WO2012160559A1 - Pharmaceutical compositions of d-alpha-tocopheryl acetate - Google Patents

Pharmaceutical compositions of d-alpha-tocopheryl acetate Download PDF

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Publication number
WO2012160559A1
WO2012160559A1 PCT/IL2012/050181 IL2012050181W WO2012160559A1 WO 2012160559 A1 WO2012160559 A1 WO 2012160559A1 IL 2012050181 W IL2012050181 W IL 2012050181W WO 2012160559 A1 WO2012160559 A1 WO 2012160559A1
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Prior art keywords
alpha
pharmaceutical composition
composition
oil
tocopheryl acetate
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PCT/IL2012/050181
Other languages
French (fr)
Inventor
Shany BLUM
Andrew P. Levy
Shimon Amselem
Anna ELGART
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Rappaport Family Institute For Research In The Medical Sciences
Haptocure Ltd.
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Publication of WO2012160559A1 publication Critical patent/WO2012160559A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A61K31/355—Tocopherols, e.g. vitamin E
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4858—Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/4841—Filling excipients; Inactive ingredients
    • A61K9/4866—Organic macromolecular compounds

Definitions

  • the present invention provides pharmaceutical compositions, including, SEDDS formulations for oral administration, comprising a particular vitamin E isomer, namely d- alpha-tocopheryl acetate.
  • SEDDS formulations for oral administration comprising a particular vitamin E isomer, namely d- alpha-tocopheryl acetate.
  • the present invention further provides methods of using said pharmaceutical compositions for treating cardiovascular disorders in diabetic patients of Hp 2-2 genotype.
  • Vitamin E is an antioxidant commonly used as a dietary supplement or as a non active pharmaceutical excipient, such as, a solubilizer, in pharmaceutical compositions.
  • vitamin E' refers to a group of compounds including the many isomers and derivatives of tocopherols, tocopheryls and tocotrienols, which have vitamin E activity. Some of the compounds are advantageous over others in their activity, however they are typically used as a mixture or as a single compound randomly selected from a group of the known isomers and derivatives.
  • Formulations of fat soluble vitamins are well known in the art. To be acceptable as nutritional supplements intended for human consumption, such formulations must be reasonably palatable. In addition they must also permit, and preferably facilitate, absorption of the fat soluble vitamins or essential nutrients from the digestive system.
  • vitamin E softgel units by Allergy Research Group® (http://www.allergyresearchgroup.com/Vitamin-E-DL-alpha-Tocopheryl-Acetate-120- Softgels-p-250.html) contain the racemic vitamin E mixture, namely, dl-alpha-tocopheryl acetate, 400 IU per softgel, and further include gelatin, glycerin and water.
  • lipid based formulations for oral drug delivery typically comprise either simple mono- or binary-oil mixtures, or more complicated multi-component systems such as micelles, microemulsions, liposomes or oil-in- water emulsions.
  • vitamin E formulations contain the vitamin in an edible oil carrier medium for topical application or encapsulated into a dosage form suitable for oral administration.
  • these formulations exhibit low palatability and relatively poor absorption of the active ingredient in the human digestive system.
  • vitamin E for topical application is disclosed in US Patent Application, Publication No. 2005/0096383.
  • vitamin E may either contain racemic dl-alpha-tocopheryl acetate or the isomer d-alpha-tocopheryl acetate, wherein the concentration of vitamin E ranges from about 7,000 I.U. to 56,000 IU.
  • Fat soluble vitamins may also be provided as water insoluble droplets of an oil phase or micelles in an aqueous carrier medium.
  • U.S. Patent No. 4,572,915 discloses micellized aqueous formulations for fat soluble vitamins, essential nutrients, herb oils and pharmaceutical agents admixed with polyethoxylated castor oil and glycerol to provide a non-aqueous phase.
  • emulsion preparation is bulky and also suffers from problems of cracking and creaming on storage.
  • fat soluble vitamins such as vitamin E are known to be better absorbed in the presence of surfactants or from emulsified vehicles than from oily preparations.
  • SEDDS Self Emulsifying Drug Delivery Systems
  • TRF tocotrienol
  • SEDDS Self Emulsified Drug Delivery System
  • Haptoglobin is a plasma 2-glycoprotein synthesized primarily by hepatocytes, which binds free hemoglobin, thus preventing oxidative damage.
  • Patent No. 6,251,608 and its continuation-in-part, Patent No. 6,613,519 disclose methods of evaluating a risk of a diabetic patient to develop a vascular complication comprising determining a haptoglobin phenotype of the diabetic patient, wherein the risk is decreased in patients with haptoglobin 1 - 1 phenotype as compared to patients with haptoglobin 1 -2 or haptoglobin 2-2 phenotypes.
  • U.S. Patent Application Publication No. 2009/0137617 by one of the inventors of the present invention discloses a method of determining the potential of a subject having a cardiovascular disorder to benefit from administration of vitamin E in combination with a statin.
  • U.S. Patent Applications Publication Nos. 2009/0074740, 2009/0246770 and 2010/0041059 by one of the inventors of the present invention disclose the use of haptoglobin genotyping in diagnosis and treatment of defective reverse cholesterol transport (RCT), methods of reducing risk of developing cardiovascular complications in diabetic patients and methods of determining a potential of a diabetic patient to benefit from anti oxidant therapy for treatment of a vascular complication, respectively.
  • RCT reverse cholesterol transport
  • U.S. Patent Applications Publication Nos. 2008/0044399, 2004/0229244 and 2007/0218462 by one of the inventors of the present invention disclose methods of determining prognosis for a diabetic subject having a cardiovascular complication, to benefit from supplementation of vitamin-E, whereby a subject expressing the Hp-2-2 genotype will benefit from supplementation of vitamin-E, and methods of predicting a benefit of antioxidant therapy for prevention of cardiovascular disease in hyperglycemic patients.
  • the present invention provides for the first time SEDDS formulations for oral administration comprising a specific derivative of vitamin E, namely, d-alpha-tocopheryl acetate, as the active ingredient, at a concentration of at least 300 IU per unit dosage form.
  • vitamin E The major forms of vitamin E are tocopherols, or tocopheryl and a counterpart moiety, as in tocopheryl acetate. It is commonly known that the two forms are not greatly different. Surprisingly, the inventors of the present invention have found that d-alpha- tocopheryl acetate is more potent and exerts better therapeutic effects as inhibiting, preventing or attenuating cardiovascular events and disorders associated therewith in Hp 2-2 genotype, a subpopulation of diabetic patients.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising d-alpha-tocopheryl acetate as a lipid component; and at least one surfactant wherein the composition forms a translucent oil-in-water emulsion when dispersed in an aqueous solution.
  • the composition is a non-aqueous formulation devoid of emulsion droplets in any of its preparation stages. According to another embodiment the composition forms an emulsion comprising oil-in-water droplets only upon administration to a subject.
  • composition forms oil-in-water droplets when dispersed in an aqueous solution.
  • aqueous solution comprises water or body fluids.
  • the mean particle size of the oil-in-water droplets is less than 0.1 ⁇ .
  • the lipid component further comprises at least one additional oil.
  • said additional oil is a long-chain triglyceride (LCT).
  • said at least one additional oil is selected from, but not limited to, olive oil, sesame oil, soy bean oil, and corn oil.
  • the composition comprises at least 300 IU d- alpha-tocopheryl acetate, at least 400 IU d-alpha-tocopheryl acetate or at least 500 IU d- alp ha- tocopheryl acetate.
  • Each possibility is a separate embodiment.
  • the composition comprises at least 25% w/w of d-alpha-tocopheryl, at least 35% w/w of d-alpha-tocopheryl, at least 55% w/w of d-alpha- tocopheryl.
  • the lipid component is consisting of d-alpha- tocopheryl acetate.
  • the amount of d-alpha-tocopheryl acetate is at least 75% w/w of the total weight of the lipid component, at least about 80% w/w of the total weight of the lipid component, at least about 85% w/w of the total weight of the lipid component, or 100% w/w of d-alpha-tocopheryl acetate.
  • Each possibility is a separate embodiment.
  • the at least one surfactant is selected from the group consisting of: a nonionic surfactant, an anionic surfactant, and a cationic surfactant.
  • a nonionic surfactant an anionic surfactant
  • a cationic surfactant an anionic surfactant
  • Each possibility is a separate embodiment.
  • the at least one surfactant is a nonionic surfactant selected from the group consisting of: polysorbate 80 (Tween® 80), capric glycerides (LabrasolTM), lauroyl macrogol-32 glycerides (gelucire® 44/14), oleoyl macrogol- 6 glycerides (Labrafil® M- 1944CS), capryol, lauroglycol, lauroyl polyoxyl-6 glycerides (Labrafil® M2130 CS), ethoxylation of hydrogenated castor oil (Cremophor® RH40), polyethylated castor oil (Cremophor® EL), sorbitane monooleate (SpanTM 80) and sorbitan monolaurate (SpanTM 20).
  • polysorbate 80 Teween® 80
  • capric glycerides LabrasolTM
  • lauroyl macrogol-32 glycerides lauroyl macrogol-32 glycerides (geluci
  • the at least one surfactant has an HLB higher than 12. According to yet another embodiment the final HLB of the composition is higher than 12.
  • composition is essentially devoid of co- solvents. According to yet another embodiment the composition is essentially devoid of alcohol.
  • the composition is designed for oral administration.
  • the composition is in an oral dosage form.
  • the composition is in the form of capsules, including, but not limited to, soft gelatin capsules.
  • the composition is in the form of capsules, wherein said capsules contain about 1,000 mg, about 750 mg, or about 500 mg formulation. Each possibility is a separate embodiment.
  • the present invention provides a method for preventing, treating or attenuating cardiovascular events and disorders in a subject of Hp 2-2 genotype, comprising administering to the subject a composition comprising d-alpha-tocopheryl acetate as the pharmaceutically active ingredient, and at least one surfactant.
  • the present invention provides use of a lipid based composition comprising d-alpha-tocopheryl acetate as the pharmaceutically active ingredient and at least one surfactant for preventing, treating or attenuating cardiovascular events and disorders in a diabetic subject of Hp 2-2 genotype.
