WO 01/03668 PCT/CAOO/00805 PULMONARY DELIVERY OF LIPOSOME-ENCAPSULATED CANNABINOIDS FIELD OF THE INVENTION The present invention is related to the field of liposome-encapsulation of hydrophilic and hydrophobic agents. 5 More specifically, the present invention relates to the field of liposome-encapsulated cannabinoids. BACKGROUND OF THE INVENTION Since its discovery over 12,000 years ago, cannabis is .0 one of the most widely used drugs throughout the world. See, Adams, et al., 1996. Addiction 91: 1585-1614. Although the cannabis plant contains more than 400 chemical compounds, the main constituents of cannabis responsible for the psychoactive properties are the A 9 - tetrahydrocannabinol (A 9 .5 THC) and A 8 -tetrahydrocannabinol (A 8 -THC). Although both A 9 THC and A 8 -THC are active compounds extracted from the plant,
A
9 -THC composed of 90% of the active ingredient of the cannabis plant. Specific cannabinoid receptors (i.e., CB1 and CB2) have recently been identified and cloned and their 0 distribution throughout the entire CNS and body has been mapped. See, Herkenham, et al., 1991. Brain Res. 547:267 274; Jansen, et al., 1992. Brain Res. 575: 93-102; Gerard, et al., 1991. Biochem. J. 279: 129-134. In addition, a cannabinoid antagonist with a high affinity for the 5 cannabinoid receptor has also been characterised. See, Cook, et al., 1998. J. Pharmacol. Exp. Ther. 285: 1150-1156. The establishment of a cannabinoid receptor, antagonist, and endogenous ligand 1 SUBSTITUTE SHEET (RULE 26) WO01/03668 PCT/CA00/00805 with biosynthesis and degradation pathways suggests the presence of a distinct neurochemical system for cannabinoids. Despite substantial advances in the knowledge of cannabinoid pharmacology, its beneficial therapeutic effects 5 are mostly anecdotal, with a lack of quantitative scientific evidence. However, over the past several decades, many potential clinical applications for A 9 -THC have been suggested. These include, but are not limited to, (i) the management of patients with glaucoma (see, Ungerleider, et LO al., 1985. Int. J. Addict. 20: 691-699); pain (see, Noyes, et al., 1974. Comp. Psychiatry. 15: 531-535); seizure (see, Consroe, et al., 1975. JAMA 234: 306-307); appetite stimulation for HIV patients (see, Plasse, et al., 1991. Pharmacol. Biochem. Behav. 40: 695-700); multiple sclerosis L5 (see, Greenberg, et al., 1994. Clin. Pharmacol. Ther. 55: 324-328); and anti-emetic effect for patients receiving, e.g., chemotherapy (see, Ungerleider, et al., 1982. Cancer 50: 636-645). Similarly, however, quantitative results for these claims are lacking. 20 The lack of quantitative results is primarily due to the fact that cannabinoids (e.g., A 9 -THC and A 8 -THC) are highly lipophilic compounds with no suitable route of administration apart from smoking the cannabis leaf or resin. Unfortunately, a large number of unnecessary toxic chemicals ?5 present in the cannabis plant will also be absorbed into the circulation after smoking. Furthermore, the quality control is generally not available and the A 9 -THC, A 8 -THC, and 11-OH THC content of the cannabis leaf are highly variable, thus making it difficult to predict the bioavailability of A 9 -THC, 2 SUBSTITUTE SHEET (RULE 26) WO01/03668 PCT/CAOO/00805
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8 -THC, and 11-OH-THC, following smoking of the crude cannabis leaf. Therefore, the evaluation of the pharmacodynamic effects (i.e., the correlation between plasma cannabinoid concentrations and observed clinical effects) has 5 been extremely difficult. Although oral A 9 -THC (Dronabinol®) has been available for many years, its absorption is slow and its bioavailability is poor (i.e., 3-6% of total dose), with unpredictable absorption and high hepatic first-pass clearance effect. See, Ohlsson, et al., 1980. Clin. 0 Pharmacol. Ther. 28: 409-416. Moreover, no detectable A9-THC was found in the plasma following rectal application of several suppository formulations using carbowax, witepsol, sesame oil, cocoa butter, and Cetomacrogol. See, Perlin, et al., 1985. J. Pharm. Sci. 74: 171-174. 5 Therefore, at present, there remains an, as yet, unfulfilled need for the development of a drug delivery system for cannabinoids, including A 9 -THC, A 8 -THC, and 11-OH THC, which can provide a rapid increase and sustained therapeutic plasma cannabinoid concentration to achieve and 0 maintain a desired clinical effect. SUMMARY OF THE INVENTION The invention features a liposomal composition containing a cannabinoid or cannabimimetic agent and methods of systemic delivery by contacting pulmonary tissue of a ?5 mammal with the liposomal composition to achieve a prolonged psychoactive effect. The compositions and methods of treatment involve the use of unilamellar and multilamellar liposomes as a vehicle to provide systemic delivery of cannabinoids, for example, A -tetrahydrocannabinol (A 9 -THC), 3 Q|TRQTTITTW' H~1HW'T DYT1T ' 14, WO 01/03668 PCT/CA00/00805
