those of commercial emulsions (CLE).

[0133] Commercial emulsions are typically a dispersion of two immiscible liquids, formed in the presence of emulsifiers/surfactants, which reduce the interfacial tension between the two phases and cover the dispersed droplets to retard aggregation, flocculation, coalescence and phase separation. Since the emulsifiers do not reduce the interfacial tension to zero and the coverage is not complete, emulsions require application of relatively high shear forces of multistage homogenizer to reduce the droplets size upon preparation of the emulsion. The resulting non-uniform droplets have a strong tendency to coalesce and/or result in phase separation, thereby stabilizing the system energetically. Thus, commercial propofol emulsions show a relatively non-uniform and large droplet size, which are unstable over prolonged periods of time (i.e. the droplet size increases due to coalescence or can even result in phase separation). Moreover, the CLE droplet size is far from being homogenous (as evident from the relatively high polydispersity index), also resulting in a milky, white-opaque appearance.

[0134] Contrary to CLE, due to the zero interfacial tension, microemulsions of the invention, such as B6A and B9A microemulsions are spontaneously formed as energetically balanced systems, which are characterized by a small and uniform droplet size, resulting in transparent systems.

[0135] Similar microemulsions formulations in which glycerol was used instead of polyethylene glycol as a co-surfactant are provided in Table 4-1.

TABLE-US-00005 TABLE 4-1 Propofol-microemulsions, 1 wt % propofol PEG Solutol PG MCT 400 Ethanol PC** Glycerol Propofol Water F(I) 7.556 0.229 0.707 0 0.556 0.202 0.859 1 89.891 F(II) 7.556 0.229 0.707 0.859 0.556 0.202 0 1 89.891 * All quantities in wt % **phosphatidyl-choline

[0136] Further suitable microemulsions were obtained when replacing Solutol HS15 with Tween 80, as shown in Table 4-2.

TABLE-US-00006 TABLE 4-2 Propofol-microemulsions, 1 wt % propofol Tween Etha- Pro- 80 PG MCT nol PC** Glycerol pofol Water F(III) 8.593 0.261 0.805 0.632 0.23 0.977 1 87.502 F(IV) 7.578 0.409 0.675 0.675 0 0.772 1 89.891 * All quantities in wt % **phosphatidyl-choline

Example II: Propofol-Microemulsions Compared to Shear-Mixed Emulsions

[0137] Due to the poor solubility of propofol, the majority of propofol emulsions currently under research are produced by utilizing high-shear forces. It is important to note that although such emulsions are often inappropriately named microemulsions or nanoemulsions in literature (see, for example [1]), such emulsions are significantly different from those of the present invention.

[0138] In order to demonstrate the differences between shear-mixed emulsions and microemulsions of the present invention, the following comparative example was carried out.

Shear Mixed Emulsion

[0139] Example 1 in [1] was selected as a representative example of a typical shear-mixed emulsion.

[0140] Unloaded Shear-Mixed Emulsion (without Propofol)

[0141] 785 mg Labrafac? CC (caprylic/capric triglyceride) and 527 mg Macrogol 15 hydroxystearate (also known as Solutol HS15) were precisely weighted into 20 ml glass vial. The mixture was heated to 40? C. for 15 minutes under agitation at 630 rpm and then cooled down to room temperature for 5 minutes.

[0142] A dispersing phase (0.9% w/v NaCl in water, i.e. physiological saline) was added to the mixture under agitation at 630 rpm until a final volume of 15 ml was obtained. The formulation was heated and mixed at 40? C. for an additional 10 minutes at 840 rpm. At this stage, prior to application of shear forces, the diameter of the droplets and PDI of the premix was determined by dynamic light scattering (Malvern Instrument, MAL500572, model ZEN1600).

[0143] The premix was then homogenized with a high-pressure homogenizer (IKA Labortechnik, Type T25B) at 10,000 psi for 105 seconds. After homogenization, the mean droplet diameter and PDI was measured again.

[0144] Propofol-Loaded Shear-Mixed Emulsion (1 wt %)

[0145] 785 mg Labrafac? CC and 527 mg Macrogol 15 hydroxystearate were precisely weighted into 20 ml glass vial. The mixture was heated to 40? C. for 15 minutes under agitation at 630 rpm and then cooled down to room temperature for 5 minutes.

[0146] 150 mg of propofol was added to the mixture and mixed at 630 rpm for 5 minutes.

[0147] A dispersing phase (0.9% w/v NaCl in water, i.e. physiological saline) was added to the mixture under agitation at 630 rpm until a final volume of 15 ml was obtained. The formulation was heated and mixed at 40? C. for an additional 10 minutes at 840 rpm. At this stage, prior to application of shear forces the diameter of the droplets and PDI of the mixture was determined by dynamic light scattering (Malvern Instrument, MAL500572, model ZEN1600).

[0148] The mixture was then homogenized with a high-pressure homogenizer (IKA Labortechnik, Type T25B) at 10,000 psi for 105 seconds. After homogenization, the mean droplet diameter and PDI was measured again.

Unloaded and Loaded Propofol Microemulsions

[0149] B6A and B9A propofol microemulsions (prepared from B6 and B9 concentrates) were prepared according to Example I above. Similar unloaded microemulsions were also prepared (i.e. maintaining the same components ratio, however without addition of propofol).

[0150] Average droplet size and PDI values for all samples are shown in Table 5.

TABLE-US-00007 TABLE 5 Average droplet size and PDI values Formulation Homogenization Droplet size (nm) PDI Unloaded shear-mixed No 196 0.126 emulsion Yes 166.2 0.1613 (Example 1 from [1]) 1 wt % propofol shear- No 267.5 0.183 mixed emulsion Yes 27.9, 97.1, 320.3 0.396 (Example 1 from [1]) B6A unloaded No 13.02 0.091 microemulsion B6A 1 wt % propofol No 15.29 0.084 microemulsion B9A unloaded No 13.01 0.035 microemulsion B9A 1 wt % propofol No 16.29 0.049 microemulsion

[0151] As can clearly be seen, contrary to the small and uniform droplet size spontaneously obtained (without homogenization) for B6A and B9A propofol-microemulsions, the shear-mixed emulsions show a significantly larger droplet size (about an order of magnitude larger) prior to high-pressure homogenization. Moreover, after shearing the emulsions by employing a high-pressure homogenization, the emulsions' droplet size does not decrease significantly, and for the propofol-loaded emulsion a three-population distribution of droplet sizes was observed. Namely, the shear-mixed 1 wt % propofol emulsion is far from being mono-dispersed.