  • a lipid based composition comprising d-alpha-tocopheryl acetate as the pharmaceutically active ingredient and at least one surfactant for preventing, treating or attenuating cardiovascular events and disorders in a diabetic subject of Hp 2-2 genotype.
  • preventing, treating or attenuating cardiovascular events and disorders is selected from the group consisting of: improving serum stimulated cholesterol efflux (RCT), reducing HDL associated lipid peroxides, increasing CD 163 expression on peripheral blood monocytes (PBMs) and decreasing association of the inflammatory marker C3 with HDL.
  • RCT serum stimulated cholesterol efflux
  • PBMs peripheral blood monocytes
  • FIG. 1 is a schematic presentation of d-alpha tocopheryl acetate.
  • FIG. 2 presents the compositions listed in Table 3A.
  • FIG. 3 presents pictures of the self-emulsification test of representative compositions from Table 3A.
  • FIG. 4 shows the effect of vitamin E on HDL function in Hp 2-1 and Hp 2-2 DM individuals.
  • FIG. 5 presents the effect of vitamin E on HDL associated lipid peroxides in Hp 2-1 and Hp 2-2 DM individuals.
  • the present invention provides for the first time a composition comprising a specific derivative of vitamin E, namely, d-alpha-tocopheryl acetate, as the active ingredient.
  • the composition facilitates the encapsulation of at least 300 IU d-alpha-tocopheryl acetate in a single capsule suitable for oral administration.
  • the present invention is directed to compositions in the form of self-emulsifying drug delivery system(s) (SEDDSs) containing d-alpha-tocopheryl acetate as an active ingredient, wherein the compositions exhibit an improved drug load of d- alpha-tocopheryl acetate, an enhanced bioavailability and improved compliance.
  • the improved drug load is at least 300 IU, 400IU or more.
  • the improved drug load is of over about 25% w/w of the total composition.
  • 1 IU of vitamin E including particular isomers of vitamin E, such as, d-alpha-tocopheryl acetate, equals 0.735mg.
  • the terms "IU”, “units” and “International Units” are interchangeable.
  • the load of the d-alpha-tocopheryl acetate in the composition of the present invention is at least 25% w/w, at least 40% w/w, at least 55% w/w of the total composition.
  • Each possibility is a separate embodiment.
  • the load of the d-alpha-tocopheryl acetate is at least 65% w/w of d-alpha-tocopheryl acetate, at least 75% w/w of d-alpha-tocopheryl acetate, at least about 85% w/w of d-alpha-tocopheryl acetate, at least 95% w/w, or 100% w/w of the lipid component of the composition.
  • Each possibility is a separate embodiment.
  • compositions of the present invention enable the oral administration of a therapeutically effective amount of d-alpha-tocopheryl acetate due to the significantly high drug load of the compositions.
  • the compositions of the present invention provide improved patient's compliance achieved by use of relatively small size capsules.
  • the compositions of the present invention provide enhanced bioavailability as compared to other formulations of d-alpha-tocopheryl acetate..
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans.
  • the present invention provides a lipid based composition
  • a lipid based composition comprising a specific derivative of vitamin E, namely, d-alpha-tocopheryl acetate, at a concentration of at least 300 IU per unit dosage form and at least one surfactant, wherein the composition is in the form of SEDDS.
  • vitamin E forms are termed as either plain “tocopherol” or “tocopheryl” followed by the name of the counterpart moiety, as in “tocopheryl acetate”.
  • Tocopherols are a class of chemical compounds of which many have vitamin E activity. It is a series of organic compounds consisting of various methylated phenols.
  • Alpha-tocopherol is the main vitamin E source found in supplements and in the European diet while gamma- tocopherol is the most common form in the American diet.
  • the compound a-tocopherol, a common form of tocopherol added to food products, is denoted by the E number E307.
  • Tocotrienols which are related compounds, also have vitamin E activity, however tocotrienols suffer from low bioavailability.
  • vitamin E All of the aforementioned derivatives with vitamin E activity may correctly be referred to as “vitamin E.” Tocopherols and tocotrienols are fat-soluble antioxidants but also seem to have many other functions in the body.
  • Dl-alpha-tocopheryl acetate is a synthetic form of alpha-tocopherol (vitamin E) existing in equal amounts of eight isomers.
  • vitamin E alpha-tocopherol
  • An alternative stable form of vitamin E is d- alpha-tocopheryl acetate, which may be derived from vegetable oils and exists in the form of one isomer.
  • Dl-alpha-tocopheryl acetate is the acetate ester of dl-alpha-tocopherol.
  • Dl-alpha- tocopheryl acetate may be produced by coupling racemic isophytol with trimethylhydroquinone to form dl -tocopherol.
  • d-alpha-tocopheryl acetate over dl-alpha-tocopheryl acetate is that the former may be a better alternative to increase tissue levels and retention of vitamin E compared to the latter, which is a racemic form of tocopheryl acetate.
  • D-alpha-tocopheryl acetate is the acetate ester of d-alpha-tocopherol.
  • the ester is more stable to light and oxygen than tocopherol.
  • the shelf-life of the ester tocopheryl is greater than that of the unesterified tocopherol.
  • the acetate is a pleasant-tasting form of powdered vitamin E that can be eaten right off the spoon.
  • Tocopheryl acetate is naturally converted by the body to alpha-tocopherol.
  • the lipid based composition is in a form selected from the group consisting of: micelles, liposomes, micro-emulsions, self-emulsifying systems and macro-emulsions. Each possibility is a separate embodiment.
  • the present invention relates to a self- emulsifying drug delivery system (SEDDS) composition containing d-alpha-tocopheryl acetate.
  • SEDDSs are devoid of an aqueous phase.
  • SEDDSs are isotrophic mixtures of oil, surfactant and drug which form oil-in-water emulsions when dispersed in aqueous solution such as Gastro Intestinal (GI) fluids.
  • GI Gastro Intestinal
  • composition is a non-aqueous formulation devoid of emulsion droplets in any of its preparation stages. According to another embodiment the composition forms an emulsion only upon administration to a subject.
  • exposing the composition to an aqueous solution results in spontaneous emulsification and formation of uniform clear SEDDS solutions, comprising oil-in-water droplets, with an average particle (droplet) size smaller than 0.1 micron.
  • the aqueous solution in which emulsification occurs may comprise any aqueous solution, such as, but not limited to bodily fluids
  • a mean particle size of "less than 0.1 ⁇ " refers to mean particle size of the oil-in-water droplets that are smaller than l OOnm, for example, the mean particle size exemplified herein below is within the range of 45-95nm. According to some embodiments, less than 0.1 ⁇ refers to mean droplet size within the range of 45-95nm ⁇ a standard deviation of 20-45nm as exemplified herein below.
  • non-aqueous formulation refers to a composition devoid of water in any of its preparation stages. This term also includes formulations which are essentially devoid of water comprise less than 0.5%, 0.4%, 0.3%, or 0.2% w/w or v/v water. Each possibility is a separate embodiment. Thus, a non-aqueous formulation according to the present invention may contain trace amounts of water (up to 0.5% w/w).
  • molecular dispersion of a drug is a prerequisite for its absorption across biological membranes. After oral administration, this dictates that the drug must first dissolve within the gastrointestinal (GI) tract before partitioning into and then across the enterocyte.
  • GI gastrointestinal
  • the absorption of poorly water-soluble drugs can be limited by the dissolution rate and the extent to which the drug dissolves.
  • SEDDSs are isotropic mixtures of oils, surfactants, and optionally solvents and co-solvents/surfactants, which provide an improved oral absorption of highly lipophilic drug compounds.
  • These drug delivery systems can be orally administered, for example, in soft or hard gelatin capsules and form fine relatively stable oil-in-water (o/w) emulsions upon aqueous dilution owing to the gentle agitation of the gastrointestinal fluids.
  • o/w oil-in-water
  • the present invention provides a lipid based composition
  • a lipid based composition comprising: a lipid component comprising d-alpha-tocopheryl acetate and at least one surfactant wherein the composition forms a translucent emulsion when dispersed in an aqueous solution.
  • surfactant or “surface active agent” interchangeably refer to amphiphilic molecules which reduce the interracial tension between hydrophobic and hydrophilic liquids including emulsifiers.
  • Surfactants are able to solubilize liquids that are immiscible with water (oils), or to solubilize water in oil.
  • the efficiency of surfactants is expressed as the quantity of surfactant that is necessary to solubilize a given quantity of oil in water, or vice versa.
  • HLB hydrophilic-lipophilic balance. Compounds with a high HLB (greater than about 12) are predominantly hydrophilic and water-soluble. Those with very low HLB values are hydrophobic and water-insoluble.
  • the present invention relates to the surprising finding that the composition of the invention allows the solubilization of at least 400 IU of d-alpha- tocopheryl acetate, generating uniform clear SEDDS solutions which readily form translucent emulsion when dispersed in a liquid solution.
  • compositions of the present invention comprising 400 IU of d-alpha-tocopheryl acetate, generate uniform clear SEDDS solutions which readily form translucent emulsion when dispersed in a liquid solution.
  • composition is essentially devoid of co- solvents. According to yet another embodiment the composition is essentially devoid of alcohol.
  • the term "essentially devoid of alcohol” refers to a formulation comprising less than 0.5%, 0.4%, 0.3%, or 0.2% w/w or v/v of alcohol. It was surprisingly found that no additional co-solvent was needed in the composition in order to generate uniform clear SEDDS solutions which readily form translucent emulsion when dispersed in a liquid solution.
  • translucent refers to liquid medium which allows the transmission of light as estimated by visual inspection.
  • clear it is meant that the solution/emulsion/formulation is free from visually suspended particles.
  • composition of the present invention comprising at least about 25% w/w of d-alpha-tocopheryl acetate, at least one surfactant and, optionally, at least one additional oil, form uniform clear SEDDS solutions.
  • the lipid component of the composition comprises at least 55% w/w of d-alpha-tocopheryl acetate, or at least 75% w/w of d-alpha- tocopheryl acetate, or at least w/w of d-alpha-tocopheryl acetate 80%, or 100% w/w of d- alpha-tocopheryl acetate as the pharmaceutically active agent.
  • lipid composition As used herein, the terms “lipid composition”, “lipid based composition”, “lipid component” and “lipid phase of the composition” are used herein interchangeably to define a composition of d-alpha tocopheryl acetate alone or in combination with one or more additional lipid-based or oil-based component(s).