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8 -tetrahydrocannabinol, (A 8 -THC); and 11-hydroxy tetrahydrocannabinol (11-OH-THC), via administration to the pulmonary system. Liposomal compositions contain a cannabinoid or 5 cannabimimetic agent, and the composition is in a form that is suitable for pulmonary administration. The liposomes of the composition are relatively uniform in size. For example, the range of size of liposomes in the composition is preferably within 25%, more preferably within 20%, more .0 preferably within 15%, more preferably within 10%, and most preferably within 5% of the mean size of the liposomes. For example, at least 85% (more preferably 90%, more preferably 95%, and most preferably 99-100%) of the liposomes in the composition are with a defined size range, e.g., between 300 5 400 nm in size. In another example, the liposomes are within 450-550 nm in size. In yet another example, the size range of the liposomes is between 700-800 nm. As utilized herein, the term cannabinoid is defined as a pharmacologically-active agent producing psychoactive effects 0 which may either be derived directly from the flowering tops of the pistillate hemp plant (e.g., Cannabis sativa var. indica) or is chemically-synthesized in the laboratory. See, Stedman, Medical Dictionary, pg. 111, Williams & Wilkins,Baltimore, MD (1987). Cannabinoids synthesized by 5 the hemp plant include, but are not limited to, cannabinol, cannabidiol, cannabinolic acid, cannabigerol, cannabicyclol, and several isomers of tetrachydrocannabinol (THC). See, Goodman and Gilman, The Pharmacological Basis of Therapeutics, 6 th Ed., pp. 560-563, MacMillan Publishing, New 0 York, NY (1983). 4 qITR.TTITTTE CHWWT MITI W 9A1 WO01/03668 PCT/CA00/00805 The cannabinoid to be delivered is selected from the group consisting of cannabinol, cannabidiol, A 9 tetrahydrocannabinol, A 8 -tetrahydrocannabinol, 11-hydroxy tetrahydrocannabinol, 11-hydroxy-A 9 -tetrahydrocannabinol, 5 levonantradol, A 11 -tetrahydrocannabinol, tetrahydrocannabivarin, dronabinol, amandamide, and nabilone. A cannabimimetic agent is a composition characterized as having at least 50% of the psychoactive effect of A 8 tetrahydrocannabinol. The mimetic may differ from A 8 LO tetrahydrocannabinol in structure, pattern of side group substitution, or both. The composition contains the active psychoactive ingredient, cannabinoid or a cannabimimetic agent, in an amount of between approximately 0.01% to 10% by weight. L5 The composition may also contain a phospholipid, e.g., a phosphatidylcholine, a dipalmitoylphosphatidylcholine, a lysophosphatidylcholine, a phosphatidylserine, a phosphatidyl-ethanolamine, a phosphatidylglycerol, or a phosphatidylinositol. Cholesterol is also a component of the 20 composition, and the approximate molar ratio of phospholipid to cholesterol is altered to achieve a desired pharmacokinetic effect. The rate of cannabinoid release from the composition is indirectly proportionate to the concentration of cholesterol in the composition, i.e., a 25 higher percentage of cholesterol yields a composition with a slower pharmacokinetic release profile compared to a composition with a lower percentage of cholesterol. Increasing the amount of cholesterol in the composition results in production of liposomes with a more rigid 5 SUBSTITUTE SHEET (RULE 26) WO01/03668 PCT/CA00/00805 membrane. A more rigid membrane indicates a relatively more stable liposome. For example, the molar ratio of dipalmitoylphosphatyidylcholine:cholesterol is 7:3, 6:4, or 9:1. Therefore, a composition formulated with an approximate 5 molar ratio of dipalmitoylphosphatyidylcholine:cholesterol of 7:3 is systemically released over a longer period of time compared to formulations with a lower relative amount of cholesterol. The compositions contain at least 10% cholesterol. To tailor the kinetics of drug release, the 10 composition is formulated to contain at least 20%, 25%, 30%, 35% or 40% cholesterol. Preferably, the percentage of cholesterol in the composition does not exceed 45%. The composition contains liposomes, which are multilamellar, unilamellar, or a mixture of both multilamellar and 15 unilamellar. The invention also includes a method for delivery of a cannabinoid to the central nervous system of a mammal using the compositions described above. Pulmonary tissue of a mammal is contacted with a liposomal composition containing a 20 cannabinoid or cannabimimetic agent. The compositions are administered orally, intratracheally, intravenously, and by other standard clinical modes of administration. Mammals, e.g., humans, to be treated include those who have been identified as suffering from or at risk of developing a 25 disease or disorder selected from the group consisting of: nausea, loss of appetite, glaucoma, seizure, multiple sclerosis, or pain. Systemic delivery of the cannabinoid is multiphasic. By multiphasic is meant the pharmacokinetic pattern