[0152] This is also supported by the appearance of the samples. As can clearly be seen in FIG. 1C, the relatively large and inhomogeneous droplet size of the shear-mixed emulsion is milky in appearance, compared to the clear and transparent microemulsion (FIGS. 1A-1B).

Example III: Dilutablity of Propofol-Microemulsions

[0153] One of the advantages of propofol-concentrated of the invention (such as B9) is the ability to dilute them at various dilution ratios, without significantly affecting their droplet size.

[0154] The hydrodynamic radius of the microemulsion droplets were measured at room temperature by dynamic light scattering (DLS) using Nano-ZS Zetasizer (Malvern, UK), with water as a dispersant. The samples were examined in polystyrene disposable cuvettes. For each set of tests, the starting point was a 1 wt % propofol microemulsion, which was further diluted to obtain the samples (as listed in the table below). Average droplet size and PDI values are provided in Table 6. Size distribution curves are presented in FIGS. 2A-2B, as compared to those of commercial propofol emulsions (propofol Lipuro?) in FIG. 2C.

TABLE-US-00008 TABLE 6 Average droplet size and PDI values at different dilutions wt % Size Test series Sample Water (nm) PDI B6-based 1 83.33* 15.29 0.084 microemulsion 2 90 14.72 0.072 3 92 14.9 0.043 4 96 14.6 0.062 5 98 14.15 0.073 B9-based 1 88.88* 16.76 0.049 microemulsion 2 90 16.72 0.061 3 92 16.76 0.047 4 96 16.29 0.04 5 98 16.02 0.032 CLE N/A *1 wt % propofol microemulsion

[0155] As clearly evident from Table 6, the droplet sizes of the empty system are smaller than those measured for the loaded systems indicating that Propofol is located within the core/interface of the drop increasing its size (see also SD-NMR analysis in Example V below).

[0156] When compared to commercial propofol emulsions, it can be further observed that the propofol-microemulsions of the invention are fully dilutable without significantly altering the droplet size.

[0157] Note that comparative results for dilution of CLE cannot be obtained, as dilution of the CLE caused phase separation. In a typical experiment temperature fluctuation between 20-40? C. showed an increase of more than 20% in droplets sizes and additional storage at 40? C. showed the beginning of coalescence phenomenon that leads to phase separation after 60 days as shown in Table 7.

[0158] This suggests that propofol-microemulsions allow better control of propofol dosing, giving the care taker the possibility to further dilute the microemulsion to a desired lower concentration of propofol, without changing the microemulsion's physical structure and maintaining its beneficial properties. This is of significance, as once introduced into the bloodstream, microemulsions of the invention will not coalesce and/or form aggregates (unlike commercial emulsions).

Example IV: Long-Term Physical Stability

[0159] The stability of the propofol-microemulsion B6A and B9A, loaded with 10 mg/ml propofol (1 wt %), was evaluated for a period of 12 months, at three different temperatures and relative humidity (% RH) conditions (5? C./ambient, 25? C./60% RH and 40? C./25% RH).

[0160] Clarity, pH and droplet size was measured at each time point at triplicate samples, and compared to the initial measurements (baseline at time 0) taken immediately after preparation of the formulations. The results are presented in Tables 7-9 and FIGS. 3A-4B.

TABLE-US-00009 TABLE 7 CLE stability over time # of Temperature Time globules of storage (? C.) (months) pH >2 ?m 30 0 8.4 253 6 7.6 476 12 7.3 661 40 0 8.4 253 6 7.0 987 12 6.1 1527

TABLE-US-00010 TABLE 8 Stability analysis for B6A formulation (10 mg/ml propofol) Storage Time Droplet Conditions (months) Clarity pH Size (nm) PDI Initial 0 ? 7.03 15.35 0.044 5? C. 1 ? 7.11 15.27 0.046 Ambient 3 ? 7.07 15.72 0.042 humidity 6 ? 7.15 15.63 0.050 12 ? 7.12 15.35 0.043 25? C. 1 ? 7.01 15.42 0.046 60% RHA 3 ? 7.17 15.73 0.049 6 ? 7.20 15.27 0.051 12 ? 7.23 15.45 0.061 40? C. 1 ? 6.99 15.31 0.054 25% RHA 3 ? 7.01 15.63 0.047 6 ? 7.16 15.40 0.049 12 ? 7.03 15.23 0.050

TABLE-US-00011 TABLE 9 Stability analysis for B9A formulation (10 mg/ml propofol) Storage Time Droplet Conditions (months) Clarity pH Size (nm) PDI Initial 0 ? 7.68 16.65 0.057 5? C. 1 ? 7.69 16.07 0.064 Ambient 3 ? 7.61 16.23 0.072 humidity 6 ? 7.63 16.77 0.053 12 ? 7.59 16.32 0.043 25? C. 1 ? 7.60 16.46 0.059 60% RHA 3 ? 7.62 16.54 0.049 6 ? 7.54 16.63 0.047 12 ? 7.63 16.45 0.059 40? C. 1 ? 7.59 16.51 0.061 25% RHA 3 ? 7.64 16.43 0.043 6 ? 7.61 16.54 0.053 12 ? 7.59 16.63 0.042

[0161] As can be observed, the microemulsions maintain their clarity, pH, droplet size and PDI values over prolonged periods of time, i.e. at least up to 12 months, when stored at various storage conditions. Thus, diluted formulations of the invention may be stored for prolonged periods of time without adversely affecting their properties.

[0162] It is known from the literature that an emulsion's stability is derived from several kinetics forces: the formation of mechanical barrier between the oil and the aqueous phases and electrostatic repulsive forces between the droplets. These forces tend to be disrupted causing the emulsion to degrade and separate to the oil and water phases. Moreover, during heat sterilization of the emulsion, small quantities of free fatty acids and hydrolysis of the soybean oil leads to pH decrease, which in turn act to destabilize the emulsion. This process continues even during storage, since the emulsions are non-buffered. Therefore, propofol commercial emulsions have a relatively narrow expiration date, of two years, with specific storage condition (see Table 7).