  • the lipid component may additionally comprise at least one additional oil.
  • Optional oils might be long-chain triglycerides (LCT) or medium- chain triglycerides (MCT).
  • long-chain triglycerides and “LCTs” interchangeably refers to glycerol esters of long-chain (more than 12 carbons) fatty acids.
  • medium-chain triglycerides and “MCTs” interchangeably refer to medium-chain (6 to 12 carbons) fatty acid esters of glycerol.
  • said additional oil is a medium-chain triglyceride (MCT).
  • at least one additional oil is a long- chain triglyceride (LCT) selected from, but not limited to olive oil, sesame oil, soy bean oil, and corn oil.
  • the at least one additional oil is a medium- chain triglyceride (LCT), selected from, but not limited to, caprylic/capric triglyceride (Mygliol®), propylene gycol dicaprate or propylene glycol dicaprate derivatives (Captex ®) and propylene glycol dicaprylocaprate (LabrafacTM) or any other medium chain triglyceride (MCT).
  • LCT medium- chain triglyceride
  • the at least one additional oil is a LCT.
  • MCTs have impaired lymphatic absorption compared to LCTs. Since vitamin E is primarily absorbed by lymphatic absorption the composition of the present invention may preferably be devoid of MCT.
  • compositions devoid of additional oils generate uniform clear SEDDS solutions which readily form translucent emulsion when dispersed in a liquid solution.
  • the surfactants of the present invention are selected from the group consisting of: nonionic, anionic, and cationic surfactants.
  • Ionic surfactants include, but are not limited to, alkylammonium salts; fusidic acid salts; fatty acid derivatives of amino acids, oligopeptides, and polypeptides; glyceride derivatives of amino acids, oligopeptides, and polypeptides; lecithins and hydrogenated lecithins; lysolecithins and hydrogenated lysolecithins; phospholipids and derivatives thereof; lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acyl lactylates; mono- and di-acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and di-glycerides; citric acid esters
  • Hydrophilic non-ionic surfactants include but are not limited to alkylglucosides; alkylmalto sides; alkylthioglucosides; lauryl macrogolglycerides; polyoxyalkylene alkyl ethers such as polyethylene glycol alkyl ethers; polyoxyalkylene alkylphenols such as polyethylene glycol alkyl phenols; polyoxyalkylene alkyl phenol fatty acid esters such as polyethylene glycol fatty acids monoesters and polyethylene glycol fatty acids diesters; polyethylene glycol glycerol fatty acid esters; polyglycerol fatty acid esters; polyoxyalkylene sorbitan fatty acid esters such as polyethylene glycol sorbitan fatty acid esters; hydrophilic transesterification products of a polyol with at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids, and sterols; polyoxyethylene sterols, derivatives, and
  • Non limiting examples of hydrophilic-non-ionic surfactants include PEG- 10 laurate, PEG-12 laurate, PEG-20 laurate, PEG-32 laurate, PEG-32 dilaurate, PEG-12 oleate, PEG-15 oleate, PEG-20 oleate, PEG-20 dioleate, PEG-32 oleate, PEG-200 oleate, PEG-400 oleate, PEG-15 stearate, PEG-32 distearate, PEG-40 stearate, PEG- 100 stearate, PEG-20 dilaurate, PEG-25 glyceryl trioleate, PEG-32 dioleate, PEG-20 glyceryl laurate, PEG-30 glyceryl laurate, PEG-20 glyceryl stearate, PEG-20 glyceryl oleate, PEG-30 glyceryl oleate, PEG-30 glyceryl laur
  • surfactants are nonionic since non-ionic surfactants are less toxic than ionic surfactants.
  • Surfactants involved in the formulation of SEDDS should have a relatively high HLB and hydrophilicity so that immediate formation of o/w droplets and/or rapid spreading of the formulation in the aqueous media (good self- emulsifying performance) can be achieved.
  • the composition comprises lauroyl macrogol- 32 glycerides, also known as Gelucire® 44/14.
  • This additive was found to be advantageous over other surfactants and emulsifiers in the formation of the pharmaceutical composition of the present invention.
  • the lauroyl macrogol-32 glyceride improves the self emulsification of the composition.
  • Gelucire® 44/14 increases the bioavailability of the composition of the present invention.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a lipid component and at least one surfactant, wherein the lipid component comprises d-alpha-tocopheryl acetate, and wherein the composition forms a translucent oil-in-water emulsion when dispersed in an aqueous solution.
  • the composition comprises d-alpha-tocopheryl acetate, Tween 80, lauroyl macrogol-32 glyceride, optionally, the composition further comprises one or more of caprylocaproyl macrogol-8 glycerides and olive oil.
  • the composition comprises 400 IU d-alpha- tocopheryl acetate, 140 mg Tween 80 and 66 mg lauroyl macrogol-32 glyceride
  • the present invention provides a 500 mg capsule comprising 400 IU d-alpha-tocopheryl acetate, 140 mg Tween 80 and 66 mg lauroyl macrogol-32 glyceride.
  • the composition comprises 400 IU d-alpha- tocopheryl acetate, 270 mg Tween 80, 141 mg lauroyl macrogol-32 glyceride and 45 mg olive oil.
  • the present invention provides a 750 mg capsule comprising 400 IU d-alpha-tocopheryl acetate, 270 mg Tween 80, 141 mg lauroyl macrogol-32 glyceride and 45 mg olive oil.
  • the composition comprises 400 IU d-alpha- tocopheryl acetate, 315 mg Tween 80, 141 mg and lauroyl macrogol-32 glyceride.
  • the present invention provides a 750 mg capsule comprising 400 IU d-alpha-tocopheryl acetate, 315 mg Tween 80, 141 mg and lauroyl macrogol-32 glyceride.
  • the composition comprises 400 IU d-alpha- tocopheryl acetate, 418 mg Tween 80, 218 mg, lauroyl macrogol-32 glyceride and 70 mg olive oil.
  • the present invention provides a 1 ,000 mg capsule comprising 400 IU d-alpha-tocopheryl acetate, 418 mg Tween 80, 218 mg, lauroyl macrogol-32 glyceride and 70 mg olive oil.
  • the present invention relates to the surprising discovery that the formulations of the present invention may be used to carry and provide high capacity load of vitamin E, and in particular, d-alpha-tocopheryl acetate, which is more potent and exerts improved therapeutic effects such as inhibiting, preventing or attenuating cardiovascular events and disorders associated therewith in the Hp 2-2 genotype subpopulation of diabetic patients.
  • composition of the invention may be administered once daily or in two or more sub-doses at appropriate intervals throughout the day, in dosages sufficient to prevent, treat or attenuate cardiovascular events and disorders in a subject of Hp 2-2 genotype.
  • the pharmaceutical composition may be administered by any suitable route and treatment regimen which result with the desired therapeutic effect.
  • the preferred route of administration is systemic.
  • a suitable systemic route of administration includes, but is not limited to, oral administration.
  • the oral dosage form can be administered as, but not limited to capsules, such as but not limited to hard gelatin capsules and soft gelatin capsules. Both of these classes of capsules are made from aqueous solutions of gelling agents like: Animal protein such as gelatin; plant polysaccharides or their derivatives, such as, carrageenans and modified forms of starch and cellulose.
  • Soft-shelled capsules are primarily used for oils and for active ingredients that are dissolved or suspended in oil.
  • the capsules of the present invention can contain various compositions weights or volumes.
  • the capsule(s) can contain 1 ,000 mg or less of the composition of the present invention.
  • the capsule(s) can contain 750mg or less of the composition of the present invention.
  • the capsule(s) can contain 500mg or less of the composition of the present invention.
  • composition of the present invention comprising at least 400 IU of d-alpha-tocopheryl acetate can be formulated to fit relatively small capsules, such as 500 mg size capsules.
  • 500mg capsules containing over about 58% w/w of d-alpha-tocopheryl acetate and thus 100% of the lipid (devoid of additional oils) generate uniform clear SEDDS solutions which readily form translucent emulsion when dispersed in an aqueous solution. This is of particular interest since reduced capsule size is imperative for daily compliance.
  • the present invention provides a composition for the use of preventing, treating or attenuating cardiovascular events and disorders in a subject of Hp 2-2 genotype, comprising administering to the subject a lipid based composition comprising d-alpha-tocopheryl acetate as the pharmaceutically active ingredient and at least one surfactant.
  • preventing, treating or attenuating cardiovascular events and disorders is selected from the group consisting of: improving serum stimulated cholesterol efflux (RCT), reducing HDL associated lipid peroxides, increasing CD 163 expression on peripheral blood monocytes (PBMs) and decreasing association of the inflammatory marker C3 with HDL.
  • RCT serum stimulated cholesterol efflux
  • PBMs peripheral blood monocytes
  • Example 1 Preparation of SEDDS pre-formulations devoid of Gelucire 44/14.
  • SEDDS formulations were prepared as follows: d-alpha-tocopheryl acetate was weighed in 5ml vials. Surfactants and oils were added to the vial for preparing the compositions listed in Tables 1 and 2, stirred for about 1 hr on magnetic stirrer hot plate at 45°C.
  • Table 1 Formulation matrix (mg) of formulations devoid of Gelucire
  • a self-emulsifying drug delivery formulation was prepared as follows: d-alpha- tocopheryl acetate was weighed in 5ml vials. Surfactants and possibly oils were added to the vial according to Table 3 A and stirred for maximum of lhr on magnetic stirrer hot plate at 45°C.
  • Table 3A Formulation matrix (mg) of formulations containing Gelucire 44/14
  • D-alpha-tocopheryl solubility was determined by visually estimating the clarity of the composition and confirming absence of phase separation following overnight incubation. As seen in Figure 2, all compositions containing Gelucire 44/14 resulted in uniform clear solutions.
  • Example 3 Self-emulsifying test.
  • Example 4 Particle size distribution in the self-emulsified compositions
  • Formulations VII, VIII, and IX were further assessed for particle size distribution using a Nicomp particle size analyzer based on dynamic light scattering spectroscopy (DLS). Samples of the formulations were diluted 1 :20 with water for injection (WFI) and measured for particle size.