of systemic 30 absorption of a cannabinoid or active metabolite thereof has 6 SUBSTITUTE SHEET (RULE 26) WO01/03668 PCT/CA00/00805 at least two compartments. For example, a multiphasic delivery system results, in a fast pharmacokinetic compartment, mid-range pharmacokinetic compartment, and a sustained pharmacokinetic compartment. A first phase (or 5 rapid compartment) is characterized by rapid systemic absorption of the cannabinoid or cannabinimimetic agent. The first phase ranges from 30 seconds to 30 minutes after pulmonary tissue is contacted with the cannabinoid composition. A second (or third) phase is characterized by 0 sustained systemic absorption of the cannabinoid or cannabinimimetic agent. The second (or subsequent) phase ranges from 30 minutes to 2 days after pulmonary tissue is contacted with the cannabinoid or cannabinimimetic composition. For example, the method results in a sustained L5 systemic concentration of a cannabinoid (e.g., as measured in plasma, or other tissues such as brain) for 6 hours, 12 hours, 24 hours, and up to several days post-administration. Thus, the invention provides a method of inducing a sustained psychoactive cannabinoid effect in the central nervous system ?0 of a mammal by contacting a pulmonary tissue of the mammal with a liposome-encapsulated cannabinoid or cannabimimetic agent. The major advantages of the present invention include: (i) the rapid bioavailability and initial onset of the 25 pharmacological effect of the cannabinoids from the immediate release of liposome-encapsulated cannabinoids (e.g., approximately 10-20 % of the total A 9 -THC dose); (ii) the continuous-release properties of the liposomes to provide a sustained pharmacological effect (e.g., approximately 80-90% 30 of the total A 9 -THC dose); (iii) the non-invasiveness of the 7 STTRTIrTITTV CHEFPT IDITT r '741 WO01/03668 PCT/CAOO/00805 drug delivery method; (iv) a controlled purity of the administered cannabinoids; and (v) in comparison with the oral administration of cannabinoids, this system does not require a functioning bowel and is not be affected by hepatic 5 first-pass elimination, which can significantly affect the bioavailability of cannabinoids.. Because of the non invasive nature of this drug delivery system, it is particularly suitable for some patient populations, such as pediatric, elderly and ambulatory patients. O The rate of drug release is regulated by altering: (i) the nature of the phospholipids utilized; (ii) the phospholipid:cholesterol ratio; (iii) the hydrophilic/lipophilic properties of the active ingredients; and (iv) the method by which the liposomes are generated. 5 As illustrated by the disclosed pharmacokinetic and tissue distribution data, pulmonary administration of liposome-encapsulated cannabinoids is efficient, safe, and does not exhibit any significant adverse cardiopulmonary side effects. The effects of the cannabinoid formulation last 0 more than 24 hours following pulmonary administration of liposomal cannabinoid. Furthermore, the various experimental manipulations of the composition of the liposomes applied herein indicate that the plasma pharmacokinetic profile of cannabinoids can be tailored to provide a desired duration of 5 the drug's therapeutic effect. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a line graph which illustrates mean plasma A 9 THC concentration versus time profile. 8 QTlrTTTTT HT-'W'T IDITT r 14£ WO01/03668 PCT/CAOO/00805 FIG. 2 is a line graph which illustrates mean plasma A 9 THC concentration verses time profiles for Composition 1. FIG. 3 is a line graph which illustrates mean plasma A 9 THC concentration verses time profiles for Composition 2. 5 FIG. 4 is a line graph whichillustrates mean plasma A 9 THC concentration verses time profiles for Composition 3. FIG. 5 is a line graph which illustrates mean plasma A 9 THC concentration verses time profiles for Composition 4. FIG. 6 is a line graph which illustrates a comparison of .0 the predicted plasma A 9 -THC concentration profiles various Composition trials (Compositions 1-16). FIG. 7 is a line graph which illustrates lung A 9 -THC concentrations versus time profiles. FIG. 8 is a line graph which illustrates brain A 9 -THC 5 concentrations versus time profiles. FIG. 9 is a line graph which illustrates mean plasma A 8 THC concentration verses time profile. FIG. 10 is a line graph which illustrates mean plasma 11-OH-THC concentration versus time profile. 