Patent Application
20230147697
Cancer is presently one of the leading causes of death in developed nations. Although recent research has vastly increased our understanding of many of the molecular mechanisms of tumorigenesis and has provided numerous new avenues for the treatment of cancer, standard treatments for most malignancies remain gross resection, chemotherapy, and radiotherapy. While increasingly successful, each of these treatments may cause numerous undesired side effects. For example, surgery may result in pain, traumatic injury to healthy tissue, and scarring. Radiotherapy and chemotherapy may cause nausea, immune suppression, gastric ulceration and secondary tumorigenesis.
Improved methods for the treatment of diseases, including cancer, and compositions capable of delivering bioactive agents to aid in the treatment of diseases and other conditions remain desirable.
The present disclosure provides compositions suitable for administering lipophilic bioactive agents to a subject. The lipophilic bioactive agents may be delivered by any route of administration. In embodiments, the lipophilic bioactive agent may be contained in liposomes.
Methods for forming compositions containing liposomes possessing lipophilic bioactive agents are also provided. In embodiments the lipophilic bioactive agent may be prepared as a first phase, optionally in combination with a solubilizer, while a second phase may be prepared containing at least one phospholipid. The two phases may be combined, thereby forming liposomes possessing lipophilic bioactive agent.
In embodiments, liposomes possessing lipophilic bioactive agent may be combined with additional carriers for administration to a subject. Such carriers may include, in embodiments, oil phases, water phases, neutralizing or buffer phases, pigments, combinations thereof, and the like.
Compositions of the present disclosure including liposomes possessing lipophilic bioactive agents may also include permeation enhancers to enhance delivery of the bioactive agent.
In embodiments, compositions of the present disclosure may include a liposomal concentrate including a phospholipid such as lecithin, lysolecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidic acid, lysophosphatidylserine, PEG-phosphatidylethanolamine, PVP-phosphatidylethanolamine, and combinations thereof, at least one lipophilic bioactive agent, and at least one solubilizer. The liposomal concentrate may be in combination with at least one pharmaceutically acceptable carrier possessing at least one permeation enhancer in an amount from about 0.5% by weight to about 20% by weight of the composition. The phospholipid may present in the composition in an amount from about 2% to about 20% by weight of the composition and the bioactive agent may be present in an amount from about 0.5% to about 20% by weight of the composition.
In other embodiments, a composition of the present disclosure may include an oil phase, a water phase, a neutralization phase, a pigment phase, and a liposomal concentrate. The oil phase may include C12-15 alkyl benzoates, cetyl alcohol, stearyl alcohol, glyceryl stearate, and polyethylene glycol 100 stearate, and may be present in an amount of from about 5% to about 20% by weight of the composition. The water phase may include glycerin, propylene glycol, ethoxydiglycol, phenoxyethanol, water, and a crosslinked acrylic acid polymer dispersion including phenoxyethanol, propylene glycol, water, and a crosslinked acrylic acid polymer, and may be present in an amount of from about 60 to about 80% by weight of the composition. The neutralization phase may include water, triethanolamine, sodium lactate, and lactic acid, and may be present in an amount of from about 0.1% to about 15% by weight of the composition. The pigment may include titanium dioxide present in an amount of from about 0.2% to about 2% by weight of the composition. The liposomal concentrate may include a polyethoxylated fatty acid ester of sorbitan, coenzyme Q10, a phosphatidylcholine lecithin, phenoxyethanol, propylene glycol, and water, and may be present in an amount of from about 0.1% to about 30% by weight of the composition. In embodiments, the propylene glycol and ethoxydiglycol from the water phase may act as permeation enhancers and may be present in a combined amount of from 3% by weight to about 15% by weight of the composition, and the coenzyme Q10 may be present in an amount of from about 0.75% by weight to about 10% by weight of the composition.
Any disease or condition that would benefit from the administration of a lipophilic bioactive agent may be treated with liposomes possessing lipophilic bioactive agents as described herein, including compositions containing such liposomes possessing lipophilic bioactive agents optionally in combination with a permeation enhancer.
Various embodiments of the present disclosure will be described herein below with reference to the Figures wherein:
In accordance with the present disclosure, a formulation is provided for improved administration of lipophilic bioactive agents, which may also be referred to herein as hydrophobic bioactive agents. As used herein, a lipophilic bioactive agent includes an agent that is insoluble in water. Specifically, lipophilic bioactive agents, as used herein, will have a solubility in water that is less than about 1 part of bioactive drug in about 1000 parts of water.
In embodiments the lipophilic bioactive agents may be placed in liposomes and administered to a patient. In accordance with the present disclosure, any phospholipid and/or phospholipid derivative such as a lysophospholipid may be utilized to form a liposome for encapsulating the lipophilic bioactive agent. Suitable phospholipids and/or phospholipid derivatives include, but are not limited to, lecithin, lysolecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidic acid, lysophosphatidylserine, PEG-phosphatidylethanolamine, PVP-phosphatidylethanolamine, combinations thereof, and the like.
In some embodiments, a lecithin derived from egg or soybean may be utilized as the phospholipid. Such lecithins include those commercially available as PHOSPHOLIPON® 85G, PHOSPHOLIPON® 90G, and PHOSPHOLIPON® 90H (the fully hydrogenated version of PHOSPHOLIPON® 90G) from American Lecithin Company, Oxford, Conn. Other suitable lecithins include LECINOL S-10 lecithin from Nikko Chemicals.
The above phospholipids or derivatives thereof may be utilized to form liposomes containing the bioactive agent. In embodiments, a lecithin having a high phosphatidylcholine content may be utilized to form a liposome. In some embodiments a high phosphatidylcholine lecithin which may be utilized includes PHOSPHOLIPON® 85G, a soy-derived lecithin containing a minimum of about 85% of a linoleic acid based-phosphatidylcholine. This lecithin is easy to use and is able to produce submicron liposomes at low process temperatures (from about 20° C. to about 55° C.) without the addition of any other special additives. PHOSPHOLIPON® 85G contains, in addition to phosphatidylcholine, approximately 5-7% phosphatidic acid. The phosphatidic acid confers a negative surface charge to the resulting liposome vesicles, reduces processing time and process energy, and aids in the formation of stable liposomes.
Suitable lipophilic bioactive agents which may be enclosed in liposomes herein include, but are not limited to, analgesics, anti-inflammatory agents, anthelmintics, anti-arrhythmic agents, anti-bacterial agents, anti-viral agents, anti-coagulants, anti-depressants, anti-diabetics, anti-epileptics, anti-fungal agents, anti-gout agents, anti-hypertensive agents, anti-malarials, anti-migraine agents, anti-muscarinic agents, anti-neoplastic agents, erectile dysfunction improvement agents, immunosuppressants, anti-protozoal agents, anti-thyroid agents, anxiolytic agents, sedatives, hypnotics, neuroleptics, β-Blockers, cardiac inotropic agents, corticosteroids, diuretics, anti-parkinsonian agents, gastro-intestinal agents, histamine receptor antagonists, keratolytics, lipid regulating agents, anti-anginal agents, cox-2 inhibitors, leucotriene inhibitors, macrolides, muscle relaxants, nutritional agents, opioid analgesics, protease inhibitors, sex hormones, stimulants, muscle relaxants, anti-osteoporosis agents, anti-obesity agents, cognition enhancers, anti-urinary incontinence agents, nutritional oils, anti-benign prostate hypertrophy agents, essential fatty acids, non-essential fatty acids, external analgesics (for example aspirin, nonsteroidal antiinflammatories and the like), steroidal antiinflammatory drugs (such as hydrocortisone and the like), skin bleaching agents (such as hydroquinone, kojic acid, sodium metabisulfite, and the like), skin protectants, and combinations thereof.