  • DLS dynamic light scattering spectroscopy
  • the purpose of this study is to evaluate human blood plasma d-alpha-tocopherol levels upon administration of the composition of the present invention.
  • the study is conducted in a phase I/Bioavailability unit using a protocol approved by the Ethics committee of the medical center. All subjects sign an informed consent prior to any study related procedure.
  • Inclusion criteria are: healthy male volunteers aged over 21 y/o, and with a normal BMI (18-25). Exclusion criteria are: history of any non-communicable disease (e.g. cancer, cardiovascular disease, diabetes), any chronic infectious or inflammatory disease (e.g. HIV, viral Hepatitis), any chronic use of medication, concurrent illness during the last 2 weeks prior to enrollment, and any substance abuse including alcohol.
  • non-communicable disease e.g. cancer, cardiovascular disease, diabetes
  • any chronic infectious or inflammatory disease e.g. HIV, viral Hepatitis
  • any chronic use of medication e.g. HIV, viral Hepatitis
  • All subjects abstain from taking any medications in the 2 weeks prior to enrollment and from taking any vitamin supplements 1 month prior to enrollment. 12 subjects are enrolled in a single dose 3 -way crossover random assignment design. Each subject is tested with all 3 dosage forms (500mg, 750mg and l ,000mg). 2 weeks washout separate between each dosage form test. Each subject ingest the capsule after a full 14 hour night fast and 5 mL blood plasma is drawn at 0 h (before dosing), 1 , 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 18, 24, 30 and 36 h after dosing via an in dwelling cannula placed in a forearm vein. All samples are analyzed for d-alpha tocopherol plasma levels (since the d-alpha- tocopheryl acetate is naturally converted by the body to alpha-tocopherol).
  • Example 6 Baseline characteristics of Hp 2-1 and Hp 2-2 participants
  • HDL Structural analysis of HDL: The structural analysis of HDL consisted of the assessment of several components of the HDL particle: ApoAl , lecithin-cholesterol acyltransferase (LCAT), complement component 3 (C3), lipid peroxides, ApoE and glutathione peroxidase (GPx).
  • ApoAl lecithin-cholesterol acyltransferase
  • C3 complement component 3
  • lipid peroxides ApoE
  • GPx glutathione peroxidase
  • C3 Complement component 3
  • LCAT lecithin-cholesterol acyltransferase
  • ApoE was assessed in affinity-purified HDL by western blot.
  • Glutathione peroxidase-3 (GPx-3) was assessed in affinity-purified HDL by ELISA.
  • HDL-associated lipid peroxides and redox active iron Total lipid peroxides associated with HDL and redox active iron were measured as previously described (Asleh and Blum, ibid).
  • Serum CRP and adiponectin Serum high sensitivity C-reactive protein (hs-CRP) and adiponectin levels were assessed by ELISA.
  • CD 163 expression Expression of CD 163 by peripheral blood monocytes (PBMs) was examined by flow cytometry. Cells were treated with APC conjugated a-CD14 and PE conjugated a-CD163 antibodies and analysis was performed on a CyAN ADP analyzer.
  • Serum Lipid Profile and ApoAl were measured as previously described (Nasser et al., Spalax. PLoS One 2009;4:e4528). Nitration of ApoAl was determined by western blot with results normalized to ApoAl .
  • Serum Paraoxonase 1 (PON1) activity Catalytic activities of PON1 was determined by using 5-(thiobutyl)-butyrolactone as substrate.
  • Results are reported as means ⁇ SE. Comparison of parametric values between groups was performed using Student's t-test or paired t-test as appropriate, with a p of ⁇ 0.05 considered significant. Non-parametric values were compared with chi- square test. Interaction testing was performed as described in an online appendix. Briefly, the change in efflux and relative change in efflux (change divided by baseline) were calculated. Modeling was done with two observations for each patient (placebo and vitamin E). The least square means with 95% confidence intervals were evaluated with the PROC MIXED procedure in SAS 9.2.
  • Complement C3 - Complement component 3 LCAT - lecithin cholesterol acyltransferase, GPx-3 - Glutathione Peroxidase-3, PONi - Paraoxonase-1, CRP - C-Reactive Protein.
  • Table 5 Effect of vitamin E on lipid profile and various markers of oxidation, inflammation and HDL structure and function, in Hp 2-1 diabetic humans.
  • Complement C3 Complement component 3
  • LCAT lecithin cholesterol acyltransferase
  • the cholesterol efflux assay used in this study utilized whole serum from patients assessing the direct relationship between cholesterol efflux capacity and atherosclerotic burden. This assay integrates total efflux mediated by several pathways which have been shown to be important for the efflux of cholesterol from macrophages (i.e., ATP -binding cassette transporter Al [ABCA1] and Gl [ABCG1 ], scavenger receptor B l, and aqueous diffusion).
  • ATP -binding cassette transporter Al [ABCA1] and Gl [ABCG1 ], scavenger receptor B l, and aqueous diffusion i.e., ATP -binding cassette transporter Al [ABCA1] and Gl [ABCG1 ], scavenger receptor B l, and aqueous diffusion.
  • ATP -binding cassette transporter Al [ABCA1] and Gl [ABCG1 ], scavenger receptor B l, and aqueous diffusion i.e., ATP -bind
  • Example 7 Vitamin E improves RCT in Hp 2-2 but inhibits RCT in Hp 2-1
  • Example 8 Vitamin E reduces HDL associated lipid peroxides in Hp 2-2 but not
  • Oxidative modification has been proposed to be the mechanism by which HDL is rendered dysfunctional and antioxidant therapy appears to restore HDL functionality (Asleh and Blum, ibid). This study was intended to determine if the interaction between vitamin E and Hp genotype on RCT might be explained by a differential effect of vitamin E on HDL oxidative modification in Hp 2-1 and Hp 2-2.
  • the increase in redox active iron in Hp 2-2 DM has been attributed to the impaired clearance of Hp 2-2-Hb by the CD 163 Hp-Hb receptor.
  • Surface expression of CD 163 is regulated by oxidative stress and hyperglycemia. The purpose of this study was to determine if the decrease in redox-active iron in Hp 2-2 DM individuals who received d-a-tocopheryl acetate may have been associated with an increase in CD 163 expression on PBMs.
  • Vitamin E decreases association of the inflammatory marker C3 with HDL in Hp 2-2 but not in Hp 2-1
  • HDL has been described as having an anti-inflammatory function.
  • pro -inflammatory biomarkers such as C3 have been associated with dysfunctional HDL in individuals with CVD.
  • This study was aimed to determine whether vitamin E would decrease the association of C3 with HDL in Hp 2-2.
  • This effect of vitamin E on a HDL associated inflammatory marker was not associated with an overall change in serum markers of inflammation such as CRP and adiponectin (Tables 4 and 5).

Abstract

The present invention provides pharmaceutical compositions, particularly, SEDDS formulations for oral administration, comprising a specific vitamin E isomer, namely d-alpha- tocopheryl acetate. The present invention further provides methods of using said pharmaceutical composition for treating cardiovascular disorders in diabetic patients of Hp 2- 2 genotype.

Description

PHARMACEUTICAL COMPOSITIONS OF D-ALPHA-TOCOPHERYL
ACETATE
FIELD OF THE INVENTION
The present invention provides pharmaceutical compositions, including, SEDDS formulations for oral administration, comprising a particular vitamin E isomer, namely d- alpha-tocopheryl acetate. The present invention further provides methods of using said pharmaceutical compositions for treating cardiovascular disorders in diabetic patients of Hp 2-2 genotype.
BACKGROUND OF THE INVENTION
Vitamin E is an antioxidant commonly used as a dietary supplement or as a non active pharmaceutical excipient, such as, a solubilizer, in pharmaceutical compositions.
The term 'vitamin E' refers to a group of compounds including the many isomers and derivatives of tocopherols, tocopheryls and tocotrienols, which have vitamin E activity. Some of the compounds are advantageous over others in their activity, however they are typically used as a mixture or as a single compound randomly selected from a group of the known isomers and derivatives.
Formulations of fat soluble vitamins, such as vitamin E, are well known in the art. To be acceptable as nutritional supplements intended for human consumption, such formulations must be reasonably palatable. In addition they must also permit, and preferably facilitate, absorption of the fat soluble vitamins or essential nutrients from the digestive system.
For example, vitamin E softgel units by Allergy Research Group® (http://www.allergyresearchgroup.com/Vitamin-E-DL-alpha-Tocopheryl-Acetate-120- Softgels-p-250.html) contain the racemic vitamin E mixture, namely, dl-alpha-tocopheryl acetate, 400 IU per softgel, and further include gelatin, glycerin and water.
An advantageous alternative to the simple formulations of fat soluble vitamins are lipid based formulations for oral drug delivery. Such formulations typically comprise either simple mono- or binary-oil mixtures, or more complicated multi-component systems such as micelles, microemulsions, liposomes or oil-in- water emulsions.
Indeed most vitamin E formulations contain the vitamin in an edible oil carrier medium for topical application or encapsulated into a dosage form suitable for oral administration. However, these formulations exhibit low palatability and relatively poor absorption of the active ingredient in the human digestive system.
For example, a composition of vitamin E oil for topical application is disclosed in US Patent Application, Publication No. 2005/0096383. According to the specification, vitamin E may either contain racemic dl-alpha-tocopheryl acetate or the isomer d-alpha-tocopheryl acetate, wherein the concentration of vitamin E ranges from about 7,000 I.U. to 56,000 IU.
Fat soluble vitamins may also be provided as water insoluble droplets of an oil phase or micelles in an aqueous carrier medium. For example, U.S. Patent No. 4,572,915 discloses micellized aqueous formulations for fat soluble vitamins, essential nutrients, herb oils and pharmaceutical agents admixed with polyethoxylated castor oil and glycerol to provide a non-aqueous phase.
Typically, emulsion preparation is bulky and also suffers from problems of cracking and creaming on storage. Moreover, fat soluble vitamins such as vitamin E are known to be better absorbed in the presence of surfactants or from emulsified vehicles than from oily preparations. There is thus increased interest in the use of Self Emulsifying Drug Delivery Systems (SEDDS) for improved bioavailability of fat soluble APIs.
Ali et al., International Journal of Pharmaceutics, 2008, 352: 104-1 14, discloses an in vitro characterization of alpha-tocopherol SEDDS formulations.