[0163] To determine long term stability of formulations, a rapid measurement was carried out using LUMiFuge? analytical centrifugation. LUMiFuge analysis enables to predict the shelf-life of a formulation in its original concentration, even in cases of slow destabilization processes like sedimentation, flocculation, coalescence and fractionation. During LUMiFuge measurements, parallel light illuminates the entire sample cell in a centrifugal field; the transmitted light is detected by sensors arranged linearly along the total length of the sample-cell. Local alterations of particles or droplets are detected due to changes in light transmission over time. The results are presented in a graph plotting the percentage of transmitted light (Transmission %) as a function of local position (mm), revealing the corresponding transmission profile over time. The commercial propofol emulsion, Propofol-Lipuro?, was compared to measurements carried out on B6- and B9-based formulations over a time period of 24 hours.

[0164] The initial detection of the Propofol-Lipuro? lipid-based emulsion (having white milky appearance) scattered and absorbed the light resulting in low transmission (close to 0%). However, within time, the emulsion stability was impaired, leading to phase separation. These results demonstrate the Propofol-Lipuro? emulsion instability concern, which might be harmful and even lethal to patients going anesthetic procedures and administered with the lipid-based emulsion.

[0165] In contrast, in microemulsions of the invention (having a clear and transparent appearance) enabled light to be transmitted (100%) throughout the whole measured cell length. The transmitted light, reflecting the transparency of the sample, was even obtained over 24 hours of centrifugal forces of 3000 rpm tested during analysis. The LUMiFuge recorded transmission for the microemulsion was similar to those measured for water. These results support expectation for long shelf life stability properties of the tested microemulsions and the safety profile of such formulations used in patient even after long storage occasion. Thus, microemulsions of the invention are thermodynamically stable and therefore are expected to have broader storage conditions with less requirements for proper storage. The ability to store the microemulsion in higher and lower temperature, as well as longer time-periods, is an important advantage in the pharmaceutical industry.

Example V: Self-Diffusion NMR (SD-NMR)

[0166] In order to determine the structure of the oil droplets (or micelles) of the microemulsions, self-diffusion NMR analysis was carried out. SD-NMR is able to locate each component within the microemulsion via measurements of its diffusion coefficient. Rapid diffusion (>100?10.sup.?11 m.sup.2s.sup.?1) is characteristic of small molecules, free in solution, while slow diffusion coefficients (<0.1?10.sup.?11 m.sup.2s.sup.?1) suggest low mobility of macromolecules or bound/aggregated molecules.

[0167] NMR measurements were performed with a Bruker AVII 500 spectrometer equipped with GREAT 1/10 gradients, a 5 mm BBO and a 5 mm BBI probe, both with a z-gradient coil and with a maximum gradient strength of 0.509 and 0.544 T m.sup.?1, respectively. Diffusion was measured using an asymmetric bipolar longitudinal eddy-current delay (bpLED) experiment, or and asymmetric bipolar stimulated echo (known as one-shot) experiment with convection compensation and an asymmetry factor of 20%, ramping the strongest gradient from 2% to 95% of maximum strength in 32 steps. The spectrum was processed with the Bruker TOPSPIN software. NMR spectra were recorded at 25?0.2? C. The components were identified by their chemical shift in 1H NMR.

[0168] Table 10-1 shows the diffusion coefficients (Dx, m.sup.2/sec) of the various components for B6- and B9-based unloaded and 1 wt % propofol-loaded microemulsions.

TABLE-US-00012 TABLE 10-1 Diffusion coefficients (m.sup.2/sec), as measured by SD-NMR, Solutol as surfactant B6A microemulsion B9A microemulsion Component Unloaded Loaded Unloaded Loaded Water 1.76 ? 10.sup.?9 1.37 ? 10.sup.?9 1.52 ? 10.sup.?9 1.62 ? 10.sup.?9 Propofol 1.02 ? 10.sup.?11 1.54 ? 10.sup.?11 Solutol 1.45 ? 10.sup.?11 1.62 ? 10.sup.?11 1.94 ? 10.sup.?11 1.50 ? 10.sup.?11 Propylene 6.68 ? 10.sup.?10 5.22 ? 10.sup.?10 5.96 ? 10.sup.?10 8.45 ? 10.sup.?10 glycol Ethanol 8.83 ? 10.sup.?10 6.91 ? 10.sup.?10 7.65 ? 10.sup.?10 9.82 ? 10.sup.?10 *Note: MCT shows similar diffusion coefficients to propofol, however its low content prevents obtaining accurate calculation of the diffusivity.

[0169] As can be seen from Table 10, the diffusion coefficient of Solutol HS15 is similar to that of Propofol. These results indicate that the propofol is located within the core and the interface of the swollen micelle. This suggests that all of the propofol in the tested microemulsions is contained within the oil droplet, and no free propofol is within the aqueous continuous phase. Since free propofol in known to be the cause of pain upon injection, the fact that propofol is located within the oil droplets is expected to significantly reduce pain and irritation during and following administration.

[0170] In addition, it should be also stressed that the droplet size is increasing when propofol is introduced into the system compared to the empty system from 12.5 nm to 17 nm. This indicates that propofol resides within the core of the droplet, thereby increasing its diameter.

[0171] Similar results were obtained when replacing Solutol HS15 with Tween 60 or Tween 80, as shown in Table 10-2.