Specific, non-limiting examples of suitable lipophilic bioactive agents include, but are not limited to, acutretin, albendazole, albuterol, aminogluthemide, amiodarone, amlodipine, amphetamine, amphotericin B, atorvastatin, atovaquone, azithromycin, baclofen, beclomethsone, benezepril, benzonatate, benzoquinones, betamethasone, bicalutanide, budesonide, bupropion, busulphan, butenafine, calcifediol, calciprotiene, calcitriol, camptothecan, candesartan, capsaicin, carbamezepine, carotenes, celecoxib, cerivistatin, cetrizine, chlorpheniramine, cholecalciferol, cilostazol, cimetidine, cinnarizine, ciprofloxacin, cisapride, clarithromycin, clemastine, clomiphene, clomipramine, clopidrogel, codeine, coenzyme Q10, cyclobenzaprine, cyclosporine, danazol, dantrolene, dexchlopheniramine, diclofenac, dicoumarol, digoxin, dihydro epiandrosterone, dihydroergotamine, dihydrotachysterol, dirithromycin, donepezil, efavirenz, eposartan, ergocalciferol, ergotamine, essential fatty acid sources, etodolac, etoposide, famotidine, farnesol, fenofibrate, fentanyl, fexofenadine, finasteride, flucanazole, flurbiprofen, fluvastatin, fosphenytion, frovatriptan, furazolidone, gabapentin, gemfibrozil, glibenclamide, glipizide, glyburide, glymepride, griseofulvin, halofantrine, ibuprofen, irbesartan, irinotecan, isoprenoids, isosorbide dinitrate, isotreinoin, itraconazole, ivermectin, ketoconazole, ketorolac, lamotrigine, lanosprazole, leflunomide, lisinopril, loperamide, loratadine, lovastatin, L-thryroxine, lutein, lycopene, medroxyprogesterone, mefepristone, mefloquine, megesterol acetate, methadone, methoxsalen, metronidazole, metronidazole, miconazole, midazolam, miglitol, minoxidil, mitoxantrone, montelukast, nabumetone, nalbuphine, naratiptan, nelfinavir, nifedipine, nilsolidipine, nilutanide, nitrofurantoin, nizatidine, omeprazole, oprevelkin, osteradiol, oxaprozin, paclitaxel, paricalcitol, paroxetine, pentazocine, pioglitazone, pizofetin, pravastatin, prednisolone, probucol, progesterone, pseudo-ephedrine, pyridostigmine, rabeprazole, raloxifene, refocoxib, repaglinide, rifabutine, rifapentine, rimexolone, ritanovir, rizatriptan, rosigiltazone, saquinavir, sertraline, sibutramine, sildenafil citrate, simvastatin, sirolimus, spironolactone, sumatriptan, tacrine, tacrolimus, tamoxifen, tamsulosin, targretin, tazarotene, telmisartan, teniposide, terbinafine, terzosin, tetrahydrocannabinol, tiagabine, ticlidopine, tirofibran, tizanidine, topiramate, topotecan, toremifene, tramadol, tretinoin, troglitazone, trovafloxacin, valsartan, venlafaxine, vertoporfin, vigabatrin, vitamin A, vitamin D, vitamin E, vitamin K, zafirlukast, zileuton, zolmitriptan, zolpidem, zopiclone, and combinations thereof. Salts, isomers and/or other derivatives of the above-listed lipophilic bioactive agents may also be used, as well as combinations thereof.
In embodiments, coenzyme Q10 may be utilized as the lipophilic bioactive agent in accordance with the present disclosure. For example, coenzyme Q10 may be applied as described in International Publication No. WO 2005/069916, the entire disclosure of which is incorporated by reference herein. Coenzyme Q10, sometimes referred to herein as CoQ10, ubiquinone, or ubidecarenone, is a popular nutritional supplement and can be found in capsule form in nutritional stores, health food stores, pharmacies, and the like, as a vitamin-like supplement to help protect the immune system through the antioxidant properties of ubiquinol, the reduced form of CoQ10. CoQ10 is found throughout most tissues of the human body and the tissues of other mammals and is concentrated in the mitochondria. CoQ10 is very lipophilic and, for the most part, insoluble in water.
Other related compounds which may be administered instead of, or in combination with, CoQ10 include, but are not limited to, benzoquinones, isoprenoids, farnesols, farnesyl acetate, farnesyl pyrophosphate, 1-phenylalanine, d-phenylalanine, dl-phenylalanine, 1-tyrosine, d-tyrosine, dl-tyrosine, 4-hydroxy-phenylpyruvate, 4-hydroxy-phenyllactate, 4-hydroxy-cinnamate, dipeptides and tripeptides of tyrosine or phenylalanine, 3,4-dihydroxymandelate, 3-methoxy-4-hydroxyphenylglycol, 3-methoxy-4-hydroxymandelate, vanillic acid, phenylacetate, pyridoxine, S-adenosyl methionine, panthenol, mevalonic acid, isopentyl pyrophosphate,phenylbutyrate, 4-hydroxy-benzoate,decaprenyl pyrophosphate, beta-hydroxybutyrate, 3-hydroxy-3-methyl-glutarate, acetylcarnitine, acetoacetylcarnitine, acetylglycine, acetoacetylglycine, carnitine, acetic acid, pyruvic acid, 3-hydroxy-3-methylglutarylcarnitine, all isomeric forms of serine, alanine, cysteine, glycine, threonine, hydroxyproline, lysine, isoleucine, and leucine, even carbon number C4 to C18 fatty acids (butyric, caproic, caprylic, capric, lauric, myristic, palmitic, and stearic acids) salts of carnitine and glycine, e.g., palmitoylcarnitine and palmitoylglycine, and 4-hydroxy-benzoate polyprenyltransferase, any salts of these compounds, as well as any combinations thereof, and the like.
In embodiments, it may be desirable to form a stable, skin penetrating bioactive agent/liposomal concentrate for delivery of the lipophilic bioactive agent. Thus, in forming a liposome, it may be desirable to combine the lipophilic bioactive agent with a material that can solubilize the lipophilic bioactive agent in a suitable media, in some embodiments water, for subsequent encapsulation in a liposome. Suitable materials which may be utilized as a solubilizer for the lipophilic bioactive agent include, for example, polyoxyalkylene dextrans, fatty acid esters of saccharose, fatty alcohol ethers of oligoglucosides (e.g., akylpolyglucosides, including those commercially available as TRITON™ from Dow Chemical North America, Midland, Mich., USA), fatty acid esters of glycerol (e.g., glycerol mono/distearate or glycerol monolaurate), and polyoxyethylene type compounds (e.g., polyoxyethylene, polyethylene glycol, polyethylene oxide, and copolymers thereof, including those commercially available as SOLUTOL™ CREOMOPHOR™, MACROGOL™, CARBOWAX™, and POLYOXYL™).
Suitable solubilizers also include polyethoxylated fatty acid esters of sorbitan (e.g., polysorbates, including those commercially available as TWEEN™ and SPAN™) fatty acid esters of poly(ethylene oxide) (e.g., polyoxyethylene stearates), fatty alcohol ethers of poly(ethylene oxide) (e.g., polyoxyethylated lauryl ether), alkylphenol ethers of poly(ethylene oxide) (e.g., polyethoxylated octylphenol), polyoxyethylene-polyoxypropylene block copolymers (also known as poloxamers, including those commercially available as “PLURONICS”), and ethoxylated fats and oils (e.g., ethoxylated castor oil, or polyoxyethylated castor oil, also known as polyethylene glycol-glyceryl triricinoleate). Combinations of these solubilizers may also be utilized in embodiments. Such combinations are available from standard commercial sources.
In some embodiments, suitable solubilizers include polysorbates, e.g. those sold under the name TWEEN™. Examples of such polysorbates include polysorbate 80 (TWEEN™ 80), polysorbate 20 (TWEEN™ 20), polysorbate 60 (TWEEN™ 60), polysorbate 65 (TWEEN™ 65), polysorbate 85 (TWEEN™ 85), and the like, and combinations including these materials with other similar surfactants, including ARLACEL® surfactants commercially available from ICI Americas, as long as the HLB (Hydrophile-Lipophile Balance) of the surfactant and surfactant mixture favors the formation of an O/W type emulsion system.
To assist in solubilization, it may be desirable, in embodiments, to heat the lipophilic bioactive agent and solubilizer for a suitable period of time. The temperature of heating and time of heating may depend upon the specific lipophilic bioactive agent, the intrinsic thermal stability of the bioactive agent, and the specific solubilizer to be utilized. For example, in embodiments the lipophilic bioactive agent and solubilizer may be heated to a temperature from about 40° C. to about 65° C., in embodiments from about 50° C. to about 55° C., for a period of time from about 5 minutes to about 60 minutes, in embodiments from about 15 minutes to about 30 minutes. The heating time and solubilization of the lipophilic active agent may be reduced if the lipophilic active and solubilizer mixture is agitated. The weight ratio of lipophilic bioactive agent to solubilizer may be about 1:1, in embodiments from about 1:1 to about 4:2, in other embodiments from about 1:2 to about 3:2.
In embodiments, a solubilizer such as polysorbate 80 may be capable of dissolving lipophilic bioactive agent, in embodiments CoQ10, at high levels, with the lipophilic bioactive agent completely soluble in the solubilizer at a ratio of from about 1:2 to about 3:2, when heated to from about 50° C. to about 55° C., a temperature which exceeds the melting point of CoQ10 (which is from about 47° C. to about 48° C.).
The amount of solubilizer added to the lipophilic bioactive agent will depend upon the solubilizer, the lipophilic bioactive agent, and the phospholipids utilized to form the liposomes. In embodiments, a composition of the present disclosure possessing liposomes including a lipophilic bioactive agent therein may possess a solubilizer in an amount from about 0.2% to about 12% by weight, in embodiments from about 1.5% to about 6.5% by weight.