Alayoubi et al., International Journal of Pharmaceutics, 2012, 426: 153-161 , discloses the optimization of a novel tocotrienol (TRF)-rich Self Emulsified Drug Delivery System (SEDDS). It was observed that by substituting Tween 80 with Cremophor EL in the SEDDS it is possible to emulsify >55% TRF (by weight of the formulation) into submicron (<200 nm) emulsion.
Haptoglobin (Hp) is a plasma 2-glycoprotein synthesized primarily by hepatocytes, which binds free hemoglobin, thus preventing oxidative damage. Some of the inventors of the present invention have previously shown that in humans, a common polymorphism of the haptoglobin gene, characterized by alleles Hp 1 and Hp 2, gives rise to structurally and functionally distinct haptoglobin protein phenotypes, known as Hp 1 -1, Hp 2- 1, and Hp 2-2 (e.g. Levy AP., Pharmacol. Ther., 2006, 1 12(2):501 -12; Levy et al., Antioxid Redox Signal. 2010, 12(2):293-304). It has been further shown by some of the inventors of the present invention that individuals with both diabetes mellitus and the Haptoglobin (Hp) 2-2 genotype are at increased risk of cardiovascular disease (Blum et al., Pharmacogenomics., 2008, 9(8):989-91). In addition, it has been shown by some of the inventors of the present invention and their coworkers that vitamin E reduces the risk for cardiovascular diseases in HP 2-2 genotype individuals with diabetes mellitus (Milman et al., Artherioscler. Thromb. Vase. Biol., 2008, 28:341 -347; Blum et al., Pharmacogenomics. 2010, 1 1 (5):675-84; Blum et al., Atherosclerosis, 2010, 21 1 :25-27).
The mechanism by which vitamin E exerts its selective therapeutic effect in Hp 2-2 genotype, but not in the Hp 2-1 genotype is disclosed by some of the inventors of the present invention in a paper entitled "Vitamin E therapy results in a reduction in HDL function in individuals with Diabetes and the Haptoglobin 2-1 genotype", published after the priority date of the present invention (Farbstein et al., Atherosclerosis, 2011).
U.S. Patent No. 6,251,608 and its continuation-in-part, Patent No. 6,613,519, by one of the inventors of the present invention, disclose methods of evaluating a risk of a diabetic patient to develop a vascular complication comprising determining a haptoglobin phenotype of the diabetic patient, wherein the risk is decreased in patients with haptoglobin 1 - 1 phenotype as compared to patients with haptoglobin 1 -2 or haptoglobin 2-2 phenotypes.
U.S. Patent Application Publication No. 2009/0137617 by one of the inventors of the present invention, discloses a method of determining the potential of a subject having a cardiovascular disorder to benefit from administration of vitamin E in combination with a statin.
In addition, U.S. Patent Applications Publication Nos. 2009/0074740, 2009/0246770 and 2010/0041059 by one of the inventors of the present invention disclose the use of haptoglobin genotyping in diagnosis and treatment of defective reverse cholesterol transport (RCT), methods of reducing risk of developing cardiovascular complications in diabetic patients and methods of determining a potential of a diabetic patient to benefit from anti oxidant therapy for treatment of a vascular complication, respectively.
Furthermore, U.S. Patent Applications Publication Nos. 2008/0044399, 2004/0229244 and 2007/0218462 by one of the inventors of the present invention disclose methods of determining prognosis for a diabetic subject having a cardiovascular complication, to benefit from supplementation of vitamin-E, whereby a subject expressing the Hp-2-2 genotype will benefit from supplementation of vitamin-E, and methods of predicting a benefit of antioxidant therapy for prevention of cardiovascular disease in hyperglycemic patients.
There remains an unmet need for vitamin E formulations which have increased load capacity, improved therapeutic effect together with improved absorption characteristics. The present invention satisfies this long sought need. SUMMARY OF THE INVENTION
The present invention provides for the first time SEDDS formulations for oral administration comprising a specific derivative of vitamin E, namely, d-alpha-tocopheryl acetate, as the active ingredient, at a concentration of at least 300 IU per unit dosage form.
The major forms of vitamin E are tocopherols, or tocopheryl and a counterpart moiety, as in tocopheryl acetate. It is commonly known that the two forms are not greatly different. Surprisingly, the inventors of the present invention have found that d-alpha- tocopheryl acetate is more potent and exerts better therapeutic effects as inhibiting, preventing or attenuating cardiovascular events and disorders associated therewith in Hp 2-2 genotype, a subpopulation of diabetic patients.
According to one aspect, the present invention provides a pharmaceutical composition comprising d-alpha-tocopheryl acetate as a lipid component; and at least one surfactant wherein the composition forms a translucent oil-in-water emulsion when dispersed in an aqueous solution.
According to one embodiment the composition is a non-aqueous formulation devoid of emulsion droplets in any of its preparation stages. According to another embodiment the composition forms an emulsion comprising oil-in-water droplets only upon administration to a subject.
According to yet another embodiment the composition forms oil-in-water droplets when dispersed in an aqueous solution. According to yet another embodiment the aqueous solution comprises water or body fluids.
According to yet another embodiment the mean particle size of the oil-in-water droplets is less than 0.1 μηι.
According to yet another embodiment the lipid component further comprises at least one additional oil. According to yet another embodiment said additional oil is a long-chain triglyceride (LCT). According to yet another embodiment said at least one additional oil is selected from, but not limited to, olive oil, sesame oil, soy bean oil, and corn oil.
According to yet another embodiment the composition comprises at least 300 IU d- alpha-tocopheryl acetate, at least 400 IU d-alpha-tocopheryl acetate or at least 500 IU d- alp ha- tocopheryl acetate. Each possibility is a separate embodiment.
According to yet another embodiment, the composition comprises at least 25% w/w of d-alpha-tocopheryl, at least 35% w/w of d-alpha-tocopheryl, at least 55% w/w of d-alpha- tocopheryl. Each possibility is a separate embodiment. According to yet another embodiment, the lipid component is consisting of d-alpha- tocopheryl acetate.
According to yet another embodiment, the amount of d-alpha-tocopheryl acetate is at least 75% w/w of the total weight of the lipid component, at least about 80% w/w of the total weight of the lipid component, at least about 85% w/w of the total weight of the lipid component, or 100% w/w of d-alpha-tocopheryl acetate. Each possibility is a separate embodiment.
According to yet another embodiment, the at least one surfactant is selected from the group consisting of: a nonionic surfactant, an anionic surfactant, and a cationic surfactant. Each possibility is a separate embodiment.
According to yet another embodiment the at least one surfactant is a nonionic surfactant selected from the group consisting of: polysorbate 80 (Tween® 80), capric glycerides (Labrasol™), lauroyl macrogol-32 glycerides (gelucire® 44/14), oleoyl macrogol- 6 glycerides (Labrafil® M- 1944CS), capryol, lauroglycol, lauroyl polyoxyl-6 glycerides (Labrafil® M2130 CS), ethoxylation of hydrogenated castor oil (Cremophor® RH40), polyethylated castor oil (Cremophor® EL), sorbitane monooleate (Span™ 80) and sorbitan monolaurate (Span™ 20).
According to yet another embodiment, the at least one surfactant has an HLB higher than 12. According to yet another embodiment the final HLB of the composition is higher than 12.
According to yet another embodiment the composition is essentially devoid of co- solvents. According to yet another embodiment the composition is essentially devoid of alcohol.
According to yet another embodiment, the composition is designed for oral administration. According to yet another embodiment, the composition is in an oral dosage form. According to yet another embodiment the composition is in the form of capsules, including, but not limited to, soft gelatin capsules. According to yet another embodiment, the composition is in the form of capsules, wherein said capsules contain about 1,000 mg, about 750 mg, or about 500 mg formulation. Each possibility is a separate embodiment.
According to another aspect, the present invention provides a method for preventing, treating or attenuating cardiovascular events and disorders in a subject of Hp 2-2 genotype, comprising administering to the subject a composition comprising d-alpha-tocopheryl acetate as the pharmaceutically active ingredient, and at least one surfactant. According to another aspect, the present invention provides use of a lipid based composition comprising d-alpha-tocopheryl acetate as the pharmaceutically active ingredient and at least one surfactant for preventing, treating or attenuating cardiovascular events and disorders in a diabetic subject of Hp 2-2 genotype. Each possibility is a separate embodiment.
According to yet another embodiment, preventing, treating or attenuating cardiovascular events and disorders is selected from the group consisting of: improving serum stimulated cholesterol efflux (RCT), reducing HDL associated lipid peroxides, increasing CD 163 expression on peripheral blood monocytes (PBMs) and decreasing association of the inflammatory marker C3 with HDL. Each possibility is a separate embodiment.
Further embodiments, features, advantages and the full scope of applicability of the present invention will become apparent from the detailed description and drawings given hereinafter. However, it should be understood that the detailed description, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic presentation of d-alpha tocopheryl acetate.
FIG. 2 presents the compositions listed in Table 3A.
FIG. 3 presents pictures of the self-emulsification test of representative compositions from Table 3A.
FIG. 4 shows the effect of vitamin E on HDL function in Hp 2-1 and Hp 2-2 DM individuals.
FIG. 5 presents the effect of vitamin E on HDL associated lipid peroxides in Hp 2-1 and Hp 2-2 DM individuals.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides for the first time a composition comprising a specific derivative of vitamin E, namely, d-alpha-tocopheryl acetate, as the active ingredient. The composition facilitates the encapsulation of at least 300 IU d-alpha-tocopheryl acetate in a single capsule suitable for oral administration.
According to one embodiment, the present invention is directed to compositions in the form of self-emulsifying drug delivery system(s) (SEDDSs) containing d-alpha-tocopheryl acetate as an active ingredient, wherein the compositions exhibit an improved drug load of d- alpha-tocopheryl acetate, an enhanced bioavailability and improved compliance. According to another embodiment, the improved drug load is at least 300 IU, 400IU or more. According to yet another embodiment, the improved drug load is of over about 25% w/w of the total composition.
It should be noted that according to the present invention, 1 IU of vitamin E, including particular isomers of vitamin E, such as, d-alpha-tocopheryl acetate, equals 0.735mg. The terms "IU", "units" and "International Units" are interchangeable.