TABLE-US-00013 TABLE 10-2 Diffusion coefficients (m.sup.2/sec), as measured by SD-NMR, Tween as surfactant Formulation with Tween 80 Formulation with Tween 60 Component Unloaded Loaded Unloaded Loaded Water 1.91 ? 10.sup.?9 1.24 ? 10.sup.?9 1.86 ? 10.sup.?9 1.12 ? 10.sup.?9 Propofol 1.73 ? 10.sup.?11 1.04 ? 10.sup.?11 Tween 1.73 ? 10.sup.?11 2.08 ? 10.sup.?11 0.92 ? 10.sup.?11 1.63 ? 10.sup.?11 Propylene 5.43 ? 10.sup.?10 4.82 ? 10.sup.?10 4.96 ? 10.sup.?10 3.24 ? 10.sup.?10 glycol

[0172] It was also found that when the surfactant was replaced by a surfactant (such as sucrose ester) that does not have a diffusion coefficient similar to that of the propofol in the microemulsion, no stable microemulsion was formed. As propofol moved somewhat faster, binding between propofol and an unsuitable surfactant is less strong, and hence more of the propofol is in free form in the aqueous phase. This may suggest that incorporation of propofol in other microemulsion composition results in deviation from equilibrium and formation non-stable microemulsions, or such that cannot be fully diluted.

[0173] The relationship between the surfactant and the propofol with respect to binding in the microemulsion-composition was assessed by loading B9A with 1 wt % of species similar in their structure to propofol. The results are provided in Table 11.

TABLE-US-00014 TABLE 11 Diffusion coefficients (m.sup.2/sec), as measured by SD-NMR, for various compounds Diffusion coefficient (?10.sup.?11) Loaded Loaded-specie specie Solutol PEG400 PG EtOH Empty System 1.94 26.3 76.5 99.8 [00001] 1.54 1.50 26.3 84.5 98.2 [00002] 2.15 1.76 26.2 44.8 94.6 [00003] 1.75 1.63 26.3 73.6 96.6 [00004] 1.78 1.75 24.5 78.3 91.2 [00005] 1.58 1.75 24.3 79.1 98.2 [00006] NA NA NA NA NA

[0174] As can be seen from Table 11, diffusion coefficients of the propofol and the Solutol are almost identical, attesting to the binding between propofol and Solutol in the microemulsion composition, and the presence of the majority (if not all) of the propofol within the oil core or solubilized within the tails of the surfactant.

[0175] Although some of the examined species tested show similarity of diffusion coefficient to that of Solutol (such as the 2,4-isomer, BHA and BHT), as evident from FIGS. 5A-5D, such formulations are far from being sufficiently solubilized in the oil phase, resulting in systems which are not completely transparent. In some cases similar structures to propofol, such as TBHQ, form a classic milky emulsion.

[0176] These results attest to the uniqueness of the microemulsion formulation composition to the location of propofol within the droplets in which the diffusion coefficients are of the same order of magnitude.

Example VI: In Vitro Hemolysis

[0177] 10 ?l of diluted formulation of B9 or B6 (i.e. B6A and B9A microemulsions) were placed on Trypticase soy agar plates with 5% defibrinated sheep blood (TSA 5% DSB) and incubated up to 24 hours.

[0178] Two formulations were used as control: 10 ?l of Triton X100, known to cause blood hemolysis, was applied on one third of the plate, and 10 ?l of 0.9% NaCl which does not cause hemolysis, was spread on the remaining third.

[0179] Both formulations tested showed no blood hemolysis, similarly to physiological saline (0.9% NaCl)see FIGS. 6A-6B.

Example VII: Microorganisms Growth Contamination

[0180] Cultures of S. aureus, E. coli, P. aeruginosa and C. albicans were freshly prepared on the day before the assay (Soybean-casein digest agar for bacteria and Sabouraud dextrose agar for C. albicans) from frozen stocks of P2 (after two passages from the original ATCC cultures). Single colonies were picked and suspended in 0.9% sterile saline at a final density of 1.0?10.sup.8 CFU/ml.

[0181] B6A and B9A microemulsions (1% propofol) were aliquoted into five portions (4 gram each) in capped bacteriological test tubes in aseptic conditions to assure avoiding sample contamination. Aliquots of 0.04 ml (i.e., 1% of the volume of test article) of bacterial and fungal suspensions and saline (serving as negative control for contamination of the original formulations) were then added to each test tube to give a starting density of ?1.0?10.sup.6 CFU/ml. The resulting mixtures were vortexed thoroughly.

[0182] Similar aliquots were obtained for CLE.

[0183] As a positive control, 0.04 ml aliquots of bacterial and fungal suspensions were added to 4 ml of sterile saline. All controls were subjected to the same testing processes as the test articles.

[0184] Aliquots of 0.5 ml were removed from each tube (i.e., T.sub.0 samplein practice all the samples were removed within 30 min after mixing, T.sub.0.5 is used to reflect this). For each sample, three 0.1 ml aliquots of T.sub.0.5 mixture (without dilution) were spread on three appropriate solid agar plates. The remaining mixture was diluted 100, 1,000 and 10,000 times, and three aliquots of 0.1 ml were spread on three agar plates. For the CLE, duplicates of 0.1 ml of samples were spread on plated without dilution, 1,000 and 10,000 times diluted (see Table 11-1). The resulting bacterial plates were kept at 37? C. and 85% humidity for 24 hours, and the fungal plate was kept at room temperature for 48 hours. The CFUs were then enumerated and converted to the CFU/ml of the starting materials.

[0185] The mixtures were kept at 25? C. (with bacterial inocula) or room temperature (with fungal inoculum) without shaking. After 24 hour growth (T.sub.24 samples), aliquots of 0.5 ml were removed from each test tube. Three aliquots of 0.1 ml (without dilution) were spread on three appropriate agar plates. The undiluted and 100, 1,000 and 10,000 times diluted aliquots were also plated on three appropriate agar plates. For the CLE, duplicates of 0.1 ml of samples were spread on plated with dilution of 1,000, 10,000 and 100,000 times (Table 12-1), and the resulting CFUs were enumerated. The mixtures were returned to incubation for additional 24 hours at the same conditions.

[0186] Colony forming units were counted and the CFU/ml was calculated from each dilution. The mean CFU/ml of all dilutions was calculated; the Microbial Migration Rate (MGR) was calculated by dividing the log ratio of CFU/ml after 24 hours incubation to that measured at T.sub.0 as follows:

MGR=Log [(T.sub.24 CFU/ml)/(T.sub.0 CFU/ml)].

[0187] The results are provided in Tables 12-2 and 12-3.