The solution of lipophilic bioactive agent and solubilizer, sometimes referred to herein as a first phase, may then be combined with the phospholipid as described above, in some embodiments lecithin. In embodiments, it may be desirable to place the phospholipid in a dispersion, sometimes referred to herein as a second phase, to which the solution of lipophilic bioactive agent and solubilizer (i.e., the first phase) are added. Suitable solvents for forming a dispersion/second phase including the phospholipid include, but are not limited to, water, purified water, deionized water, ethanol, isopropanol, glycols, diglycols, polyglycols, combinations thereof, and the like. Where added, the solvent may be present in an amount from about 70% by weight to about 98% by weight of the second dispersion, in embodiments from about 78% by weight to about 93% by weight of the second dispersion, with the phospholipid being present in an amount from about 2% by weight to about 30% by weight of the second dispersion, in embodiments from about 7% by weight to about 22% by weight of the second dispersion.
In embodiments, the phospholipid may be present in an amount of from about 1% by weight to about 20% by weight of the combination of phospholipid, solubilizer, and lipophilic bioactive agent, in embodiments from about 4% by weight to about 12% by weight of the combination of phospholipid, solubilizer, and lipophilic bioactive agent.
In embodiments, solubilization of a lipophilic bioactive agent such as CoQ10 in a material that has both lipophilic and hydrophilic properties, in embodiments a polysorbate such as polysorbate 80, may assist in liposome formulation by forming water-dispersible CoQ10 for encapsulation by a high phosphatidylcholine lecithin, such as PHOSPHOLIPON® 85G.
In some embodiments, additional components may be combined with this second phase to enhance formulation of the liposomes possessing a lipophilic bioactive agent, to improve overall rheological and processing properties, and to insure microbiological integrity of the resulting liposomal concentrate during storage. Such components include, without limitation, absorbents, antifoaming agents, acidifiers, alkalizers, buffers, antimicrobial agents, antioxidants (for example tocopherols, BHT, polyphenols, phytic acid) binders, biological additives, chelating agents (for example, disodium EDTA, tetrasodium EDTA, sodium metasilicate, and the like), denaturants, preservatives (for example imidazolidinyl urea, diazolidinyl urea, phenoxyethanol, methylparaben, ethylparaben, propylparaben, and the like), reducing agents, solubilizing agents, solvents, viscosity modifiers, humectants, thickening agents, and combinations thereof. These additional components may be present in an amount from about 0.001% by weight to about 10% by weight of the second phase, in embodiments from about 0.1% by weight to about 1% by weight of the second phase.
Examples of suitable humectants which may be added to the second phase include, but are not limited to, polyols and polyol derivatives, including glycerol, diglycerol, triglycerol, ethylene glycol, propylene glycol, butylene glycol, pentylene glycol (sometimes referred to herein as 1,2-pentane diol), isopreneglycol (1,4-pentane diol), 1,5-pentane diol, hexylene glycol, erythritol, 1,2,6-hexanetriol, polyethylene glycols such as PEG-4, PEG-6, PEG-7, PEG-8, PEG-9, PEG-10, PEG-12, PEG-14, PEG-16, PEG-18, PEG-20, combinations thereof, sugars and sugar derivatives (including fructose, glucose, maltose, maltitol, mannitol, inositol, sorbitol, sorbityl silanediol, sucrose, trehalose, xylose, xylitol, glucuronic acid and salts thereof), ethoxylated sorbitol (Sorbeth-6, Sorbeth-20, Sorbeth-30, Sorbeth-40), and combinations thereof. In some embodiments, a commercially available 1,2-pentane diol such as HYDROLITE-5® pentylene glycol (commercially available from Symrise GmbH) may be utilized. In other embodiments, a propylene glycol may be utilized. Where utilized, such humectants may be present in amounts from about 0.1% by weight to about 20% by weight of the second phase, in embodiments from about 3% by weight to about 10% by weight of the second phase.
In some embodiments, a preservative such as phenoxyethanol and a humectant such as butylene glycol, hexylene glycol, pentylene glycol and/or propylene glycol may both be added to the second phase. In embodiments, the pentylene glycold and/or propylene glycol may provide humectancy and assist in the preservation of the concentrate when combined with phenoxyethanol. The phenoxyethanol and pentylene glycol and/or propylene glycol mix should be water soluble and non-volatile. This is in contrast with the use of ethanol for preservation, which is often utilized by suppliers of liposomal dispersions. Where present, such preservatives may be present in amounts from about 0.01% by weight to about 3% by weight of the second phase, in embodiments from about 0.3% by weight to about 1% by weight of the second phase.
The dispersion containing the phospholipid, sometimes referred to herein as the second phase, and the solution containing the lipophilic bioactive agent and solubilizer, sometimes referred to herein as the first phase, may be homogenized by mixing at high shear to form a liposomal concentrate utilizing homogenizers, mixers, blenders and similar apparatus within the purview of those skilled in the art. In some embodiments, commercially available homogenizers including a Silverson L4RT Homogenizer or similar types of stator/rotor homogenizers made by Gifford-Wood, Frain, IKA and others, as well as multi-stage homogenizers, colloid mills, sonolators, or other types of homogenizers, may be used to produce submicron liposomal dispersions of the lipophilic bioactive agent. The stator/rotor type homogenizers described above have an operational range of from about 100 rpm to about 12,000 rpm, and may be supplied with a range of low shear, standard shear, and/or high shear head screens.
Homogenization may occur by mixing the two phases at suitable speeds of, for example, from about 4,000 rpm to about 12,000 rpm, in embodiments from about 5,000 rpm to about 10,000 rpm, in some embodiments about 7,000 rpm. The shear rate of the homogenizer may also be increased or decreased independent of the speed of the homogenizing shaft by increasing or decreasing the size of the processing screen surrounding the homogenizer head. In embodiments, liposomes may be made with both a standard emulsification screen and a high shear screen, for example, those screens supplied with the Silverson L4RT homogenizer. Mixing may occur for a suitable period of time of less than about 90 minutes, in embodiments from about 2 minutes to about 60 minutes, in embodiments from about 5 minutes to about 45 minutes. The resulting liposomes may have a particle size of less than about 600 nm, in embodiments from about 100 nm to about 500 nm, in other embodiments from about 200 nm to about 400 nm, in some embodiments about 300 nm.
In embodiments, the two phases may be separately heated to a temperature of from about 45° C. to about 65° C., in some embodiments from about 50° C. to about 55° C., and mixed with high shear homogenization at speeds and for periods of time described above to form submicron liposomes of CoQ10. Where the lipophilic bioactive agent is CoQ10, the processing temperature for the CoQ10 phase, the water/phospholipid phase, and the combined phases should not exceed about 55° C. in order to avoid oxidative degradation of the CoQ10. Processing the mixture at a temperature of from about 45° C. to about 55° C. may be useful to obtain a desired viscosity of the concentrate from about 5,000 cP to about 100,000 cP, in embodiments from about 15,000 cP to about 40,000 cP at a temperature of from about 35° C. to about 45° C. In some embodiments, processing for extended periods, e.g., for up to about 60 minutes at the speeds noted above within this temperature range, should not adversely impact the integrity of the resulting liposomes.
The bioactive agent may be present in the resulting concentrate in an amount of from about 10% by weight of the concentrate to about 30% by weight of the concentrate, in embodiments from about 18% by weight of the concentrate to about 26% by weight of the concentrate, in some embodiments from about 21% by weight of the concentrate to about 22% by weight of the concentrate. The amount of phospholipids in the concentrate may be from about 1% by weight of the concentrate to about 20% by weight of the concentrate, in embodiments from about 4% by weight of the concentrate to about 12% by weight of the concentrate, with the balance being the solubilizer, solvent, humectant and preservative.
In embodiments, it may be desirable to include a permeation enhancer in any composition including the liposomes described above. The permeation enhancer may increase the bioavailability of the resulting liposomes containing the lipophilic bioactive agent. While liposomes of the present disclosure may be administered by any route within the purview of those skilled in the art, the addition of a permeation enhancer may be especially beneficial for topical routes of administration.