According to yet another embodiment the load of the d-alpha-tocopheryl acetate in the composition of the present invention is at least 25% w/w, at least 40% w/w, at least 55% w/w of the total composition. Each possibility is a separate embodiment.
According to yet another embodiment the load of the d-alpha-tocopheryl acetate is at least 65% w/w of d-alpha-tocopheryl acetate, at least 75% w/w of d-alpha-tocopheryl acetate, at least about 85% w/w of d-alpha-tocopheryl acetate, at least 95% w/w, or 100% w/w of the lipid component of the composition. Each possibility is a separate embodiment.
According to yet another embodiment, the compositions of the present invention enable the oral administration of a therapeutically effective amount of d-alpha-tocopheryl acetate due to the significantly high drug load of the compositions. Thus, the compositions of the present invention provide improved patient's compliance achieved by use of relatively small size capsules. Moreover, the compositions of the present invention provide enhanced bioavailability as compared to other formulations of d-alpha-tocopheryl acetate..
The term "pharmaceutically acceptable" as used herein means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans.
According to yet another embodiment, the present invention provides a lipid based composition comprising a specific derivative of vitamin E, namely, d-alpha-tocopheryl acetate, at a concentration of at least 300 IU per unit dosage form and at least one surfactant, wherein the composition is in the form of SEDDS.
Typically, vitamin E forms are termed as either plain "tocopherol" or "tocopheryl" followed by the name of the counterpart moiety, as in "tocopheryl acetate". Tocopherols are a class of chemical compounds of which many have vitamin E activity. It is a series of organic compounds consisting of various methylated phenols.
Alpha-tocopherol is the main vitamin E source found in supplements and in the European diet while gamma- tocopherol is the most common form in the American diet. The compound a-tocopherol, a common form of tocopherol added to food products, is denoted by the E number E307.
Tocotrienols, which are related compounds, also have vitamin E activity, however tocotrienols suffer from low bioavailability.
All of the aforementioned derivatives with vitamin E activity may correctly be referred to as "vitamin E." Tocopherols and tocotrienols are fat-soluble antioxidants but also seem to have many other functions in the body.
Dl-alpha-tocopheryl acetate is a synthetic form of alpha-tocopherol (vitamin E) existing in equal amounts of eight isomers. An alternative stable form of vitamin E is d- alpha-tocopheryl acetate, which may be derived from vegetable oils and exists in the form of one isomer. Dl-alpha-tocopheryl acetate is the acetate ester of dl-alpha-tocopherol. Dl-alpha- tocopheryl acetate may be produced by coupling racemic isophytol with trimethylhydroquinone to form dl -tocopherol.
Although the common practice is that the various vitamin E forms of tocopherols and tocopheryls (paired to a counterpart moiety, as in tocopheryl acetate) do not differ in terms of therapeutic effect, the inventors of the present invention have found that the particular isomer of vitamin E, d-alpha-tocopheryl acetate, is advantageous over other forms of vitamin E and mixtures thereof.
Another advantage of d-alpha-tocopheryl acetate over dl-alpha-tocopheryl acetate is that the former may be a better alternative to increase tissue levels and retention of vitamin E compared to the latter, which is a racemic form of tocopheryl acetate.
Furthermore, although plain tocopherol may be absorbed a little better, advantageously, tocopheryl have a better shelf life.
D-alpha-tocopheryl acetate is the acetate ester of d-alpha-tocopherol. The ester is more stable to light and oxygen than tocopherol. The shelf-life of the ester tocopheryl is greater than that of the unesterified tocopherol. The acetate is a pleasant-tasting form of powdered vitamin E that can be eaten right off the spoon. Tocopheryl acetate is naturally converted by the body to alpha-tocopherol. According to yet another embodiment, the lipid based composition is in a form selected from the group consisting of: micelles, liposomes, micro-emulsions, self-emulsifying systems and macro-emulsions. Each possibility is a separate embodiment.
According to yet another embodiment, the present invention relates to a self- emulsifying drug delivery system (SEDDS) composition containing d-alpha-tocopheryl acetate. In contrast to micro emulsions, micelle and liposome formulations, SEDDSs are devoid of an aqueous phase. SEDDSs are isotrophic mixtures of oil, surfactant and drug which form oil-in-water emulsions when dispersed in aqueous solution such as Gastro Intestinal (GI) fluids. SEDDS formulations spread readily in the GI tract, and the digestive motility of the stomach and the intestine provide the agitation necessary for self- emulsification.
According to yet another embodiment the composition is a non-aqueous formulation devoid of emulsion droplets in any of its preparation stages. According to another embodiment the composition forms an emulsion only upon administration to a subject.
According to yet another embodiment, exposing the composition to an aqueous solution results in spontaneous emulsification and formation of uniform clear SEDDS solutions, comprising oil-in-water droplets, with an average particle (droplet) size smaller than 0.1 micron.
According to yet another embodiment, the aqueous solution in which emulsification occurs may comprise any aqueous solution, such as, but not limited to bodily fluids
As used herein, a mean particle size of "less than 0.1 μηι" refers to mean particle size of the oil-in-water droplets that are smaller than l OOnm, for example, the mean particle size exemplified herein below is within the range of 45-95nm. According to some embodiments, less than 0.1 μΜ refers to mean droplet size within the range of 45-95nm ± a standard deviation of 20-45nm as exemplified herein below.
As used herein, the term "non-aqueous formulation" refers to a composition devoid of water in any of its preparation stages. This term also includes formulations which are essentially devoid of water comprise less than 0.5%, 0.4%, 0.3%, or 0.2% w/w or v/v water. Each possibility is a separate embodiment. Thus, a non-aqueous formulation according to the present invention may contain trace amounts of water (up to 0.5% w/w).
With few exceptions, molecular dispersion of a drug is a prerequisite for its absorption across biological membranes. After oral administration, this dictates that the drug must first dissolve within the gastrointestinal (GI) tract before partitioning into and then across the enterocyte. The absorption of poorly water-soluble drugs can be limited by the dissolution rate and the extent to which the drug dissolves. Without wishing to be bound to any theory or mechanism, a mechanism by which lipid-based drug formulations enhance drug solubilization within the GI tract are by presentation as a solubilized formulation. SEDDSs are isotropic mixtures of oils, surfactants, and optionally solvents and co-solvents/surfactants, which provide an improved oral absorption of highly lipophilic drug compounds. These drug delivery systems can be orally administered, for example, in soft or hard gelatin capsules and form fine relatively stable oil-in-water (o/w) emulsions upon aqueous dilution owing to the gentle agitation of the gastrointestinal fluids.
The present invention provides a lipid based composition comprising: a lipid component comprising d-alpha-tocopheryl acetate and at least one surfactant wherein the composition forms a translucent emulsion when dispersed in an aqueous solution.
As used herein, the terms "surfactant" or "surface active agent" interchangeably refer to amphiphilic molecules which reduce the interracial tension between hydrophobic and hydrophilic liquids including emulsifiers. Surfactants are able to solubilize liquids that are immiscible with water (oils), or to solubilize water in oil. The efficiency of surfactants is expressed as the quantity of surfactant that is necessary to solubilize a given quantity of oil in water, or vice versa.
As used herein, the term "HLB" stands for hydrophilic-lipophilic balance. Compounds with a high HLB (greater than about 12) are predominantly hydrophilic and water-soluble. Those with very low HLB values are hydrophobic and water-insoluble.
Furthermore, the present invention relates to the surprising finding that the composition of the invention allows the solubilization of at least 400 IU of d-alpha- tocopheryl acetate, generating uniform clear SEDDS solutions which readily form translucent emulsion when dispersed in a liquid solution.
It was surprisingly found that the compositions of the present invention comprising 400 IU of d-alpha-tocopheryl acetate, generate uniform clear SEDDS solutions which readily form translucent emulsion when dispersed in a liquid solution.
According to yet another embodiment the composition is essentially devoid of co- solvents. According to yet another embodiment the composition is essentially devoid of alcohol.
As used herein, the term "essentially devoid of alcohol" refers to a formulation comprising less than 0.5%, 0.4%, 0.3%, or 0.2% w/w or v/v of alcohol. It was surprisingly found that no additional co-solvent was needed in the composition in order to generate uniform clear SEDDS solutions which readily form translucent emulsion when dispersed in a liquid solution.
As used herein, the term "translucent" refers to liquid medium which allows the transmission of light as estimated by visual inspection. Moreover, by "clear", it is meant that the solution/emulsion/formulation is free from visually suspended particles.
It was surprisingly found that the composition of the present invention comprising at least about 25% w/w of d-alpha-tocopheryl acetate, at least one surfactant and, optionally, at least one additional oil, form uniform clear SEDDS solutions.
According to yet another embodiment, the lipid component of the composition comprises at least 55% w/w of d-alpha-tocopheryl acetate, or at least 75% w/w of d-alpha- tocopheryl acetate, or at least w/w of d-alpha-tocopheryl acetate 80%, or 100% w/w of d- alpha-tocopheryl acetate as the pharmaceutically active agent.
As used herein, the terms "lipid composition", "lipid based composition", "lipid component" and "lipid phase of the composition" are used herein interchangeably to define a composition of d-alpha tocopheryl acetate alone or in combination with one or more additional lipid-based or oil-based component(s).
According to yet another embodiment, the lipid component may additionally comprise at least one additional oil. Optional oils might be long-chain triglycerides (LCT) or medium- chain triglycerides (MCT).
As used herein, the term "long-chain triglycerides" and "LCTs" interchangeably refers to glycerol esters of long-chain (more than 12 carbons) fatty acids.
As used herein, the term "medium-chain triglycerides" and "MCTs" interchangeably refer to medium-chain (6 to 12 carbons) fatty acid esters of glycerol.
According to yet another embodiment said additional oil is a long-chain triglyceride
(LCT). According to yet another embodiment said additional oil is a medium-chain triglyceride (MCT). According to yet another embodiment at least one additional oil is a long- chain triglyceride (LCT) selected from, but not limited to olive oil, sesame oil, soy bean oil, and corn oil. According to yet another embodiment the at least one additional oil is a medium- chain triglyceride (LCT), selected from, but not limited to, caprylic/capric triglyceride (Mygliol®), propylene gycol dicaprate or propylene glycol dicaprate derivatives (Captex ®) and propylene glycol dicaprylocaprate (Labrafac™) or any other medium chain triglyceride (MCT).