0 DETAILED DESCRIPTION OF THE INVENTION Liposomes are used as a vehicle to deliver cannabinoids (e.g., tetrachydrocannabinol) and other cannabimimetic agents to improve the pharmacokinetic profiles of the cannabinoid and cannabimimetic agents. Liposomes are microscopic 5 vesicles composed of one or more aqueous compartments alternating with phospholipid bilayers. The liposomes 9 qTTRTITT1TTEW Q14TET iDYIT r 90 WO01/03668 PCT/CA00/00805 described herein are formulated to provide a controlled, sustained release system. The rate of drug release by the liposome is primarily determined by its physicochemical properties. Liposomes are tailored by the modification of 5 size, composition, and surface charge to provide the desired rate of drug delivery. The primary advantages of the present invention include, but are not limited to: (i) the rapid onset of drug effect; (ii) the slow release properties of the liposomes to provide LO a sustained drug effect; and (iii) the non-invasive method of drug delivery through the pulmonary system. The methods provide a systemic drug effect for humans. The sustained release property of the liposomal product is regulated by the lipid and other excipient composition of L5 the liposomal products. The methods described herein permit accurate and reproducible prediction of the overall rate of drug release, based upon the specific composition of the liposome formulation. The rate of drug release is primarily dependent upon: (i) the nature of the specific phospholipids 20 (e.g., hydrogenated (-H) or unhydrogenated (-G)); (ii) the phospholipid:cholesterol ratio (i.e., the higher the ratio, the faster the rate of release); (iii) the hydrophilic/lipophilic properties of the active ingredients; and (iv) the method utilized in the production of the of 25 liposomes. I. Quantitation of Cannabinoids by Gas Chromatography-Mass Spectrometry Plasma cannabinoid concentrations were determined using a gas chromatography-mass spectrometry technique (GC-MS; see, 10 SUBSTITUTE SHEET (RULE 261 WO01/03668 PCT/CA00/00805 e.g., Wilkins, et al., 1995. J. Anal. Toxicol. 19: 483-491). For A 9 -THC, a measured volume of plasma (approximately 0.4 1.0 ml), with labelled-A 9 -THC added as an internal standard, was deproteinized by the addition of 2-volumes of 5 acetonitrile, and centrifuged at 2500 r.p.m. The majority of the acetonitrile was removed from the supernatant by a stream of nitrogen gas. The remaining aqueous layer was then extracted with 3-volumes of hexane:ethylacetate (9:1 v/v). The organic layer was dried under a stream of nitrogen gas 10 and derivatized with 50 ml of trifluoroacetic anhydride in 50 ml of chloroform for 30 minutes at 450C. The sample was then analyzed by GC/MS (Finnigan Voyager) using Negative Ion Chemical Ionization (methane CI gas) in SIM mode (m/z 410 and m/z 413). The quantitation was performed using a 5-point L5 calibration curve (i.e., blank plasma "spiked" with 0.1, 0.5, 5, 10, 100 ng/ml of A 9 -THC). Three QC samples (i.e., blank plasma aliquots "spiked" with 0.5, 5 and 50 ng/ml of A 9 -THC) were analyzed with every batch of 45 samples. This assay method was also used to determine the plasma concentrations ?0 of other cannabinoids, including, but not limited to, A 8 tetrahydrocannabinol (A 8 -THC), and 11-hydroxy tetrahydrocannabinol (11-OH-THC), using different internal standards. II. Preparation of Liposomal Cannabinoids 5 The lipids used for the preparation of liposomes to entrap cannabinoids primarily consisted of dipalymitoylphosphatidylcholine (DPPC) and cholesterol in a molar ratio of 9:1, 7:3, or 6:4, however, other bilayer forming lipids may also be utilized for the same purpose. 11 qTT TTQTITT' HWWT (DITT 1 1 WO01/03668 PCT/CA00/00805 The selected lipids were dissolved in a minimal volume of chloroform in a round-bottomed glass vessel, followed by the addition of a defined amount of cannabinoids (Sigma Aldrich Canada, Ltd.; Oakville, ON, Canada). Chloroform was 5 then evaporated under a stream of helium gas at 40 0 C, and the glass vessel was placed under vacuum overnight to remove any residual solvent. The dried lipid-cannabinoid mixture was then hydrated at 51 0 C in phosphate-buffered saline (0.15 M, pH 7.2) and kept at this temperature with periodic vortexing LO for the next 30 minutes. The liposomes with entrapped cannabinoid were extruded a total of 10-times with an extruder (Lipex Biomolecules; Vancouver, BC) fitted with doubly-stacked polycarbonate filters of 400 nm or 1000 nm pore size, using a helium pressure of 100-200 lb/in 2 . .5 Liposomal vesicle size was determined with a Coulter N4SD particle-size analyzer (see Table 1). Unlike other methods of liposome manufacture (which method yields a heterogeneous population of liposomes which vary widely in size), extrusion yields a population of liposomes that are relatively uniform 0 in size. Uniformity of size allows more reproducible pharmacokinetics than other methods in the art. The materials and procedures for liposome encapsulation are well-known. Many other liposome manufacturing techniques can be used to make the final liposomal product containing 5 the appropriate active ingredient, lipids, and other excipient composition. The pharmacologically-active cannabinoid ingredients include, but are not limited to, A 9 THC, A 8 -THC, and 11-OH-THC. Lipid components include, but are not limited to, phospholipids and cholesterol. The 0 excipients include, but are not limited to, tocopherol, 12 QITRTTTTITTV