Suitable permeation enhancers include, but are not limited to, ethoxydiglycol (also known as diethylene glycol monoethyl ether, commercially available as TRANSCUTOL and TRANSCUTOL P from Gattefosse and TRIVALIN CG from Tri-K Industries), 1,3-butylene glycol, isopentyl diol, 1,2-pentane diol, propylene glycol, 2-methyl propan-2-ol, propan-2-ol, ethyl-2-hydroxypropanoate, hexan-2,5-diol, di(2-hydroxypropyl) ether, pentan-2,4-diol, acetone, polyoxyethylene(2) methyl ether, 2-hydroxypropionic acid, 2-hydroxyoctanoic acid, propan-1-ol, 1,4 dioxane, tetrahydrofuran, butan-1,4-diol, propylene glycol dipelargonate, polyoxypropylene 15 stearyl ether, octyl alcohol, polyoxyethylene ester of oleyl alcohol, oleyl alcohol, lauryl alcohol, dioctyl adipate, dicapryl adipate, diisopropyl adipate, diisopropyl sebacate, dibutyl sebacate, diethyl sebacate, dimethyl sebacate, dioctyl sebacate, dibuyl suberate, dioctyl azelate, dibenzyl sebacate, dibutyl phthalate, dibutyl azelate, ethyl myristate, dimethyl azelate, butyl myristate, dibutyl succinate, didecyl phthalate, decyl oleate, ethyl caproate, ethyl salicylate, isopropyl palmitate, ethyl laurate, 2-ethyl-hexyl pelargonate, isopropyl isostearate, butyl laurate, benzyl benzoate, butyl benzoate, hexyl laurate, ethyl caprate, ethyl caprylate, butyl stearate, benzyl salicylate, 2-hyroxyoctanoic acid, dimethyl sulphoxide, methyl sufonyl methane (MSM), n,n-dimethyl acetamide, n,n-dimethyl formamide, 2-pyrrolidone, 1-methyl-2-pyrrolidone, 5-methyl-2-pyrrolidone, 1,5-dimethyl-2-pyrrolidone, 1-ethyl-2-pyrrolidone, phosphine oxides, sugar esters, tetrahydrofurfural alcohol, urea, diethyl-m-toluamide, 1-dodecylazacyloheptan-2-one, combinations thereof, and the like.
The amount of permeation enhancer in the compositions of the present disclosure, including any combination of any and all phases described herein, may be less than about 25% by weight of the composition, in embodiments from about 0.5% by weight to about 20% by weight of the composition, in other embodiments from about 3% by weight to about 15% by weight of the composition, in yet other embodiments from about 5% by weight of the composition to about 10% by weight of the composition.
In embodiments, a suitable permeation enhancer may include ethoxydiglycol. In other embodiments, a suitable permeation enhancer may include ethoxydiglycol in combination with another permeation enhancer such as propylene glycol, pentylene glycol, or any other permeation enhancer described above.
Surprisingly, and as detailed below in the Examples, it has been discovered that, in some embodiments, lower amounts of ethoxydiglycol, rather than higher amounts of ethoxydiglycol, optionally in combination with other permeation enhancers, may provide enhanced bioavailability of a lipophilic bioactive agent, in embodiments CoQ10, when administered topically. In embodiments, suitable amounts of ethoxydiglycol may be from about 0.5% to about 10% by weight of the composition, in embodiments from about 2% to about 8% by weight of the composition, in embodiments from about 4% to about 6% by weight of the composition.
Once formed, the resulting liposomes, which may be in a concentrate, may be administered to a patient or, in embodiments, may be combined with any pharmaceutically acceptable carrier. As used herein the terms “pharmaceutically acceptable carrier” and “pharmaceutically acceptable carriers” refers to those compounds which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as salts and biocompatible derivatives of those compounds. As used herein, a pharmaceutically acceptable carrier includes any and all solvents, including water, dispersion media, coatings, antibacterial and antifungal agents, stabilizing excipients, absorption enhancing or delaying agents, polymers, including polymeric binders and polymeric adhesives, combinations thereof, and the like. Such materials should be non-toxic to the recipients at the dosages and concentrations employed, and may include buffers such as TRIS HCl, phosphate, citrate, acetate and other organic acid salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) peptides such as polyarginine, proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidinone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium and/or nonionic surfactants such as TWEEN, PLURONICS and/or polyethylene glycol.
The use of such media and agents for pharmaceutically active substances is within the purview of those skilled in the art. Supplementary active ingredients can also be incorporated into the compositions.
In embodiments, the above carriers may be utilized alone or in combination to form a carrier system. Suitable pharmaceutically acceptable carrier systems are within the purview of those skilled in the art and may include, but are not limited to, lotions, creams, gels, emulsions, dispersions, solids, solid sticks, semi-solids, aerosol or non-aerosol foams, sprays, serums, transdermal adhesive patch systems, combinations thereof, and the like. In embodiments, the liposomes may be in a liposomal concentrate and may be introduced to a patient with a permeation enhancer as described above. In embodiments, the permeation enhancer may be present in a water phase added to the liposomal concentrate to form a composition of the present disclosure. In embodiments, the formulation may be used for transdermal delivery.
Lotions or creams including the liposomes described above may include additional phases for formation of the lotion and/or cream. For example, in some embodiments, a composition of the present disclosure may include a lotion formed by combining the liposomes described above and permeation enhancer with additional oil phases, water phases, neutralizing phases, pigments, combinations thereof, and the like. In embodiments, these combined phases may form the pharmaceutically acceptable carrier described above.
As also noted above, in embodiments the permeation enhancer may be in one of the additional phases, for example, the water phase.
Similarly, the concentrate described above may be placed in any suitable solvent, including water, for administration, or combined with a polymeric binder and/or adhesive for administration as a solid, semi-solid, and the like. Emulsions and/or dispersions may be formed by combining the liposomal concentrate with surfactants utilizing any means within the purview of those skilled in the art.
Where additional phases are present in the formation of a composition of the present disclosure in the form of a lotion or cream, the liposome concentrate or dispersion may be present in an amount of from about 0.1% to about 30% by weight of the lotion or cream, in embodiments from about 5 to about 25% by weight of the lotion or cream.
The bioactive agent may thus be present in the final composition, in embodiments a lotion, cream or any other suitable form described above, in amounts of from about 0.5% by weight to about 20% by weight of the composition, in embodiments from about 0.75% by weight to about 10% by weight of the composition, in other embodiments from about 1% by weight to about 7.5% by weight of the composition, in other embodiments from about 1.25% by weight to about 5% by weight of the composition, in other embodiments from about 1.5% by weight to about 3% by weight of the composition.
For example, in some embodiments a lotion or cream including the liposome concentrate described above may include an oil phase which, in turn, may include emollients, fatty alcohols, emulsifiers, combinations thereof, and the like. For example, an oil phase could include emollients such as C12-15 alkyl benzoates (commercially available as FINSOLV™ TN from Finetex Inc. (Edison, N.J.)), capric-caprylic triglycerides (commercially available from Huls as MIGLYOL™ 812), and the like. Other suitable emollients which may be utilized include vegetable derived oils (corn oil, safflower oil, olive oil, macadamian nut oil, etc.); various synthetic esters, including caprates, linoleates, dilinoleates, isostearates, fumarates, sebacates, lactates, citrates, stearates, palmitates, and the like; synthetic medium chain triglycerides, silicone oils or polymers; fatty alcohols such as cetyl alcohol, stearyl alcohol, cetearyl alcohol, lauryl alcohol, combinations thereof, and the like; and emulsifiers including glyceryl stearate, PEG-100 stearate, Glyceryl Stearate, Glyceryl Stearate SE, neutralized or partially neutralized fatty acids, including stearic, palmitic, oleic, and the like; vegetable oil extracts containing fatty acids, Ceteareth-20,Ceteth-20, PEG-150 Stearate, PEG-8 Laurate, PEG-8 Oleate, PEG-8 Stearate, PEG-20 Stearate, PEG-40 Stearate, PEG-150 Distearate, PEG-8 Distearate, combinations thereof, and the like; or other non-polar cosmetic or pharmaceutically acceptable materials used for skin emolliency within the purview of those skilled in the art, combinations thereof, and the like.
The emollients, in embodiments C12-15 alkyl benzoates, may be included for emolliency and spreadability. Where present, the emollient may be present in an amount from about 0.2% by weight to about 15% by weight of the total composition, in embodiments from about 2% by weight to about 6% by weight of the total composition. Alcohols such as cetyl alcohol and stearyl alcohol may be added to impart body or texture to a cream. Where both cetyl alcohol stearyl alcohol are utilized, the ratio of cetyl alcohol to stearyl alcohol may be from about 2:1 to about 1:2, with the waxy alcohols making up from about 1 to about 6 weight percent of the total composition, in embodiments from about 2% by weight to about 4% by weight of the total composition.
As noted above, this oil phase may also include emulsifiers. Suitable emulsifiers include, but are not limited to, stearates including glyceryl stearate, PEG-100 stearate, glyceryl stearate SE, glyceryl stearate citrate, combinations thereof, and the like. In embodiments, a combination of stearates may be utilized in the oil phase as an emulsifier. For example, a glyceryl stearate and PEG-100 stearate mixture (in embodiments, a mixture of glyceryl stearate and polyethylene glycol 100 stearate commercially available as ARLACEL® 165 from ICI Americas) may be used as an emulsifier to form an oil-in-water (o/w) emulsion. In such a combination, the PEG-100 stearate may act as the primary emulsifier and the glyceryl stearate may be a co-emulsifier. The emulsifier may be present in an amount from about 2% by weight to about 8% by weight of the total composition, in embodiments from about 3% by weight to about 5% by weight of the total composition.