TABLE-US-00015 TABLE 12-1 Samples for colony forming activities Inocula S. aureus* E. coli* P. aeruginosa* C. albicans* Saline**** Microem. B6A (1%)** T.sub.0.5 T.sub.0.5 T.sub.0.5 T.sub.0.5 T.sub.0.5 Formulation (12 plates).sup.a (12 plates).sup.a (12 plates).sup.a (12 plates).sup.a (2 plates).sup.d T.sub.24 T.sub.24 T.sub.24 T.sub.24 T.sub.0.5 (12 plates).sup.a (12 plates).sup.a (12 plates).sup.a (12 plates).sup.a (2 plates).sup.d T.sub.48 T.sub.48 T.sub.48 T.sub.48 T.sub.48 (12 plates).sup.a (12 plates).sup.a (12 plates).sup.a (12 plates).sup.a (2 plates).sup.d B9A (1%)** T.sub.0.5 T.sub.0.5 T.sub.0.5 T.sub.0.5 T.sub.0.5 (12 plates).sup.a (12 plates).sup.a (12 plates).sup.a (12 plates).sup.a (2 plates).sup.d T.sub.24 T.sub.24 T.sub.24 T.sub.24 T.sub.0.5 (12 plates).sup.a (12 plates).sup.a (12 plates).sup.a (12 plates).sup.a (2 plates).sup.d T.sub.48 T.sub.48 T.sub.48 T.sub.48 T.sub.48 (12 plates).sup.a (12 plates).sup.a (12 plates).sup.a (12 plates).sup.a (2 plates).sup.d CLE** T.sub.0.5 T.sub.0.5 T.sub.0.5 T.sub.0.5 T.sub.0.5 (8 plates).sup.b (8 plates).sup.b (8 plates).sup.b (8 plates).sup.b (2 plates).sup.d T.sub.24 T.sub.24 T.sub.24 T.sub.24 T.sub.0.5 (8 plates).sup.b (8 plates).sup.b (8 plates).sup.b (8 plates).sup.b (2 plates).sup.d T.sub.48 T.sub.48 T.sub.48 T.sub.48 T.sub.48 (8 plates).sup.b (8 plates).sup.b (8 plates).sup.b (8 plates).sup.b (2 plates).sup.d Saline*** T.sub.0.5 T.sub.0.5 T.sub.0.5 T.sub.0.5 T.sub.0.5 (8 plates).sup.c (8 plates).sup.c (8 plates).sup.c (8 plates).sup.c (2 plates).sup.d T.sub.24 T.sub.24 T.sub.24 T.sub.24 T.sub.24 (8 plates).sup.c (8 plates).sup.c (8 plates).sup.c (8 plates).sup.c (2 plates).sup.d T.sub.48 T.sub.48 T.sub.48 T.sub.48 T.sub.48 (8 plates).sup.c (8 plates).sup.c (8 plates).sup.c (8 plates).sup.c (2 plates).sup.d Inoculum/volume *~1.0 ? 108 CFU/ml, 0.04 ml; **4 gram; ***4.0 ml; ****0.04 ml Dilution schemes: .sup.aundiluted, 1/100, 1/1,000 and 1/10,000 dilutions (0.1 ml each, triplicated); .sup.bundiluted 1/1,000 and 1/10,000 and 1/100,000 dilutions (0.1 ml each, duplicated); .sup.cundiluted, 1/100, 1/1,000 and 1/10,000 dilutions (0.1 ml each, duplicated); .sup.dundiluted (0.1 ml each duplicated)

TABLE-US-00016 TABLE 12-2 Mean CFU/ml Sampling Mean CFU/ml (?10.sup.6) Sample time (hr) S. aureus E. coli P. aeruginosa C. albicans Saline 0.5 3.75 7.53 16.63 8.90 24 2.00 6.30 6.33 2.05 B6A 0.5 17.60 5.68 2.53 8.65 24 19.50 0.28 0.40 2.238 B9A 0.5 17.32 2.04 0.04 9.85 24 9.58 2.07 0.01 7.87 CLE 0.5 13.23 13.53 13.50 9.85 24 59.75 207.5 NA 12.68

TABLE-US-00017 TABLE 12-3 Microbial migration rate (MGR) Sample S. aureus E. coli P. aeruginosa C. albicans Saline 0.53 0.83 0.38 0.23 B6A 1.10 0.04 0.15 0.25 B9A 0.55 1.01 0.25 0.79 CLE 4.51 15.33 NA 1.28

[0188] Bacterial growth of B6A and B9A formulations decreased or has shown almost no change in growth rate after 24 hours. These results indicate that both formulations do not support microorganism growth. The calculated MGR value of the tested bacterial strains was negative or lower than 0.05. In contrast to the microemulsions, the commercial lipid-based emulsion showed an increase in the growth in all tested microorganisms with an MGR value above 0.5. The MGR calculated value was extremely high (1.19) for the emulsion tested for the growth of E. coli. The results indicate that emulsion provides a good and supportive environment for bacterial and fungal growth, while the microemulsions do not.

Example VIII: Pharmacological Tests

[0189] Pharmacokinetics following single intravenous bolus (IV) dose of commercial Propofol-Lipuro? (referred to herein as prototype), and B6A and B9A propofol-containing diluted microemulsions, all with a concentration of 10 mg/ml, was assessed at 6 mg/kg in male beagle dogs. The test article was monitored in plasma up to 8 hours.

Test System and Study Design

[0190] Study 1:

[0191] 3 non-naive male beagle dogs (8.06-9.07 kg, supplied by Marshall Bioresources, Beijing, China) were assigned to the study with 3 males per group. Each animal had a unique skin tattoo number on ear as the identification. The dogs in each group received a single intravenous dose of propofol formulation at a nominal dose of 6 mg/kg. Blood samples were harvested according to each sampling time. The study design is presented in Table 13-1.

[0192] Study 2:

[0193] 18 naive male beagle dogs (7.08-11.16 kg in weight, supplied by Marshall Bioresources, Beijing, China) were assigned to 3 groups with 6 males per group. Each animal had a unique skin tattoo number on ear as the identification. The dogs in each group received a single intravenous dose of propofol formulation at a nominal dose of 6 mg/kg. Blood samples were harvested according to each sampling time. The study design is presented in Table 13-1.