QT4r17T (DTTT V 741 WO01/03668 PCT/CA00/00805 antioxidants, viscosity-inducing agents, and/or preservatives. For disclosure of preferred methodology for liposome preparation in the present invention, see, United States Patent No. 5,451,408, incorporated herein by reference 5 in its entirety. Table 1 Compositi Cholesterol/D Cannabinoid Filter Particle on Number PPC Molar Concentration Size size (nm) Ratio 1 0.11 A 9 -THC 0.3 400 nm 366 ± 21 mg/ml 2 0.42 Ag-THC 0.3 400 nm 368 ± 42 mg/ml 3 0.66 Ag-THC 0.3 400 nm 378 ± 214 mg/ml 4 0.66 A 9 -THC 0.3 1000 nm 888 ± 32 mg/ml 15 0.42 A 8 -THC 0.3 400 nm 634 ± 31 mg/ml 16 0.42 11-OH-THC 400 nm 539 ± 134 0.3 mg/ml 13 IPTHTTTTT CHWET (DITT W 10 WO01/03668 PCT/CA00/00805 The following compositions are merely illustrative of the compositions of present invention, and are not to be regarded as limiting. A 9 -THC, A 8 -THC, and 11-OH-THC were encapsulated into both uni- and multi-lamellar liposomes. 5 III. Cannabinoid Compositions for Inhalation Composition 1 (for each 10 ml):
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9 -THC 3.0 mg Dipalmitoyl phosphatidylcholine 944.7 mg Cholesterol 55.3 mg .0 Phosphate-Buffered Saline q.s 10 ml Extruded through 400 nm filter Composition 2 (for each 10 ml):
A
9 -THC 3.0 mg Dipalmitoyl phosphatidylcholine 815.8 mg 5 Cholesterol 184.2 mg Phosphate-Buffered Saline q.s 10 ml Extruded through 400 nm filter Composition 3 (for each 10 ml): A'-THC 3.0 mg 0 Dipalmitoyl phosphatidylcholine 740.1 mg Cholesterol 259.9 mg Phosphate Buffered Saline q.s 10 ml Extruded through 400 nm filter Composition 4 (for each 10 ml): 5 A 9 -THC 3.0 mg Dipalmitoyl phosphatidylcholine 740.1 mg Cholesterol 259.9 mg 14 SUBSTITUTE SHEET (RULE 26) WO 01/03668 PCT/CA00/00805 Phosphate Buffered Saline q.s 10 ml Extruded through 1000 nm filter Composition 5 (for each 5 ml):
A
9 -THC 1.5 mg 5 Soy lecithin (hydrogenated) 250.0 mg Phosphate Buffered Saline q.s 5 ml Extruded through 400 nm filter Composition 6 (for each 5 ml):
A
9 -THC 1.5 mg 3 Soy lecithin (hydrogenated) 225.0 mg Cholesterol 25 mg Phosphate Buffered Saline q.s 5 ml Extruded through 400 nm filter Composition 7 (for each 5 ml): 5 A 9 -THC 1.5 mg Soy lecithin (unhydrogenated) 250.0 mg Phosphate Buffered Saline q.s 5 ml Extruded through 400 nm filter Composition 8 (for each 5 ml): ) A 9 -THC 1.5 mg Soy lecithin (unhydrogenated) 225.0 mg Cholesterol 25 mg Phosphate Buffered Saline q.s 5 ml Extruded through 400 nm filter Composition 9 (for each 5 ml):
A
9 -THC 1.5 mg Phospholipon 80 (hydrogenated) 250.0 mg 15 ITRTTTTITV IF.H T (IIIET. 9 WO01/03668 PCT/CA00/00805 Phosphate Buffered Saline q.s 5 ml Extruded through 400 nm filter Composition 10 (for each 5 ml):
A
9 -THC 1.5 mg 5 Phospholipon 80 (hydrogenated) 225.0 mg Cholesterol 25 mg Phosphate Buffered Saline q.s 5 ml Extruded through 400 nm filter Composition 11 (for each 10 ml): L0 A 9 -THC 3 mg Dipalmitoyl Phosphatidylcholine 1000 mg Phosphate Buffered Saline q.s 10 ml Extruded through 400 nm filter Composition 12 (for each 30 ml): .5 A'-THC 200.0 mg Dipalmitoylphosphatidylcholine 2834.1 mg Cholesterol 166.2 mg Lactose 4500 mg Phosphate Buffered Saline q.s 30 ml 0 Extruded through 400 nm filter Composition 13 (for each 30 ml): A'-THC 200.0 mg Dipalmitoyl phosphatidylcholine 2447.4 mg Cholesterol 552.6 mg 5 Lactose 4500 mg Phosphate Buffered Saline q.s 30 ml Extruded through 400 nm filter 16 SUBSTITUTE SHEET (RULE 26) WO01/03668 PCT/CAOO/00805 Composition 14 (for each 30 ml):
A
9 -THC 200.0 mg Dipalmitoyl phosphatidylcholine 2220.3 mg Cholesterol 779.7 mg 5 Lactose 4500 mg Phosphate Buffered Saline q.s 30 ml Extruded through 400 nm filter Composition 15 (for each 5 ml):
A
8 -tetrahydrocannabinol 1.5 mg 0 Dipalmitoyl phosphatidylcholine 407.92 mg Cholesterol 92.08 mg Phosphate Buffered Saline q.s 5 ml Extruded through 400 nm filter Composition 16 (for each 5 ml): 5 11-hydroxy-tetrahydrocannabinol 1.5 mg Dipalmitoyl phosphatidylcholine 407.92 mg Cholesterol 92.08 mg Phosphate Buffered Saline q.s 5 ml Extruded through 400 nm filter 0 IV. Pharmacokinetics and Tissue Distribution of Liposomal
A
9 -THC To allow for a comparison of the bioavailabilities and pharmacokinetic parameters of pulmonary delivery of different liposomal A9-THC preparations, a comparative study with 5 intravenous administration was conducted in rabbits. 1. Pharmacokinetics of Intravenous A 9 -THC 17 STITRSTITITTE .TIFFT tPIT F M WO01/03668 PCT/CAOO/00805 Five New Zealand White rabbits were used to study the plasma A 9 -THC concentration-time profiles following intravenous administration of A 9 -THC (100 pg/ml) in alcohol. Anesthesia was induced by intramuscular injection of ketamine 5 and was maintained by halothane in a mixture of nitrous oxide and oxygen. Under anaesthesia, the central ear artery was cannulated using a #22 catheter for blood sampling. A 9 -THC in alcohol (100 gg) was then administered intravenously to the marginal ear vein of the contra lateral ear. Arterial blood 10 samples (1 ml, each) were drawn at nominal times of 5, 10 15, 20, 25, and 30 minutes, and at 1, 2, 4, 6, and 8 hours post administration of A 9 -THC. Venous samples were also collected at 24 hours post-administration. The plasma was separated immediately following the blood collection and stored at L5 200C until analyzed. Plasma A9-THC concentrations were determined using a gas chromatography-mass spectrometry technique as described, supra. The mean plasma A 9 -THC concentration verses time profile is illustrated in Table 2 (see, I.V.) and also in FIG. 1. 