The weight ratio of emulsifier to emollients as described above in this oil phase may be from about 10:1 to about 1:2, in some embodiments from about 2:1 to about 1:1.
Where present, an oil phase may be present in an amount of from about 5% to about 20% by weight of a lotion or cream, in embodiments from about 8% to about 15% by weight of a lotion or cream. Lotions or creams formed with the above liposomes may also include a water phase, which may, in embodiments, include the permeation enhancer described above as well as those items combined to form the second phase described above, including humectants and preservatives. Thus, in embodiments, the water phase utilized in formation of a lotion or cream possessing liposomes as described herein may include the second phase described above. In addition, in embodiments it may be desirable to add a viscosity modifier, sometimes referred to herein as a viscosity agent, to provide the lotion and/or cream with a desired viscosity.
Suitable viscosity agents which may be added to the water phase include water soluble polymers, including anionic polymers and nonionic polymers. Useful polymers include vinyl polymers such as cross linked acrylic acid polymers with the CTFA name CARBOMER, pullulan, mannan, scleroglucans, polyvinylpyrrolidone, polyvinyl alcohol, guar gum, hydroxypropyl guar gum, xanthan gum, acacia gum, arabia gum, tragacanth, galactan, carob gum, karaya gum, locust bean gum, carrageenin, pectin, amylopectin, agar, quince seed (Cydonia oblonga Mill), starch (rice, corn, potato, wheat), algae colloids (algae extract), microbiological polymers such as dextran, succinoglucan, starch-based polymers such as carboxymethyl starch, methylhydroxypropyl starch, alginic acid-based polymers such as sodium alginate, alginic acid propylene glycol esters, acrylate polymers such as sodium polyacrylate, polyethylacrylate, polyacrylamide, polyethyleneimine, and inorganic water soluble materials such as bentonite, aluminum magnesium silicate, laponite, hectonite, and anhydrous silicic acid. Combinations of the foregoing may also be used in embodiments. In some embodiments, a CARBOMER such as CARBOMER 940 may be added as a viscosity agent to control the rheological properties of the cream formulas and add stability to the primary emulsion.
Where utilized, a viscosity agent may be present in an amount from about 0.1% to about 2% by weight of the composition, in embodiments from about 0.25% to about 0.6% of the composition.
Alternatively, the water phase may contain other soluble humectants such as glycols, polyols, lactate salts, amino acids, peptides, sugars, urea, sodium PCA, hyaluronic acid, or salts thereof, or any other suitable humectant or water soluble or water-dispersible moisturizer within the purview of those skilled in the art. The weight ratio of humectants to permeation enhancer to preservative to viscosity agent may be from about 20:10:1:1 to about 10:20:1:1, in some embodiments from about 15:10:2:1 to about 10:15:1:1.
Thus, as noted above, the water phase utilized to form a lotion and/or cream of the present disclosure may include water, humectants, preservatives, viscosity agents, and permeation enhancers. For example, in embodiments a suitable water phase may include a combination of glycerine, pentylene glycol and/or propylene glycol, ethoxydiglycol, phenoxyethanol, water, and CARBOMER 940. Such a water phase may contain glycerine for skin moisturization and humectancy; propylene glycol for humectancy and to aid in skin penetration and to improve the microbiological preservation profile; ethoxydiglycol to enhance CoQ10 skin penetration of the liposomes; phenoxyethanol for microbiological preservation; purified water as the phase solvent, and CARBOMER 940 to control the rheological properties of the cream formulas and to add stability to the primary emulsion.
In some embodiments, the viscosity agent may be added to the water phase as a dispersion in a humectant as described above, optionally in combination with water, optionally in combination with a preservative as described above. For example, in embodiments CARBOMER 940 may be added as a dispersion such as a 2% dispersion containing CARBOMER 940 dispersed in a mixture of water, propylene glycol, and phenoxyethanol. This CARBOMER 940 dispersion may be made separately in a batch manufacturing process. Where a viscosity agent such as CARBOMER 940 is added as a separate dispersion to the water phase, the weight ratio of viscosity agent to humectant to preservative to water may be from about 0.3:2:0.05:10 to about 0.5:1:0.2:10, in some embodiments from about 0.1:0.5:0.05:9 to about 0.2:1:0.1:9.
Where present, a water phase may be present in an amount of from about 60% to about 80% by weight of a lotion or cream, in embodiments from about 63% to about 71% by weight of a lotion or cream.
In some embodiments, a third phase, which may be referred to herein as a neutralization phase or buffer phase, may also be added in the formation of a cream or lotion. The components of such a phase may include, but are not limited to, water, amines including triethanolamine, triisopropanolamine, 2-amino-2methyl-1,3-propanediol, tris(hydroxymethyl)amine, 2-aminobutanol, sodium hydroxide, potassium hydroxide, salts such as sodium lactate, potassium lactate, sodium citrate, potassium citrate, sodium or potassium mono-, di, or tri-phosphate, sodium borate, potassium borate, acids such as lactic acid, citric acid, phosphoric acid, boric acid, combinations thereof, and the like. The water may act as a solvent and a diluent for the other ingredients in this phase. The amine such as triethanolamine may act as a neutralizer of an acid component in the water phase, such as the CARBOMER acrylic acid copolymer; additional salts such as a sodium lactate solution (60% w/w in water) and additional acids such as lactic acid may be added as a buffer system to adjust and maintain the final pH of the cream at from about 4.8 to about 6, in some embodiments from about 5 to about 5.5 (within the natural pH range of the skin). In embodiments, a pH of about 5 or higher may be useful, as the CARBOMER 940 acrylic copolymer of the water phase or similar material should be fully neutralized and develop its full viscosity potential.
In embodiments a suitable amount of amine such as triethanolamine may be added so that it is present in an amount from about 0.5% to about 2% by weight of the final composition, in embodiments from about 1% to about 1.5% by weight of the final composition. A suitable amount of salt such as sodium lactate may be added so that it is present in an amount from about 0.5% to about 3% by weight of the final composition, in embodiments from about 1% to about 1.5% by weight of the final composition. In embodiments, a suitable amount of acid such as lactic acid may be added so that it is present in an amount from about 0% to 1% by weight of the final composition, in some embodiments about 0.25% to about 0.75% by weight of the final composition, in some embodiments about 0.5% by weight of the final composition. The neutralizer and/or buffer may be added so that it is present in an amount from about 0.01% to about 10% by weight of the final composition, in embodiments from about 2% to about 4% by weight of the final composition.
Where present, the neutralizing phase may be present in an amount of from about 0.1% to about 15% by weight of a lotion or cream, in embodiments from about 5% to about 8% by weight of a lotion or cream.
In embodiments, where the lipophilic bioactive agent is CoQ10, a cream without a pigment may have a yellow-orange color. Thus, it may be desirable to add a pigment to any lotion or cream to cosmetically mask the color imparted by the drug. Any pigment suitable for cosmetic or pharmaceutical formulations may be combined with the liposomes of the present disclosure. Such pigments include, but are not limited to, titanium dioxide, iron oxides, zinc oxide, combinations thereof, and the like. In embodiments, a water-dispersible grade of titanium dioxide powder may be used for lightening the color of the final cream. The yellow-orange color of the cream, imparted by CoQ10, may be substantially reduced and may be cosmetically improved by the addition of titanium dioxide in an amount of up to about 1% by weight of the lotion or cream, in embodiments from about 0.2% to about 2% by weight of the lotion or cream. In addition to the inorganic pigments, water-soluble or water dispersible FD&C or D&C dyes, and/or pearlescent opacifiying agents based on glyceryl stearate, or mixtures of glyceryl stearate and other pearlescent agents suitable for use in topical pharmaceutical compositions, may be utilized.
In some embodiments, the amount of preservatives utilized in a composition of the present disclosure including a lipophilic bioactive agent in liposomes may be reduced by the inclusion of additional additives including those described above. For example, the amount of preservatives may be reduced in a composition of the present disclosure by the addition of multifunctional diols including, but not limited to, 1,2-pentane diol, 1,4-pentane diol, hexylene glycol, propylene glycol, 1,3-butylene glycol, glycerol, diglycerol, combinations thereof, and the like. In addition, the amount of preservatives may be reduced by lowering the water activity, Aw, of the composition by the addition of humectants described above and through the addition of soluble salts, e.g., sodium lactate and lactic acid which are present in the buffer and neutralization phase of the embodiments described above.