TABLE-US-00018 TABLE 13-1 Pharmacokinetic test design (IV Bolus) Dose Dose # of Dose volume concentration Study # males Formulation (mg/kg) (mL/kg) (mg/mL) 1 3 Prototype 6 0.6 10 3 B6Abased 6 0.6 10 3 B9Abased 6 0.6 10 2 6 Prototype 6 0.6 10 6 B6A 6 0.6 10 6 B9A 6 0.6 10

Dose Preparation and Administration:

[0194] Study 1:

[0195] B6A and B9A formulations and Prototype were provided as ready to use solutions (in their diluted state, 10 mg/mL). Prior to administration, solution was mixed by slightly shaking the vial, the center disc was opened and the septum rubber cleaned with an alcohol pad. Next, the required volume was retrieved using a sterile syringe going through the septum stopper. Air bubbles were removed before IV injection.

[0196] Study 2:

[0197] formulations were supplied as concentrated solutions (60 or 90 mg/mL; 6 or 9 wt %) and were diluted to the desired concentration (10 mg/mL) prior to administration. Dose preparation procedure was carried out as follows: water for injection (WFI) was added using a sterile pipette. After the addition of WFI, the cap was closed and the test items was thoroughly mixed by shaking the vial, until a transparent, uniform and clear formulation was formed. The formulation was then left to stand for about 15 min at room temperature to release most bubbles and decrease foam formation. The required volume was retrieved using a sterile syringe, while avoiding taking any foam or bubbles. Each dog out of the six received the required volume-dose from a separate freshly prepared vial. Prototype formulation was supplied as ready to use formulation and was not further diluted prior to administration.

[0198] Doses preparations are provided in Table 13-2.

TABLE-US-00019 TABLE 13-2 Dose preparation parameters Volume Formulation Concentrated Dilution after Study # type dose with WFI dilution Number of vials Total 1 Prototype 20 mL 4 80 mL 1% propofol (800 mg) B6A 9 mL 4 36 mL 1% propofol (360 mg) B9A 9 mL 4 36 mL 1% propofol (360 mg) 2 Prototype 20 mL 4 80 mL 1% propofol (800 mg) B6 1.5 mL 7.5 mL 9.0 mL 7 63 mL 6% propofol (630 mg) B9 1 mL 8.0 mL 9.0 mL 7 63 mL 9% propofol (630 mg)

Sample Collection and Preparation:

[0199] Serial blood samples (approximately 0.8 mL into K.sub.2EDTA anticoagulant tube) were collected via a cephalic vein. Blood samples were collected at Predose (0 minute, only for study 2), 0.0333 (2 minutes), 0.0833 (5 minutes), 0.167 (10 minutes), 0.333 (20 minutes), 0.5 (30 minutes), 1, 2, 4, 6 and 8 hours post dose from all phases.

[0200] After collection, all blood samples were transferred into pre-labeled plastic micro-centrifuge tubes containing K.sub.2EDTA (10 ?L, 0.5 M) and placed on wet ice immediately upon collection. After blood was collected, the samples were processed for plasma by centrifugation at approximately 4? C., 3000 g for 10 minutes within 60 minutes of collection. The plasma was transferred into labeled polypropylene micro-centrifuge tubes and then quickly frozen over dry ice and stored frozen in a freezer set to maintain ?60? C. or lower until bio-analysis.

Clinical Observation:

[0201] Cage-side observations for general health and appearance were done twice daily. Animals were given a physical examination prior to study initial to confirm animals' health. On dosing days, the animals were observed before and after each sample collection time point. General condition, behavior, activity, excretion, respiration or other unusual observations noted throughout the study were recorded.

Sample Analysis:

[0202] Dog plasma samples were analyzed for propofol using a qualified bioanalytical method based on protein precipitation followed by LC-MS/MS analysis. The lower limit of quantification (LLOQ) for propofol was 5.00 ng/mL or 10.0 ng/mL and the upper limit of quantification (ULOQ) was 2000 ng/mL.

[0203] Plasma concentration data of propofol was subjected to a non-compartmental pharmacokinetic analysis using a Phoenix WinNonlin software program (version 6.2.1, Pharsight, Mountain View, Calif.).

[0204] Terminal half-life (T.sub.1/2), volume of distribution at steady state (Vd.sub.ss), total body clearance (Cl), mean residence time (MRT) from time zero to the last quantifiable concentration (MRT.sub.0-last) and from time zero to infinity (MRT.sub.0-inf), the area under the plasma concentration-time curve (AUC) from time zero to the last quantifiable concentration (AUC.sub.0-last) and AUC from time zero extrapolated to infinity (AUC.sub.0-inf) were calculated using the linear/log trapezoidal rule.

[0205] Individual plasma concentrations below the lower limit of quantification (BQL) were excluded when performing pharmacokinetic analysis. Nominal sampling times were used to calculate all pharmacokinetic parameters. For samples collected within the first hour of dosing, a?1 minute was acceptable; for the remaining time points, samples that were taken within 5% of the scheduled time were acceptable and were not considered as protocol deviation.

Results:

[0206] Individual and Mean (n=3) plasma concentrations of propofol are presented graphically in FIGS. 7A-7B. The mean C.sub.0, T.sub.1/2, V.sub.dss, Cl, AUC.sub.0-last, AUC.sub.0-inf, MRT.sub.0-last and MRT.sub.0-inf values of propofol after single intravenous dosing are provided in Table 13-3.