According to yet another embodiment, the at least one additional oil is a LCT. MCTs have impaired lymphatic absorption compared to LCTs. Since vitamin E is primarily absorbed by lymphatic absorption the composition of the present invention may preferably be devoid of MCT.
According to the present invention it was surprisingly found that even compositions devoid of additional oils generate uniform clear SEDDS solutions which readily form translucent emulsion when dispersed in a liquid solution.
The surfactants of the present invention are selected from the group consisting of: nonionic, anionic, and cationic surfactants. Ionic surfactants include, but are not limited to, alkylammonium salts; fusidic acid salts; fatty acid derivatives of amino acids, oligopeptides, and polypeptides; glyceride derivatives of amino acids, oligopeptides, and polypeptides; lecithins and hydrogenated lecithins; lysolecithins and hydrogenated lysolecithins; phospholipids and derivatives thereof; lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acyl lactylates; mono- and di-acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and di-glycerides; citric acid esters of mono- and di-glycerides; and mixtures thereof.
Hydrophilic non-ionic surfactants include but are not limited to alkylglucosides; alkylmalto sides; alkylthioglucosides; lauryl macrogolglycerides; polyoxyalkylene alkyl ethers such as polyethylene glycol alkyl ethers; polyoxyalkylene alkylphenols such as polyethylene glycol alkyl phenols; polyoxyalkylene alkyl phenol fatty acid esters such as polyethylene glycol fatty acids monoesters and polyethylene glycol fatty acids diesters; polyethylene glycol glycerol fatty acid esters; polyglycerol fatty acid esters; polyoxyalkylene sorbitan fatty acid esters such as polyethylene glycol sorbitan fatty acid esters; hydrophilic transesterification products of a polyol with at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids, and sterols; polyoxyethylene sterols, derivatives, and analogues thereof; polyoxyethylated vitamins and derivatives thereof; polyoxyethylene-polyoxypropylene block copolymers; and mixtures thereof.
Non limiting examples of hydrophilic-non-ionic surfactants include PEG- 10 laurate, PEG-12 laurate, PEG-20 laurate, PEG-32 laurate, PEG-32 dilaurate, PEG-12 oleate, PEG-15 oleate, PEG-20 oleate, PEG-20 dioleate, PEG-32 oleate, PEG-200 oleate, PEG-400 oleate, PEG-15 stearate, PEG-32 distearate, PEG-40 stearate, PEG- 100 stearate, PEG-20 dilaurate, PEG-25 glyceryl trioleate, PEG-32 dioleate, PEG-20 glyceryl laurate, PEG-30 glyceryl laurate, PEG-20 glyceryl stearate, PEG-20 glyceryl oleate, PEG-30 glyceryl oleate, PEG-30 glyceryl laurate, PEG-40 glyceryl laurate, PEG-40 palm kernel oil, PEG-50 hydrogenated castor oil, PEG-40 castor oil, PEG-35 castor oil, PEG-60 castor oil, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, PEG-60 corn oil, PEG-6 caprate/caprylate glycerides, PEG-8 caprate/caprylate glycerides, polyglyceryl- 10 laurate, PEG-30 cholesterol, PEG-25 phyto sterol, PEG-30 soya sterol, PEG-20 trioleate, PEG-40 sorbitan oleate, PEG-80 sorbitan laurate, polysorbate 20, polysorbate 40, polysorbate 80, POE-9 lauryl ether, POE-23 lauryl ether, POE- 10 oleyl ether, POE-20 oleyl ether, POE-20 stearyl ether, tocopheryl PEG- 100 succinate, PEG-24 cholesterol, polyglyceryl- lOoleate, Tween 40, Tween 60, Tween 80, lauroyl macrogol-32 glycerides sucrose monostearate, sucrose monolaurate, sucrose monopalmitate, PEG 10-100 nonyl phenol series, PEG 15-100 octyl phenol series, and poloxamers.
Most preferably the surfactants are nonionic since non-ionic surfactants are less toxic than ionic surfactants. Surfactants involved in the formulation of SEDDS should have a relatively high HLB and hydrophilicity so that immediate formation of o/w droplets and/or rapid spreading of the formulation in the aqueous media (good self- emulsifying performance) can be achieved.
According to yet another embodiment the composition comprises lauroyl macrogol- 32 glycerides, also known as Gelucire® 44/14. This additive was found to be advantageous over other surfactants and emulsifiers in the formation of the pharmaceutical composition of the present invention. In particular, the lauroyl macrogol-32 glyceride improves the self emulsification of the composition.
Furthermore, Gelucire® 44/14 increases the bioavailability of the composition of the present invention.
According to yet another embodiment, the present invention provides a pharmaceutical composition comprising a lipid component and at least one surfactant, wherein the lipid component comprises d-alpha-tocopheryl acetate, and wherein the composition forms a translucent oil-in-water emulsion when dispersed in an aqueous solution.
According to yet another embodiment, the composition comprises d-alpha-tocopheryl acetate, Tween 80, lauroyl macrogol-32 glyceride, optionally, the composition further comprises one or more of caprylocaproyl macrogol-8 glycerides and olive oil.
According to yet another embodiment, the composition comprises 400 IU d-alpha- tocopheryl acetate, 140 mg Tween 80 and 66 mg lauroyl macrogol-32 glyceride, According to yet another embodiment, the present invention provides a 500 mg capsule comprising 400 IU d-alpha-tocopheryl acetate, 140 mg Tween 80 and 66 mg lauroyl macrogol-32 glyceride.
According to yet another embodiment, the composition comprises 400 IU d-alpha- tocopheryl acetate, 270 mg Tween 80, 141 mg lauroyl macrogol-32 glyceride and 45 mg olive oil.
According to yet another embodiment, the present invention provides a 750 mg capsule comprising 400 IU d-alpha-tocopheryl acetate, 270 mg Tween 80, 141 mg lauroyl macrogol-32 glyceride and 45 mg olive oil.
According to yet another embodiment, the composition comprises 400 IU d-alpha- tocopheryl acetate, 315 mg Tween 80, 141 mg and lauroyl macrogol-32 glyceride.
According to yet another embodiment, the present invention provides a 750 mg capsule comprising 400 IU d-alpha-tocopheryl acetate, 315 mg Tween 80, 141 mg and lauroyl macrogol-32 glyceride.
According to yet another embodiment, the composition comprises 400 IU d-alpha- tocopheryl acetate, 418 mg Tween 80, 218 mg, lauroyl macrogol-32 glyceride and 70 mg olive oil.
According to yet another embodiment, the present invention provides a 1 ,000 mg capsule comprising 400 IU d-alpha-tocopheryl acetate, 418 mg Tween 80, 218 mg, lauroyl macrogol-32 glyceride and 70 mg olive oil.
The present invention relates to the surprising discovery that the formulations of the present invention may be used to carry and provide high capacity load of vitamin E, and in particular, d-alpha-tocopheryl acetate, which is more potent and exerts improved therapeutic effects such as inhibiting, preventing or attenuating cardiovascular events and disorders associated therewith in the Hp 2-2 genotype subpopulation of diabetic patients.
The pharmaceutical composition of the invention may be administered once daily or in two or more sub-doses at appropriate intervals throughout the day, in dosages sufficient to prevent, treat or attenuate cardiovascular events and disorders in a subject of Hp 2-2 genotype.
The pharmaceutical composition may be administered by any suitable route and treatment regimen which result with the desired therapeutic effect. The preferred route of administration is systemic. A suitable systemic route of administration includes, but is not limited to, oral administration. The oral dosage form can be administered as, but not limited to capsules, such as but not limited to hard gelatin capsules and soft gelatin capsules. Both of these classes of capsules are made from aqueous solutions of gelling agents like: Animal protein such as gelatin; plant polysaccharides or their derivatives, such as, carrageenans and modified forms of starch and cellulose.
Soft-shelled capsules, are primarily used for oils and for active ingredients that are dissolved or suspended in oil.
In one embodiment, the capsules of the present invention can contain various compositions weights or volumes. For example, the capsule(s) can contain 1 ,000 mg or less of the composition of the present invention. For example, the capsule(s) can contain 750mg or less of the composition of the present invention. For example, the capsule(s) can contain 500mg or less of the composition of the present invention.
It has advantageously been found that the composition of the present invention comprising at least 400 IU of d-alpha-tocopheryl acetate can be formulated to fit relatively small capsules, such as 500 mg size capsules. 500mg capsules containing over about 58% w/w of d-alpha-tocopheryl acetate and thus 100% of the lipid (devoid of additional oils) generate uniform clear SEDDS solutions which readily form translucent emulsion when dispersed in an aqueous solution. This is of particular interest since reduced capsule size is imperative for daily compliance.
According to another aspect, the present invention provides a composition for the use of preventing, treating or attenuating cardiovascular events and disorders in a subject of Hp 2-2 genotype, comprising administering to the subject a lipid based composition comprising d-alpha-tocopheryl acetate as the pharmaceutically active ingredient and at least one surfactant.
In one embodiment, preventing, treating or attenuating cardiovascular events and disorders is selected from the group consisting of: improving serum stimulated cholesterol efflux (RCT), reducing HDL associated lipid peroxides, increasing CD 163 expression on peripheral blood monocytes (PBMs) and decreasing association of the inflammatory marker C3 with HDL.
The following examples are presented to provide a more complete understanding of the invention. The specific techniques, conditions, materials, proportions and reported data set forth to illustrate the principles of the invention are exemplary and should not be construed as limiting the scope of the invention. Examples
Example 1: Preparation of SEDDS pre-formulations devoid of Gelucire 44/14.
SEDDS formulations were prepared as follows: d-alpha-tocopheryl acetate was weighed in 5ml vials. Surfactants and oils were added to the vial for preparing the compositions listed in Tables 1 and 2, stirred for about 1 hr on magnetic stirrer hot plate at 45°C.