In embodiments, other soluble ingredients may also be added to compositions of the present disclosure to reduce the level of preservatives necessary. Such additional soluble ingredients include, but are not limited to, pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like. Other buffers which may be added include sodium hydroxide, potassium hydroxide, ammonium hydroxide, monoethanolamine, diethanolamine, triethanolamine, diisopropanolamine, aminomethylpropanol, trimethamine, tetrahydroxypropyl ethylenediamine, citric acid, acetic acid, lactic acid, and salts of lactic acid including sodium lactate, potassium lactate, lithium lactate, calcium lactate, magnesium lactate, barium lactate, aluminum lactate, zinc lactate, sodium citrate, sodium acetate, silver lactate, copper lactate, iron lactate, manganese lactate, ammonium lactate, combinations thereof, and the like. These additives may be added to any phase described above utilized in forming a cream or lotion, including the oil phase, water phase, neutralizing phase, pigment, combinations thereof, and the like.
In embodiments the use of the liposome concentrate described above in forming the compositions of the present disclosure may permit tailoring the production of various compositions having the bioactive agent at varying concentrations. For example, in embodiments, the liposome concentrate may have the bioactive agent at a concentration of from about 10 to about 15 times greater than the amount of bioactive agent in a final composition for administration to a patient. For manufacturing, a large batch of concentrate may be produced, and then multiple portions of the concentrate may be utilized to produce multiple compositions having the bioactive agent at varying concentrations. This permits great flexibility in tailoring the concentration of a bioactive agent in a composition of the present disclosure.
For example, in embodiments, a submicron liposome concentrate may be utilized to create a dosage range of treatment creams possessing a lipophilic bioactive agent. In embodiments, the liposome concentrate may be a CoQ10-solubilized, fluidized or emulsified within a high linoleic acid-phosphatidyicholine multilamella liposome. There are a few reasons for creating a liposome concentrate of lipophilic drug actives. For example, the creation of a drug-liposome-concentrate in its nascent form, without the addition of a cream, lotion, or other vehicles, may permit direct measurement of the drug active, the liposome particle size, and particle size distribution without interference from other additives, typically present in the final product form. For example, cream and lotion emulsions formed by the homogenization of an oil phase, a water phase, and suitable emulsifiers and coemulsifiers, as described herein may include oil-in-water emulsion particles ranging from submicron size to a preponderance of particles above one micron. Once a liposome dispersion or liposome concentrate, as described, is added to the other components or phases of the final cream or lotion vehicle, measurement of the drug-liposome particle size and particle size distribution as distinguished from the particles of O/W emulsion or other additives (e.g., pigment) becomes impractical, if not impossible.
The preparation of a drug-liposome concentrate may also help maintain the intrinsic stability and initial particle size distribution of the drug-liposome. In the embodiments described for Coenzyme Q10 liposome concentrates, the concentrate can be stored at a controlled room temperature (59-86° F.) for several months until needed for manufacture. Once required for production, the liposome concentrate may be added after the formation of the emulsion vehicle and at a temperature that will not adversely affect the drug-liposome particles.
The preparation of the drug-liposome concentrate may also allow for assay of the drug and confirmation of stability of the drug in the liposome prior to incorporation of the liposome concentrate in the final vehicle. Moreover, the formulation of a drug-liposome concentrate at a single concentration allows this concentrate to be used to create a wide range of final dosage concentrations of the drug. In embodiments, the drug-liposome concentrate of Coenzyme Q10 may contain about 21% or about 22% Coenzyme Q10 and may be used to make final compositions, sometimes referred to herein as products, containing Coenzyme Q10 in amounts from about 0.5% up to about 20% by weight of the final composition, in embodiments from about 0.75% by weight to about 10% by weight of the final composition. This range may be widened substantially depending on the dose requirements of the lipophilic active. As noted in the Examples below, in some embodiments the bioactive, such as Coenzyme Q10, may be present in amounts of from about 1.25% by weight of the final composition to about 5% by weight of the final composition, in other embodiments from about 1.5% by weight to about 5% by weight of the composition.
The resulting creams, lotions, and the like may have a long shelf-life; i.e., they may remain stable during storage for at least about 2 years, in embodiments from about 2 to about 10 years.
Such lotions and creams may be packaged in any suitable packaging within the purview of those skilled in the art, including metal or glaminate tubes. The resulting creams have acceptable patient use characteristics for aesthetic considerations of product application, e.g., acceptable “rub-in”, skin feel, product odor, product color, and product transfer.
Compositions of the present disclosure may be utilized to administer lipophilic bioactive agents for the treatment of any disease or condition which may benefit from the application of the lipophilic bioactive agent, including those disclosed in International Publication No. WO 2005/069916, the entire disclosure of which is incorporated by reference herein. While the instant disclosure has discussed topical/transdermal formulations in some detail, depending on the specific conditions being treated, the liposomes containing lipophilic bioactive agents described above may also be formulated and administered by other systemic and/or local routes. Suitable routes of administration include, but are not limited to, other topical routes of administration, oral, rectal, inhalation, vaginal, transmucosal, intestinal, parenteral including intramuscular, subcutaneous, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, intratumoral, combinations thereof, and the like.
Where the compositions are administered by injection, the compositions may be administered in a single bolus, multiple injections, or by continuous infusion (for example, intravenously or by peritoneal dialysis). For parenteral administration, the compositions may be formulated in a sterilized pyrogen-free form. Compositions of the present disclosure can also be administered in vitro to a cell (for example, to induce apoptosis in a cancer cell in an in vitro culture) by simply adding the composition to the fluid in which the cell is contained.
In some embodiments, compositions of the present disclosure may be utilized in the treatment of cancer. As used herein, “cancer” refers to all types of cancer or neoplasm or malignant tumors found in mammals, including, but not limited to: leukemias, lymphomas, melanomas, carcinomas and sarcomas.
As used herein, the terms “cancer,” “neoplasm,” and “tumor,” are used interchangeably and in either the singular or plural form, refer to cells that have undergone a malignant transformation that makes them pathological to the host organism.
Primary cancer cells (that is, cells obtained from near the site of malignant transformation) can be readily distinguished from non-cancerous cells by well established techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.
When referring to a type of cancer that normally manifests as a solid tumor, a “clinically detectable” tumor is one that is detectable on the basis of tumor mass, e.g., by procedures such as CAT scan, MR imaging, X-ray, ultrasound or palpation, and/or which is detectable because of the expression of one or more cancer-specific antigens in a sample obtainable from a patient.
Examples of cancers include cancer of the brain, breast, pancreas, cervix, colon, head and neck, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus and Medulloblastoma.
The term “sarcoma” generally refers to a tumor which is made up of a substance such as embryonic connective tissue and is generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Examples of sarcomas which can be treated with the compositions of the present disclosure include, but are not limited to, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorine carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, telangiectatic sarcoma, and the like.
The term “melanoma” is taken to mean a tumor arising from the melanocytic system of the skin and/or other organs. Melanomas which can be treated with the compositions of the present disclosure include, but are not limited to, for example, acrallentiginous melanoma, amelanotic melanoma, benign juvenile I melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungual melanoma, superficial spreading melanoma, and the like.
The term “carcinoma” refers to a malignant new growth made up of epithelial cells tending to infiltrate the surrounding tissues which give rise to metastases. Carcinomas which can be treated with the compositions of the present disclosure include, but are not limited to, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of the adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma moue, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solenoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and the like.
Additional cancers which can be treated with the compositions of the present disclosure include, for example, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary cancer, bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, prostate cancer, and the like.
In addition, and as noted above, however, the compositions of the present disclosure may also be utilized to administer a lipophilic bioactive agent for the treatment of any disease or condition that may benefit from the application of a lipophilic bioactive agent.
The following Examples are being submitted to illustrate embodiments of the present disclosure. These Examples are intended to be illustrative only and are not intended to limit the scope of the present disclosure. Also, parts and percentages are by weight unless otherwise indicated.
A concentrate was produced with CoQ10 as the lipophilic bioactive agent. About 10 kilograms (kg) of polysorbate 80 was placed in a vacuum kettle and heated to a temperature of from about 50° C. to about 55° C. About 8.8 kg of CoQ10 was combined with the PHOSPHOLIPON® 85G, a vacuum was applied with the temperature maintained at from about 50° C. to about 55° C., and the contents mixed for about 15 minutes. The resulting material may be referred to herein as the CoQ10 phase or the first phase. The CoQ10 was dissolved in the polysorbate 80 with the vacuum kettle sealed, vacuum on, and temperature of the mix of polysorbate/CoQ10 from about 50° C. to about 55° C.