TABLE-US-00020 TABLE 13-3 Mean pharmacokinetic values after single intravenous dosing Study # 1 2 Phase 2 3 4 2 1 5 Formulation Prototype B6A* B9A** Prototype B6A* B9A** C.sub.0 (ng/mL) 8867 9030 9223 12008 9400 7532 T.sub.1/2 (h) 2.19 2.03 3.74 3.48 3.60 3.00 Vd.sub.ss (L/kg) 4.67 5.76 8.40 6.41 7.22 5.37 C1 (ml/min/kg) 67.1 70.7 74.6 58.0 63.2 70.5 AUC.sub.0-last (ng .Math. h/ml) 1560 1377 1267 1660 1495 1384 AUC.sub.0-inf (ng .Math. h/ml) 1600 1423 1360 1773 1607 1446 MRT.sub.0-last (h) 0.88 1.04 1.02 1.03 1.04 0.822 MRT.sub.0-inf (h) 1.15 1.36 1.87 1.92 1.90 1.30 AUC.sub.0-last/AUC.sub.0-inf 103 103 107 107 107 104 (%) *designated in FIGS. 7A-7B as 811(B6) **designated in FIGS. 7A-7B as 801(B9)

[0207] Study 1:

[0208] following Prototype administration to the 3 non-naive males beagle dogs, C.sub.0 (initial plasma concentration) value (mean?S.D.) was observed at 8867?2844 ng/mL, AUC.sub.0-inf (the area under the concentration vs. time curve from time zero to the infinity) value (mean?S.D.) was obtained at 1600?541 ng/mL.Math.hr and Cl (total body clearance) value (mean?S.D.) was obtained at 67.1?20.7 mL/min/kg.

[0209] Following 811(B6) administration to the 3 non-naive males beagle dogs, C.sub.0 value (mean?S.D.) was observed at 9030?2080 ng/mL, AUC.sub.0-inf value (mean?S.D.) was obtained at 1423?136 ng/mL.Math.hr and Cl value (mean?S.D.) was obtained at 70.7?7.03 mL/min/kg. The C.sub.0, AUC.sub.0-inf and Cl values were comparable with those derived from commercial product Prototype dosing at the same dosage with the ratios of 1.02, 0.889 and 1.05, respectively. In terms of pharmacodynamics, all of the dogs fell into the state of anesthesia upon completing of the injection that lasted for 8 to 13 minutes without any adverse effects observed.

[0210] Following 801(B9) administration to the 3 non-naive male beagle dogs, C.sub.0 value (mean?S.D.) was observed at 9223?5071 ng/mL, AUC.sub.0-inf value (mean?S.D.) was obtained at 1360?190 ng/mL.Math.hr and Cl value (mean?S.D.) was obtained at 74.6?10.6 mL/min/kg. C.sub.0, AUC.sub.0-inf and Cl values were comparable with those derived from commercial product Prototype dosing at the same dosage with the ratios of 1.04, 0.850 and 1.11, respectively. In terms of pharmacodynamics, all of the dogs fell into the state of anesthesia upon completing of the injection that lasted for 8 to 10 minutes without any adverse effects observed.

[0211] Study 2:

[0212] following Prototype (Propofol commercial emulsion) administration to the 6 na?ve male beagle dogs, C.sub.0 value (mean?S.D.) was observed at 12008?5932 ng/mL, AUC.sub.0-inf value (mean?S.D.) was obtained at 1773?324 ng/mL.Math.hr and Cl value (mean?S.D.) was obtained at 58.0?10.7 mL/min/kg. The pharmacodynamics showed that all of the 6 dogs fell into the state of anesthesia smoothly at the end of injection and the anesthesia status lasted for about 5 to 7 min with mild degree of adverse effects such as swimming stroking limbs observed in 1 of the 6 dogs (D203).

[0213] Following 811(B6) administration to the 6 naive male beagle dogs, C.sub.0 value (mean?S.D.) was observed at 9400?2572 ng/mL, AUC.sub.0-inf value (mean?S.D.) was obtained at 1607?219 ng/mL.Math.hr and Cl value (mean?S.D.) was obtained at 63.2?8.17 mL/min/kg. AUC.sub.0-inf and Cl values were comparable with those derived for Prototype with the ratios of 0.906 and 1.09 respectively. The ratio of C.sub.0 in 811(B6) to Prototype was 0.783, a bit lower than that of Prototype. All of the dogs fell into the state of anesthesia smoothly at about 20 seconds upon injection and the anesthesia status lasted for 10 to 11 minutes.

[0214] Following 801(B9) administration to the 5 naive male beagle dogs, C.sub.0 value (mean?S.D.) was observed at 7532?749 ng/mL, AUC.sub.0-inf value (mean?S.D.) was obtained at 1446?218 ng/mL.Math.hr and Cl value (mean?S.D.) was obtained at 70.5?11.8 mL/min/kg. AUC.sub.0-inf and Cl value was comparable with that derived from Prototype with the ratio to Prototype 0.816 and 1.22, while C.sub.0 was lower than that of Prototype with a ratio to Prototype 0.627. 2 out of the 6 dogs fell into the state of anesthesia upon completion of injection.

Example VIX: Toxicology

[0215] Toxicokinetics (TK) was assessed after once every-other-day intravenous (IV) bolus administration of diluted formulations 811 (B6) and 811 (B9), and the commercial emulsion Propofol-? Lipuro 1% (10 mg/mL) to male and female beagle dogs for 7 days.

[0216] Dosing:

[0217] 18 (9/sex) male and female dogs were randomly assigned to 3 groups (3/sex/group, Groups 1, 2 and 3) Animals were administered once every other day by IV bolus with 1 wt % propofol microemulsions 811 (B6) and, 801 (B9) and Propofol-? Lipuro 1% emulsion (10 mg/mL) at 6 mg/kg for 7 days, respectively.

[0218] The following parameters were examined during the study: viability, clinical observations, body weight, food consumption, respiratory rate, ECG, heart rate, clinical pathology (hematology and serum chemistry), gross pathology and histopathology and toxicokinetics.

Blood Sampling, Plasma Preparation and Analysis:

[0219] On Day 1 and Day 5, blood samples (approximately 1 mL into K.sub.2EDTA anticoagulant tube) were collected from all study animals at 0 (predose), 0.0333, 0.0833, 0.167, 0.333, 0.5, 1, 4, 8, 12 and 24 hours postdose via the cephalic vein from all animals Blood samples were collected into appropriately labeled tubes, inverted several times to ensure mixing and placed on wet ice. Plasma was obtained within 2 hours of collection by centrifugation at 3200?g and 4? C. for 15 minutes. Plasma was transferred into uniquely labeled polypropylene tubes (Eppendorf), covered by aluminum foil and frozen in the upright position immediately over dry ice and stored in a freezer set to maintain ?60? C. to ?80? C. on dry ice until analysis.