18 SUBSTITUTE SHEET (RULE 26) WO 01/03668 PCT/CA00/00805 -C, 0o '.O , r-n-q C - II 0 CCO" r- o') C - O *+ ,4 . . - 0 7 . . . . . 0 -H- -H +1C- +1- N 0 r -I - O r-i o- -i -H 0 O1 ) 0 0 a 0 o 0 0 o Oa-H o . LO 0 * 1 CD .0 .0 UJ - E L +1 C +10- +O N +10 mC M -H OD 410D~ +10 0 n C+ i1m I O I0 c) L +r-1i m + | m* 1 CC) > O 4 OC m 0 -CN C Co ( . • - . .o S M-u - +1o o +1 -1 H4 r +1 LO 'T-HM0 -H LO + Io cl II o 0 0 0 0 0 0 o 0 0 * 0 * o * 0 C . 0 0 OC-H 0 *L0 *(0 * 10 .0 .0 * O 9-~0+10 n-+Ior-+0oN41O N"+I0om9040 om1o S * - 0 > H - C -- NO + - m -H) - OaLt -H- ,- oCN o. . . . * (J C NCO Lr- nI N v 0 NJ - ' +1
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**. OD m+1m t0 C3H1m mH 000 mm LCO O+'- r+COj LOH~ 0-H LO OD OC0 g o0 0 * (N N 0 [ ~ t- 0 Oa If C * o '0 ~* O w- t"0 (N * * .IlCOmMN (N+I(N .+ILO LO-N 000 0 O C1 0 D 21 RTTRSTITTITTF RHET (IRTTIN I' WO 01/03668 PCT/CAOO/00805 C4 C) r. Nr- 'HCi r- +1 mf r-~ +1 "a'o H~ 1 H C HO +1 r- C), r- +1 C:, 10 CN zll q:3 m ZI (Y)~ *C C H dou C) 'H * m *H ma) C -H CO C-H C) - H C) HC) 10) OC14H C H H C *n CD m'*' ~ 1 'CD *U) 'HM) 14 N+1 C)-+HC) '+1 C)+-HC) w 11 N ()I' CN 'H C- - N1 mO *- N ' * 'H CD 10 0) O N * * ko CD -H O - H ~ C +i C) CN+1N C)+-HC; OD k.0 '1D C) '-O mO m N- C~j co C) t* U-) 10 T -H' m 1 O+I' C)+IC)l ' +1 -) C;- C ; ) mO OD C) N O CD COU-1)1 CD 'HA U- CD *- C) C ) r -HC N4-H C; "N-H lC) -H C; (N 1O 00 mO 'Hi O L 1O OD tl CC) 00 D - H m H CD +1 N; + CD I r tr-i >1 ~ H-H 413 0 U E 22 SUBSTITUTE SHEET (RULE 26) WO01/03668 PCT/CA00/00805 Table 3 illustrates in tabular form the Mean ± SD of A 9 THC dosage (Jg/-kg) and pharmacokinetic parameters for 2 compartments based on the regressions of the individual animal data within each trial. A = intercept (ng/-ml; a = 5 reciprocal of the time constant (h-'); AUC = area under the plasma drug concentration curve (ng.h.ml-1); Kel = drug elimination rate constant (h-'); Cl = total systemic clearance (ml-min-1kg-1); Vd = volume of distribution (L.kg- 1 ); T, = time to a drug concentration level of 1 ng/ml; and bioavailability 0 (kg.h.L- 1 ). Significant differences between trials (Composition 15 and 16 were excluded due to low sample numbers) were in Dosage (I.V. vs Composition 1, 2, 3, and 4), a, (Composition 1 verses 4); A 2 (Composition 1 verses 2 and I.V.); a 2 (Composition 1 verses 4; I.V. verses Composition 3 5 and 4); Kel (Composition 1 verses 4); AUC (Composition 1 verses 2); Vd (Composition 2 verses 1 and I.V.), and T, (Composition 2 verses 1, 4, and, I.V.). For detailed explanation of the parameters, see, Klassen, Distribution, excretion, and absorption of toxicants. in: Toxicology: The 0 Basic Science of Poisons, pp. 33-63, Klassen and Amdur (eds), Macmillan, NY (1986). 2. Phamacokinetics of Inhaled Liposome-Encapsulated A'-TEC New Zealand White rabbits (4 to 6 per composition 5 tested) were used to study the A 9 -THC concentration-time profiles following pulmonary administration of several compositions (Compositions 1, 2, 3, and 4) of liposome encapsulated A 9 -THC. Under similar experimental conditions as utilized in the intravenous study, the central ear artery 23 SUBSTITUTE SHEET (RULE 26) WO01/03668 PCT/CAOO/00805 of the rabbit was cannulated using a #22 catheter for blood sampling. Under deep anaesthesia, tracheal intubation was performed using a #1 laryngoscope. A 9 -THC (150 gg) in 0.5 ml of liposome preparation (Composition 1, 2, 3, and 4) was 5 instilled into the trachea through the endotracheal tube. Arterial blood samples (1 ml, each) were then drawn at nominal times of 5, 10 15, 20, 25, and 30 minutes, and at 1, 2, 4, 6, and 8 hours post-administration. Venous blood samples were also collected at 24 hours post-administration. 10 The mean plasma A 9 -THC concentration verses time profiles for Compositions 1, 2, 3, and 4 are shown in tabular form in Table 2, and also illustrated in FIGS. 2, 3, 4, and 5, respectively. The data shown in FIG. 1 through FIG. 5 indicates that 15 the mean drug clearance data can segregates into a 3 compartment pharmacokinetic model of systemic drug absorption. The "slow" compartment corresponds primarily to the plasma A 9 -THC measured beyond 300 minutes following pulmonary A 9 -THC administration. The "mid" and "fast" 20 compartments correspond to A 9 -THC concentrations measured from 30 to 300 minutes inclusive, and within 30 minutes after
A