In a separate kettle, about 15.8 kg of water was heated to a temperature of from about 50° C. to about 55° C., and about 0.2 kg of phenoxyethanol and about 2 kg of HYDROLITE-5@ pentylene glycol were added and mixed until clear and uniform. About 8 kg of PHOSPHOLIPON® 85G was then added until dispersed. The resulting material may be referred to herein as the water phase or the second phase. The water phase achieved a uniform dispersion and hydration of the lecithin and was added to the CoQ10/Polysorbate liquid as described below at a temperature from about 50° C. to about 55° C.
A Silverson in-line production scale homogenizer, similar to the Silverson L4RT model used for laboratory scale batches, was utilized to combine the two phases described above, i.e., the CoQ10 phase and the water phase. Homogenization occurred using the Silverson standard emulsion head screen by mixing at full capacity (from about 7000 rpm to about 10,000 rpm) for a total of about 5 minutes through a closed recirculating loop and under vacuum (from about 18 mm to about 20 mm Hg) at temperatures of from about 50° C. to about 55° C. with sweep agitation until the solubilized CoQ10 was completely encapsulated and uniformly dispersed thereby creating a thick, uniform liposomal dispersion. The resulting CoQ10 concentrate possessed CoQ10 at a concentration of about 22% by weight. The PHOSPHOLIPON® 85G concentration was about 8% by weight of the total composition, that is, of the combination of the two phases described above.
In separate experiments, a one kg laboratory batch of the 22% CoQ10 concentrate described above was produced and samples were taken at 5 minute intervals during homogenization. The particle size of the liposomes at the various sampling times was determined utilizing laser diffraction equipment (Malvern 2000) following the manufacturer's directions. Details of the homogenization process and the particle sizes obtained during homogenization are set forth below in Table 1.
As can be seen from Table 1, the CoQ10 concentrate formula and process described above was capable of producing liposomes with an average diameter of 107 nm and a particle distribution that included 85% of all liposomes produced within a size of from about 59 nm to about 279 nm. A short process time (about 5 minutes) produced a liposome dispersion of CoQ10 just as efficiently as a long process time (about 45 minutes). As can also be seen from the above, optimal liposome particles were obtained where the CoQ10 was not exposed to temperatures above about 55° C.
A cross linked acrylic acid polymer dispersion was prepared for use as a viscosity agent in a cream composition. The acrylic acid utilized, CARBOMER 940, was prepared in a 2% dispersion with the following components set forth below in Table 2:
The manufacturing process was conducted as follows. The equipment was first cleaned and sanitized. On a benchtop, the phase 1 ingredients were mixed until clear and uniform. The required amount of water (phase 2) was weighed and added to a phase vessel kettle of the homogenizer described above in Example 1. The water was heated with a hot water/steam jacket to a temperature of from about 60° C. to about 65° C. Phase 1 was then added to the phase 2 water with moderate agitation until clear and uniform. The phase 1 container was rinsed with process water and the temperature was maintained at from about 60° C. to about 65° C. The agitator was then turned on high and CARBOMER 940 powder (phase 3) was added.
The temperature was maintained at from about 60° C. to about 65° C. and mixing continued at medium-high speed of from about 500 rpm to about 800 rpm until all the CARBOMER 940 powder was added. The CARBOMER powder was added slowly to the vortex of the mixture of phases 1 and 2. The powder was hand sifted slowly so that the total amount of CARBOMER was added in no less than about 10 minutes.
Mixing continued at medium-high agitation until all powder was thoroughly dispersed and no “fish-eyes” were present. The manufacturing process was conducted so that all of the unneutralized CARBOMER 940 powder was completely dispersed to create a smooth translucent dispersion of fully hydrated CARBOMER polymer. Agitation of the batch was high enough to create a visible vortex, but not so high to cause splashing of the batch. Adequate mixing of the batch occurred at a high speed of from about 800 rpm to about 1300 rpm over a period of time from about 60 minutes to about 90 minutes. The batch temperature was maintained at from about 60° C. to about 65° C. at the start of mixing and from about 55° C. to about 65° C. during mixing. The elevated temperature assisted in dispersion of the CARBOMER polymer and helped prevent agglomeration.
The batch was cooled to from about 25° C. to about 30° C. with chilled water through a jacket and mixing continued with medium-high agitation. Samples were taken to determine microquality, pH, specific gravity and viscosity.
A cream emulsion base was formed utilizing several phases for combination with the CoQ10 concentrate possessing liposomes of Example 1. Phases A, B, C and D were combined to form the base cream. Phase E was the CoQ10 concentrate of Example 1 (22% w/w CoQ10). Details of the preparation of the cream emulsion base and the subsequent addition of the CoQ10 concentrate of Example 1 are set forth below.
For preparation of the cream possessing CoQ10 at 1.5% by weight, the procedure for combining the various phases was as follows with the ingredients set forth below in Tables 3-7:
Phase A (the “Oil Phase”) included C12-15 alkyl benzoates, which are light esters added for emolliency and spreadability. The cetyl alcohol and stearyl alcohol were waxes added to impart body or texture to the cream and the glyceryl stearate and PEG-100 stearate mixture was a primary emulsifier included to form an oil-in-water (o/w) emulsion. On a benchtop, the Phase A ingredients were weighed in a vacuum kettle and heated to from about 70° C. to about 75° C. in water bath.
Phase B (the “Water Phase”), contained glycerine for skin moisturization and humectancy; pentylene glycol for humectancy, to aid in skin penetration and to improve the microbiological preservation profile; ethoxydiglycol to enhance CoQ10 skin penetration of the liposomes; phenoxyethanol for microbiological preservation; purified water as the phase solvent, and CARBOMER 940 dispersion of Example 2 above to control the rheological properties of the cream formulas and to add stability to the primary emulsion.
Phase B ingredients were placed in a separate vacuum mixing kettle. The ingredients were mixed with moderate sweep mixing while heating to from about 70° C. to about 75° C. (no vacuum). When the Phase B ingredients reached from about 70° C. to about 75° C., Phase A ingredients were added at from about 70° C. to about 75° C. with moderate sweep mixing. The mixture of Phases A and B was recirculated through a Silverson homogenizer as described above in Example 1 (standard head) and continued to the next part of the process.
In Phase C (the “Neutralization and Buffer Phase”), purified water acted as a solvent and a diluent for the other ingredients in this phase. Triethanolamine was the primary neutralizer of the CARBOMER acrylic acid copolymer in the water phase (Phase B); sodium lactate solution (60% w/w in water) and lactic acid were added as a buffer system to adjust and maintain the final pH of the cream from about 5 to about 5.5, which is within the natural pH range of the skin.
On a benchtop, Phase C ingredients were weighed and mixed until uniform and heated to from about 60° C. to about 65° C. The Phase C mixture was then added to the vacuum mixing kettle containing Phases A and B with sweep mixer on medium-high.
Mixing continued while moving to the next part of the process.
Phase D (the “Pigment Phase”). A water-dispersible grade of Titanium Dioxide powder was used in the formula solely for the purpose of lightening the color of the final cream color. The yellow-orange color of the cream, imparted by CoQ10, was substantially reduced and cosmetically improved by the addition of about 1% w/w Titanium Dioxide.
For Phase D of the process, weighed TiO2 was added to the batch (Phases A, B and C) and mixed and recirculated through the Silverson homogenizer (high shear head) for about 10 minutes or until completely uniform and fully extended (color was checked to confirm).
It was important to insure there was no agglomeration or clumping of the titanium dioxide on the sweep mixing blades; this was confirmed by visual inspection. A Silverson in line homogenizer as described above in Example 1 was used with a high shear screen to insure maximum deagglomeration and grinding of the titanium dioxide. The final dispersion of the titanium dioxide was checked with a Hegman PH-175 fineness of grind gauge.
Recirculation was stopped and the batch was cooled to from about 50° C. to about 55° C. with the sweep mixer on medium, at a speed of about 30 rpm. The previously weighed CoQ10 concentrate (Phase E) from Example 1 was warmed to from about 45° C. to about 50° C. and added to the batch (Phases A, B, C and D).
All phases were mixed with sweep agitation at about 60 rpm with a vacuum applied until uniform. Temperature was maintained at about 50° C.
The batch was cooled to from about 35° C. to about 45° C. with mixing at about 60 rpm and the application of a vacuum.
The resulting material was placed into holding containers.
For preparation of a cream possessing CoQ10 at 3% by weight, the exact same procedure described above for forming the cream possessing CoQ10 at 1.5% by weight was followed. The materials for each phase, and the amounts utilized, are set forth below in Tables 8-12:
A similar cream was prepared by using the 22% CoQ10 concentrate from Example 1 in an amount of about 25% by weight to create a cream having CoQ10 at a concentration of about 5% by weight.