[0220] Dog plasma samples were analyzed for propofol using a validated bioanalytical method based on protein precipitation followed by ultra-performance liquid chromatographic triple quadrupole mass spectrometric (UPLC-MS/MS) analysis. Using 30 ?L aliquot of dog plasma, the lower limit of quantification (LLOQ) was 10.0 ng/mL, and the higher limit of quantification was 6000 ng/mL.

[0221] Plasma concentration vs. time profiles of propofol were analyzed using a non-compartmental model by a validated WinNonlin? program (Pharsight, Version 6.2.1). The initial plasma concentration (C.sub.0) and the area under the plasma concentration vs. time curve (AUC) from time zero to 24 hours post dose (AUC.sub.0-24 h) were calculated using the linear up/log down trapezoidal rule.

[0222] Plasma concentration below LLOQ (BLQ) was set to zero for toxicokinetic analysis, however, when more than half (>50%) of the individual values at a single time point are BLQ, mean values will be reported as BLQ. AUC.sub.0-24 h and C.sub.0 values were reported to 3 significant digits. AUC.sub.0-24 h and C.sub.0 ratios were reported to 2 significant digits.

Results:

[0223] Two formulations B6-based and B9-based microemulsions, and one commercial emulsion product (Propofol-? Lipuro 1% (10 mg/mL)) administered by intravenous (IV) bolus to Beagle dogs once every other day (QOD) at 6 mg/kg dose level for a 7-day study period was well tolerated and all animals survived to the end of study. The mean?SD for C.sub.0 and AUC.sub.0-24h values for propofol are presented in Table 14.

TABLE-US-00021 TABLE 14 Mean toxicokinetic values AUC.sub.0-24 h Group Study day Sex C.sub.0 (ng/mL) (h .Math. ng/mL) 1 1 Male 4560 ? 1740 1170 ? 133 Female 12800 ? 9260 1660 ? 478 5 Male 10700 ? 1720 1790 ? 239 Female 23600 ? 21800 3020 ? 2430 2 1 Male 14800 ? 540 1580 ? 117 Female 10800 ? 5350 1640 ? 326 5 Male 14100 ? 3630 1890 ? 413 Female 9120 ? 5870 1830 ? 668 3 1 Male 7630 ? 3080 1180 ? 290 Female 10700 ? 6760 1180 ? 462 5 Male 9500 ? 5580 1420 ? 358 Female 8810 ? 4470 1290 ? 456

[0224] Tested article related clinical signs such as slight skin redness discolored and swelling of the ears were observed only after the third day of administration. These clinical signs were reversible after few hours and noted.

[0225] There were no test article-related abnormal changes noted in body weight, food consumption, respiratory rate, ECG, heart rate and clinical pathology. Gross necropsy and organ weight findings were considered as not treatment related changes according to the histopathology evaluations.

[0226] No marked sex difference in systemic exposure was observed. No marked drug accumulation was observed.

[0227] The histopathology evaluations found no differences between the 2 tested formulations and the commercial emulsion groups. No treatment related changes were noted. A range of histopathological findings were noted in different organs, all are considered as incidental findings, characteristic for Beagle dogs of the same age. Injection site lesions were seen in some animals from all groups, consisting of focal or multifocal arterial wall hemorrhage, subchronic inflammation and endothelial hyperplasia, as well as periartherial subchronic inflammation. All these changes are considered to be related to needle trauma, and are not suggestive of potential local irritation, because of the sporadic incidence among the animals.

[0228] In conclusion, no obvious and clear difference was noted between the two tested formulations (B6 and B9 propofol-microemulsions) and the commercial lipid propofol emulsions, indicating the safety (lack of adverse effects) of the tested propofol-microemulsions.

Example VIIX: Pain Upon Administration Assessment

[0229] Pain and local irritation are known side-effects during administration of commercial propofol formulations. As noted above, the unique interaction between Solutol and propofol in formulations of the invention maintains propofol bound within the oil core of the microemulsion until sufficiently diluted within the bloodstream, enabling relatively pain-free administration of the formulation.

[0230] In order to assess the effectivity in formulating propofol into microemulsions of the invention with respect to administration-associated pain, a paw-licking test was carried out. B9A (1 wt % propofol) microemulsion was tested in comparison to Diprivan? (a commercially available injectable propofol emulsion) as positive control and 0.9% saline as negative control.

[0231] Forty-four Wealing male Sprague Dawley Rats were randomized to one of the four treatment groups (11 animals per group). The injected dose volume was titrated from 0.1 mL to 0.3 mL (corresponding to approximately 20 mg propofol/kg bodyweight) until the expected effect in the positive control group (Diprivan?) was observed. The first 4 animals in each dose group received 0.1 mL or 0.2 mL, and the data were not used for analysis in this study.

[0232] The total number of paw licks were recorded for 12 minutes, following sub-plantar injection. Total scores and means were compared using two-sample t-test. Scores were further evaluated in 2 subsets: Grade 1 (single paw lick episode) and Grade 2 (5 sec of uninterrupted licking).

[0233] No paw-licks were observed in the saline control animals. The mean combined score of rats injected with the positive control Diprivan?, had a total paw lick score of 14.7.

[0234] Animals treated with B9A displayed a mean combined score of 1.57 with no Grade 2 (5 sec of uninterrupted licking) observed. Statistically significant differences (p?0.05) from the Diprivan? group were observed with respect to total mean licking scores and subset Grade 1 and 2 responses. The results are summarized in Table 15.

TABLE-US-00022 TABLE 15 Paw-lick scores - group totals Grade 1 Grade 2 Combined Group response response score Saline Total 0 0 0 control Mean 0 0 0 SD 0 0 0 Diprivan? Total 53 50 103 Mean 7.57 7.14 14.71 SD 4.76 8.49 4.90 B9A Total 11 0 11 Mean 1.57 0 1.57 SD 2.15 0 0.81

[0235] One animal in the B9A group had a broken nail on the dosed paw which began bleeding. This likely contributed to the lick response. In case this animal would be excluded from the analysis, the number of licks in the B9A group would be as low as 5. Thus, the pain on injection observed with B9A was significantly lower than that observed with Diprivan?.