9 -THC administration, respectively. Application of the compartmental fitting procedure resulted in the predicted profiles superimposed on FIG. 1 to FIG. 5. The mean (± SD) 25 A 9 -THC dosage (pg/kg) and pharmacokinetic parameters based upon the regression, are summarized in Table 3. The clearance of Composition 2 (i.e., a 7:3 ratio of DPPC:Cholesterol) appeared to be considerably slower than the other compositions. 24 SIBRSTITITTE SHRET (RITI'F 76 WO01/03668 PCT/CAOO/00805 An additional parameter was also introduced that is particularly relevant to the present invention. This is the computed time to a drug concentration level of 1 ng/ml (T 1 ) which is close to the minimum effective concentration. Its 5 value was determined iteratively by solving the fitted drug clearance equation with concentration equal to 1 ng/ml. There was a significant increase in the value of Ti for Composition 2, as compared to other compositions administered through the lungs (except for Composition 3) as well as 0 following intravenous A 9 -THC administration, suggesting that Composition 2 possesses a potentially prolonged therapeutic value. FIG. 6 shows a comparison of the predicted plasma A 9 THC concentration profiles of all the various Composition trials where the clearance of Composition 2 is seen to be 5 considerably longer than any of the other compositions. This is consistent with the significantly higher value of T, found for Composition 2 among all trials (see, Table 3). The results disclosed herein have demonstrated that pulmonary administration of liposome-encapsulated A 9 -THC has Several distinct advantages over I.V.-based administration. These advantages include, but are not limited to: (i) Intravenous administration of A 9 -THC dissolved in alcohol (A 9 -THC is highly lipophilic and is not soluble in water) is invasive and painful upon injection. In fact, 2 of 5 the 5 rabbits had evidence of thrombophlebitis at the site of I.V. injection site 24 hours after the I.V. administration of
A
9 -THC. In contrast, pulmonary administration is a non invasive method of A 9 -THC delivery and appeared to be well tolerated by the rabbits; and 25 STRSTITTIT SHFT'T tTIIT V "M WO01/03668 PCT/CA00/00805 (ii) Pulmonary delivery of liposome-encapsulated A 9 -THC provides a rapid onset of drug effect with a peak A 9 -THC concentration occurred within 5 minutes after administration (comparable to intravenous administration; see, Table 2) and 5 a sustained plasma A 9 -THC concentration to provide a prolonged A 9 -THC drug effect. 3. Tissue Concentrations of A 9 -THC Following Pulmonary Administration of Liposome-Encapsulated A 9 -THC 0 A total of 25 New Zealand White rabbits were used to study the tissue A 9 -THC concentrations following pulmonary administration of liposome-encapsulated A 9 -THC (Composition 2). Under similar experimental conditions as described supra, the central ear artery of the rabbit was cannulated 5 using a #22 catheter for blood sampling. Under deep anaesthesia, tracheal intubation was performed, and 0.5 ml volume of liposome preparation (Composition 2; 150 pg of A 9 THC) was instilled into the trachea through the endotracheal tube. Immediately after the instillation of the liposomal D A 9 -THC, 5 rabbits were sacrificed and the lungs and brains of these animals were immediately removed and excess blood was removed by dry, sterile gauze. The A 9 -THC concentrations of the lungs and brains were determined, and these values were considered as the baseline. Similarly, 5 rabbits were 5 sacrificed at 1, 4, 12, and 24 hours following pulmonary administration of liposomal A 9 -THC and the lungs and brain were removed to determine the tissue A 9 -THC concentrations. In addition, for these rabbits, arterial blood samples (1 ml each) were collected where applicable at 5, 10, 15, 20, 25, 26 qTTRNTITITTT1P SET (PTRITT i1 WO01/03668 PCT/CA00/00805 and 30 minutes post-administration, and also at 1, 2, 4, 6, 8 hour intervals. Venous blood samples were collected at 18 and 24 hours. The organs were weighed and finely minced. One gram of 5 the tissue (either brain or lung) was then homogenized. To facilitate extraction of the A 9 -THC from the tissue, an equal volume of acetonitrile was added to the homogenate and vortexed. Following centrifugation at 9000 x g for 20 minutes in a refrigerated (4 0 C) centrifuge, the supernatant 0 was separated. The A 9 -THC concentration of the supernatant was then determined using the GC/MS as described, supra. The lung and brain A 9 -THC concentrations versus time profiles are shown in FIG. 7 and FIG. 8, respectively. This data is described by a 2-compartment model. The half-times 5 for both the "fast" and "slow" compartments for the brain are 0.28 and 13.9 hours, respectively; whereas the half-times for both the "fast" and "slow" compartments for the lungs are 0.09 and 31.4 hours, respectively. Although no A 9 -THC was detected in the plasma at 24 hours following pulmonary 0 administration of liposomal A 9 -THC, the mean (± SD) A 9 -THC concentration present in the lungs at 24 hours was found to be 1.5 ± 0.8 ng/gm of tissue. The retention of A 9 -THC within the lung tissues 24 hours after intratracheal administration is likely due to the liposomal encapsulation, which delayed 5 the clearance of A 9 -THC from the lungs. See, Tan, et al., 1996. Drug Delivery 3: 251-254. Although the endogenous lipase present in the lung parenchyma would continuously break down the liposomes present in the lungs and release the entrapped A9-THC for systemic absorption, the highly lipid 27 SUBSTITUTE SHEET (RULE 26) WO01/03668 PCT/CAOO/00805 soluble A 9 -THC was distributed extensively in the body immediately after absorption. Although there is A 9 -THC retained within the lung tissues, this rapid redistribution of the A9-THC together 5 with the dilution effect from the large plasma volume may account for the undetectable A 9 -THC in the plasma at 24 hours. This is in direct contrast to those values obtained for the brain, which decreased significantly after 1 hour. The time constant of 20 hours, suggests that it would take 0 54.3 hours for THC in the brain to decrease to a concentration of less than 0.1 ng/gm of tissue. Although no pharmacodynamic effects were measured during the study, the small amount of A 9 -THC present within the brain (0.5 ± 0.1 ng/gm of tissue 24 hours after the pulmonary administration) 5 may indicate a long-lasting A 9 -THC drug effect within the CNS following pulmonary administration of liposomal A 9 -THC. V. Pharmacokinetics of Liposome-Encapsulated A8-TEC Two New Zealand White rabbits were used to study the A 8 THC concentration versus time profiles following pulmonary 0 administration of liposome-encapsulated
A
8 -THC. Under similar experimental conditions as described for liposome encapsulated A 9 -THC, supra, the central ear artery of the rabbit was cannulated for blood sampling. Under deep anaesthesia, tracheal intubation was performed, and A 8 -THC 5 (150 jg) in 0.5 ml of liposome preparation (Composition 15) was instilled into the trachea. Arterial blood samples (1 ml, each) were then drawn at nominal times of 5, 10 15, 20, 25, 30, 60, and 90 minutes, and at 2, 4, 6, 8, and 10 hours. 28 qITRHTTTITTW -ET'FT (DTT UI K WO01/03668 PCT/CAOO/00805 Two venous blood samples were also collected at approximately 24 hours post-administration. The plasma A 8 -THC concentrations were determined by the GC-Mass spectrometry as described, supra. 5 The mean plasma A 8 -THC concentration verses time profile is shown in FIG. 9 and illustrated in tabular form in Table 2 (Composition 15). A two-compartment model was used to fit the mean results of the A 8 -THC. The pharmacokinetic parameters were consistent with those derived from other 0 studies described herein. Furthermore, the results indicate that the drug concentrations of A 8 -THC exceeded 1 ng/ml well beyond 1 hour post pulmonary administration. For example, the
A
8 -THC concentrations were still measurable in blood samples after 24 hours following pulmonary administration. 5 VI. Pharmacokinetics of Liposome-Encapsulated 11-OH-TEC Two New Zealand White rabbits were also used to study the 11-OH-THC concentration-time profiles following pulmonary administration of liposome-encapsulated 11-OH-THC. Under similar experimental conditions to those utilized for 0 liposome-encapsulated A 8 -THC and A 9 -THC, as described supra, the central ear artery of the rabbit was cannulated for blood sampling. Under deep anaesthesia, tracheal intubation was performed and 11-OH-THC (150 jg) in 0.5 ml of liposome preparation (Composition 16) was instilled into the trachea. 5 Arterial blood samples (1 ml, each) were drawn at nominal times of 5, 10 15, 20, 25, 30, 60, and 90 minutes, and at 2, 4, 6, 8, and 10 hours post-administration. Two venous blood samples were also collected at approximately 24 hours. The 29 SIRSTITITE STERT (PRITIF 61 WO01/03668 PCT/CAOO/00805 plasma 11-OH-THC concentrations were then determined by the GC-Mass spectrometry, as described supra. The mean plasma 11-OH-THC concentration verses time profile is shown in FIG. 10 and illustrated in tabular form in Table 2 (Composition 5 16). A two-compartment model was then used to fit the mean results of the 11-OH-THC. Similarly, the estimated pharmacokinetic parameters were not markedly different from those results obtained for the A 8 -THC. The 11-OH-THC concentration verses time profile indicates that the drug D concentrations of 11-OH-THC exceeded 1 ng/ml well beyond 1 hour post pulmonary administration. Moreover, the 11-OH-THC concentrations were also measurable in the blood samples greater than 24 hours post pulmonary administration. Equivalents 5 Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims which follow. In particular, it is contemplated by the ) inventor that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims. Other embodiments are within the following claims. 30 Q1TrTTTIT1' T -Pr Q T (DTTT 17 14't