A summary of the contents of CoQ10 creams having 1.5% CoQ10 by weight, 3% CoQ10 by weight, and 5% CoQ10 by weight are set forth below in Tables 13, 14 and 15 respectively. Note that in all the formulation examples given above and below for CoQ10 creams, the amount of concentrate used would actually yield a final theoretical concentration of about 10% above the target concentration. So, for “CoQ10 Cream, 1.5%”, the actual batch amount used was 7.5% by weight of a 22% by weight concentrate that yielded 1.65% w/w CoQ10. The “CoQ10 Cream, 3%” was made with 15% by weight of the 22% by weight CoQ10 concentrate that yielded a theoretical content of 3.3% CoQ10 by weight. The 10% excess drug was added to extend the overall shelf life of the product and maintain the drug content from about 90% to about 110% of the label or expected drug content.
Note: 10% manufacturing overage of CoQ10 was added to the 1.5%, 3% and 5% batches (i.e., 1.5% plus 0.15%, 3% plus 0.3%, and 5% plus 0.5%).
Creams possessing CoQ10 produced in Example 3 (i.e., 1.5%, 3%, and 5%) above were applied to porcine skin. The topical dose study was conducted on two pigs each, one male and one female. Each animal had 6 test areas; three test areas on each side. For each pig, one side (3 sites) was dosed once per day for 7 days, while the opposite test side (3 test areas) for each pig was dosed only one time on day 1. The creams from Example 3, prepared with ethoxydiglycol, were used on the male animals. The female animals received 3 test formulas that contained the same ingredients as the samples produced in Example 3 above, except they contained 5% 1,3-butylene glycol instead of 5% ethoxydiglycol. Details of these formulations made with 1,3-butylene glycol, which possessed 1.5% CoQ10 by weight, 3% CoQ10 by weight and 5% CoQ10 by weight, are set forth below in Tables 16, 17, and 18 respectively.
All animals received the same dose of each formulation, which was 200 mg, to a 121 cm2 application area applied once or daily for 7 days.
After application, skin samples were obtained and analyzed as follows. The skin test area was gently washed with a mild soap and water mixture (e.g., 1% Ivory Soap in water or equivalent) to remove any residual topical test formulation. If the area to be excised was larger than the dosed area, the dosed area was demarked with indelible ink to delineate the skin area that was dosed. A full thickness skin section was removed by scalpel with a size approximating 10 cm×10 cm, to the depth and including the adipose layer. Following excision, the skin section was laid flat and wrapped in two layers of plastic wrap (SARAN WRAP™ or equivalent), and frozen to about −70° C. or colder in a timely manner. Each skin section was identified as appropriate (e.g. animal identification, study number, date, etc.). Samples were maintained at about −70° C. or lower until examined.
Each skin section was placed in a watertight plastic bag and thawed in from about 30° C. to about 35° C. water baths. Once thawed, each skin section was gently rinsed with distilled deionized water to remove any residual surface dose and blood. All subcutaneous tissue (e.g. adipose) was removed by scalpel to the level of the papular dermis.
Each skin section was then tape stripped (TRANSPORE™, from 3M) from about 10 to about 20 times until approximately 10-25% surface glistening was observed. This process removed the stratum corneum and any residual surface dose.
On each full skin sheet, 6 areas were demarcated with ink. The demarcated areas were 1 cm2 in area.
Each skin section was placed in a watertight plastic bag and immersed in a ˜65° (±3°) C water bath to initiate the separation process of the epidermis from the dermis. The test sites were then excised from the skin sheet by punch, and the epidermis removed from the dermis by forceps. The individual skin sections were weighed and the weight recorded. The individual skin sections were minced with a scalpel, placed into pre-labeled tubes, and saved for subsequent analysis.
The skin samples were extracted in isopropanol (IPA) on a shaker for about 47 hours, then stored at about −20° C. until further processed. The samples were then centrifuged at about 13,500 rpm for about 10 minutes and the supernatant was collected into 2 mL amber vials.
Quantification of CoQ10 was performed by High Performance Liquid Chromatography (HPLC-UV). Briefly, HPLC was conducted on a Hewlett-Packard 1100 Series HPLC system with an Agilent 1100 Series LC/MSD. A solvent system including about 65% Ethanol and about 35% Methanol was run through an Aquasil C18 column (about 3 mm×about 100 mm, 5p) at a flow rate of about 1 mL/min. Ten microliters of sample were injected. Peak areas were quantified to concentration using an external standard curve prepared from the neat standard. The curve was spiked into IPA due to solubility issues of CoQ10 in water.
The results for the content of CoQ10 in mini-pig skin are summarized in
The data indicated that measurable amounts of CoQ10 were observed in all epidermal samples and in selected dermal samples.
All dosed sites for the epidermis were found to contain CoQ10 at levels that were significantly greater than the non-dosed sites (p<0.001).
There were no significant differences between the epidermal contents for CoQ10 across the three dosing concentrations in either the male or female pig skin sections (p>0.02)
Between the male and female pig, for the sites from the animal's right side (1-day dosing), the epidermal content for the 1.5% CoQ10 and 5% CoQ10 applied doses from the male's skin was significantly greater than that seen in the female's skin (p<0.003), but not for the 3% CoQ10 dose (p=0.0329). Thus, as can be seen from the data, the penetration of the CoQ10 on a single dose basis was significantly greater for the ethoxydiglycol formula vs. the butylene glycol formula (p<0.003 for the 1.5% and 5% doses and p=0.0329 for the 3% dose).
The epidermal levels for both male and female skin sections, for all three dose applications, for the 7-day dosing period (left side), were statistically identical.
Dermal content was only observed in the male skin sections for the 1.5% CoQ10 and 5% CoQ10 dose applications from the 7-day dosing period (left side), and the 1.5% CoQ10 dose application from the 1-day dosing period (right side).
A summary of the data is provided as follows in Table 21:
If one were to extrapolate the data from Table 21 to the total area of skin, the penetration of the CoQ10 would be as set forth below in Table 22.
A single application of the CoQ10 cream formulation delivered an average of 12%, 17%, or 70% of the applied dose for the respective 5%, 3%, and 1.5% CoQ10 cream formulations. In general, the penetration of the CoQ10 on a single dose basis was significantly greater for the ethoxydiglycol formula vs. the butylene glycol formula (p<0.003 for the 1.5% and 5% doses and p=0.0329 for the 3% dose). The data indicated that there was a rise in epidermal content with applied concentration to 3% CoQ10 with the 5% CoQ10 dose being essential equal to the 3% CoQ10 dose. This suggests that the skin became saturated with CoQ10 at the 3% CoQ10 dose, or that the vehicle was unable to deliver more CoQ10 above the 3% CoQ10 concentration. It can be seen that the levels achieved in the skin following 7 days of topical application were identical between the 2 animals.
For the ethoxydiglycol formulations, and for the single application data, average penetration of 73.8%, 16.6%, and 13.3% for the respective 1.5%, 3% and 5% ethoxydiglycol containing creams was obtained.
An interesting and unexpected finding was the disproportional amount of CoQ10 found in the epidermis for the 1.5% cream, the lowest dose of CoQ10 tested. Without wishing to be bound by any theory, this enhanced penetration of CoQ10 may be a function of the ratio of CoQ10 to ethoxydiglycol in the cream formulations, or may possibly be related to the ratio of ethoxydiglycol to CoQ10 and the phospholipid liposome. The relatively higher ratio of ethoxydiglycol to CoQ10 used in the cream containing a lower concentration of CoQ10 may be responsible for the higher amounts of CoQ10 found in the epidermis.
The 1.5% cream and 3% cream also successfully completed 9 weeks accelerated testing (storage at about 35° C. and about 50° C.); passed 5 freeze-thaw cycles packaged in both plastic jar and metal tube packaging; and passed USP microbiological challenge testing. Results were confirmed for the same system with multiple development batches and at 1.5%, 3% and 5% by weight concentrations of CoQ10 in the cream prototype formulation base.
Creams were produced as described in Example 3 above, except propylene glycol was utilized instead of pentylene glycol. A concentrate was first produced as described in Example 1 above, with the components listed below in Table 23:
The resulting CoQ10 concentrate possessed CoQ10 at a concentration of about 21% by weight.
A CARBOMER dispersion was prepared as described in Example 2 above for use in forming the cream with the components listed below in Table 24:
A cream having 1.5% by weight CoQ10 and another cream having 3% by weight CoQ10 were prepared as described above in Example 3, with the components listed below in Tables 25 and 26:
It will be appreciated that various of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also that various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.
This application claims the benefit of and priority to U.S. Provisional Application Ser. No. 60/919,554, filed Mar. 22, 2007, the entire disclosure of which is incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 60919554 | Mar 2007 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16785814 | Feb 2020 | US |
| Child | 17847577 | US | |
| Parent | 13751769 | Jan 2013 | US |
| Child | 16785814 | US | |
| Parent | 12052825 | Mar 2008 | US |
| Child | 13751769 | US |