Table 1: Formulation matrix (mg) of formulations devoid of Gelucire
Figure imgf000017_0001
Table 2: Formulation matrix (mg) of formulations devoid of Gelucire
Ingredient Compositions
Ul U2 U3 VI Wl XI Yl Zl V2 d-alpha- 588 588 588 588 588 588 588 588 588 tocopheryl
Tween 80 836 836 764 836 836 836 836 836 836
Labrafil M 2130 436 364 436 436 436
CS
Labrafil M 1944 436 436 436 576
CS
Olive Oil 140 212 212 140
Sesame Oil 140 140
Soy Bean Oil 1400 1400
Total 2000 2000 2000 2000 2000 2000 2000 2000 2000 D-alpha-tocopheryl solubility was determined by visually estimating the clarity of the composition and confirming absence of phase separation following overnight incubation. None of the above formulations resulted in uniform translucent solutions. Example 2: Preparation of SEDDS formulation containing Gelucire 44/14
A self-emulsifying drug delivery formulation was prepared as follows: d-alpha- tocopheryl acetate was weighed in 5ml vials. Surfactants and possibly oils were added to the vial according to Table 3 A and stirred for maximum of lhr on magnetic stirrer hot plate at 45°C.
Table 3A: Formulation matrix (mg) of formulations containing Gelucire 44/14
Figure imgf000018_0001
D-alpha-tocopheryl solubility was determined by visually estimating the clarity of the composition and confirming absence of phase separation following overnight incubation. As seen in Figure 2, all compositions containing Gelucire 44/14 resulted in uniform clear solutions.
Example 3: Self-emulsifying test.
The self-emulsifying formulation properties were tested as follows: 0.1 mL of each the formulations described in Table 3 A was added to separate scintillation vials containing 10 mL preheated (37°C) water for injection (WFI). The scintillation vial was placed in a shaker- water bath preheated to 37°C at 150rpm for 30min. Emulsification properties were subsequently assessed by visual inspection of emulsion translucency. As seen in Figure 3 all formulations were classified as translucent. Based on the visual inspection, the rank order for l ,000mg capsules was VII>II>I. The rank order for 750mg capsules was: V=IV>VI=VIII>III. Example 4: Particle size distribution in the self-emulsified compositions
Formulations VII, VIII, and IX were further assessed for particle size distribution using a Nicomp particle size analyzer based on dynamic light scattering spectroscopy (DLS). Samples of the formulations were diluted 1 :20 with water for injection (WFI) and measured for particle size.
The particle size distribution results (Table 3B) indicate the formation of oil-in- water emulsion nanodroplets in all tested formulations with a mean particle size in the range of 50- 100 nm was obtained.
Table 3B. Particle size distribution in the self-emulsified compositions
Figure imgf000019_0001
Example 5: Bioavailability of the formulations
The purpose of this study is to evaluate human blood plasma d-alpha-tocopherol levels upon administration of the composition of the present invention. The study is conducted in a phase I/Bioavailability unit using a protocol approved by the Ethics committee of the medical center. All subjects sign an informed consent prior to any study related procedure.
Inclusion criteria are: healthy male volunteers aged over 21 y/o, and with a normal BMI (18-25). Exclusion criteria are: history of any non-communicable disease (e.g. cancer, cardiovascular disease, diabetes), any chronic infectious or inflammatory disease (e.g. HIV, viral Hepatitis), any chronic use of medication, concurrent illness during the last 2 weeks prior to enrollment, and any substance abuse including alcohol.
All subjects abstain from taking any medications in the 2 weeks prior to enrollment and from taking any vitamin supplements 1 month prior to enrollment. 12 subjects are enrolled in a single dose 3 -way crossover random assignment design. Each subject is tested with all 3 dosage forms (500mg, 750mg and l ,000mg). 2 weeks washout separate between each dosage form test. Each subject ingest the capsule after a full 14 hour night fast and 5 mL blood plasma is drawn at 0 h (before dosing), 1 , 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, 18, 24, 30 and 36 h after dosing via an in dwelling cannula placed in a forearm vein. All samples are analyzed for d-alpha tocopherol plasma levels (since the d-alpha- tocopheryl acetate is naturally converted by the body to alpha-tocopherol).
Example 6: Baseline characteristics of Hp 2-1 and Hp 2-2 participants
Crossover study design: the study was registered as clinical trial NCT01 1 13671 and approved by the institutional ethics committee of the Rambam Medical Center, Haifa, Israel. All participants (n=59) provided informed consent. Subjects were recruited from the ICARE cohort. Targeted enrollment was achieved only for Hp 2-1 and Hp 2-2 (42% and 49% of population) but not for Hp 1- 1 individuals (9% of population) and therefore analyses are reported only for Hp 2-1 and Hp 2-2. 36 Hp 2- 1 and 32 Hp 2-2 DM individuals were consented and enrolled in the study.
Participants were randomized to receive either placebo or vitamin E (400IU natural source d-a-tocopheryl acetate in oil per day) for 3 months followed by crossover. Serum was collected at baseline and after each treatment and stored at -80°C. Analyses are reported on 31 Hp 2-1 and 28 Hp 2-2 participants who completed the protocol.
Isolation of HDL by affinity chromatography: Serum was diluted in an equal volume of 0.5M NaCl in PBS and loaded onto a polyclonal anti-ApoAl sepharose column. After washing with PBS the HDL was eluted with tris-glycine (0.1 M, pH 2.5) followed immediately by neutralization with 1 M tris, pH 9.0.
Cholesterol efflux: Serum was assessed for its ability to promote the efflux of
3H-cholesterol from J774 A. l macrophages as previously described (Asleh and Blum et al.,. Diabetes 2008; 57:2794-2800). Results presented are normalized to HDL levels and are expressed as the percentage of cholesterol efflux.
Structural analysis of HDL: The structural analysis of HDL consisted of the assessment of several components of the HDL particle: ApoAl , lecithin-cholesterol acyltransferase (LCAT), complement component 3 (C3), lipid peroxides, ApoE and glutathione peroxidase (GPx).
Assessment of protein association with HDL: Complement component 3 (C3), lecithin-cholesterol acyltransferase (LCAT) and ApoE were assessed in affinity-purified HDL by western blot. Glutathione peroxidase-3 (GPx-3) was assessed in affinity-purified HDL by ELISA.
HDL-associated lipid peroxides and redox active iron: Total lipid peroxides associated with HDL and redox active iron were measured as previously described (Asleh and Blum, ibid).
Serum CRP and adiponectin: Serum high sensitivity C-reactive protein (hs-CRP) and adiponectin levels were assessed by ELISA.
CD 163 expression: Expression of CD 163 by peripheral blood monocytes (PBMs) was examined by flow cytometry. Cells were treated with APC conjugated a-CD14 and PE conjugated a-CD163 antibodies and analysis was performed on a CyAN ADP analyzer.
Serum Lipid Profile and ApoAl : HDL, total cholesterol and ApoAl were measured as previously described (Nasser et al., Spalax. PLoS One 2009;4:e4528). Nitration of ApoAl was determined by western blot with results normalized to ApoAl .
Serum Paraoxonase 1 (PON1) activity: Catalytic activities of PON1 was determined by using 5-(thiobutyl)-butyrolactone as substrate.
Statistical analysis: Results are reported as means ±SE. Comparison of parametric values between groups was performed using Student's t-test or paired t-test as appropriate, with a p of <0.05 considered significant. Non-parametric values were compared with chi- square test. Interaction testing was performed as described in an online appendix. Briefly, the change in efflux and relative change in efflux (change divided by baseline) were calculated. Modeling was done with two observations for each patient (placebo and vitamin E). The least square means with 95% confidence intervals were evaluated with the PROC MIXED procedure in SAS 9.2.
Study participants with Hp 2-1 and Hp 2-2 genotypes did not differ in their baseline demographics and in the characteristics or management of their DM (Table 4). At baseline, both ApoAl and serum stimulated cholesterol efflux (RCT) were significantly increased in Hp 2-1 as compared to Hp 2-2 study participants (ApoAl : Hp 2-1 , 156.8±3.4 vs. Hp 2-2, 146.7 ±3.7 p=0.05; RCT: Hp 2-l , 15.4±1.1% vs Hp 2-2, 1 1.8±0.8%, p=0.01 ; Table 5). Table 4: Effect of vitamin E on lipid profile and various markers of oxidation, inflammation and HDL structure and function, in Hp 2-2 diabetic humans.
Figure imgf000022_0001
Complement C3 - Complement component 3, LCAT - lecithin cholesterol acyltransferase, GPx-3 - Glutathione Peroxidase-3, PONi - Paraoxonase-1, CRP - C-Reactive Protein.
Table 5: Effect of vitamin E on lipid profile and various markers of oxidation, inflammation and HDL structure and function, in Hp 2-1 diabetic humans.
Figure imgf000023_0001
Complement C3 - Complement component 3, LCAT - lecithin cholesterol acyltransferase,
GPx-3 - Glutathione Peroxidase-3, PONi - Paraoxonase-1, CRP - C-Reactive Protein.
The cholesterol efflux assay used in this study utilized whole serum from patients assessing the direct relationship between cholesterol efflux capacity and atherosclerotic burden. This assay integrates total efflux mediated by several pathways which have been shown to be important for the efflux of cholesterol from macrophages (i.e., ATP -binding cassette transporter Al [ABCA1] and Gl [ABCG1 ], scavenger receptor B l, and aqueous diffusion). The effect of Hp genotype on these mediators of cholesterol efflux is unknown although some of the inventors of the present invention have previously reported a relationship between LCAT activity and Hp genotype (Asleh et al., Circ Res, 2006, 99: 1419- 1425). Moreover, the changes in HDL function upon administration of d-alpha-tocopheryl acetate as reported by the inventors of the present invention is fundamental with respect to the effect of vitamin E on the risk of cardiovascular diseases (CVD). Modest changes in cholesterol efflux as reported herein were shown to be directly associated with atherosclerotic burden and CVD (Khera et al., N Engl J Med, 201 1, 364: 127-135). Khera demonstrated that cholesterol efflux is related to intima media thickness and the likelihood of angiographic coronary artery disease independent of HDL cholesterol. The difference in efflux between the groups with and without CVD in that study was less than 10%. However, the changes in efflux with d-alpha-tocopheryl acetate observed by the inventors of the present invention were about 10% (11% reduction in Hp 2- 1 and 9% increase in Hp 2-2).
Example 7: Vitamin E improves RCT in Hp 2-2 but inhibits RCT in Hp 2-1
The Hp 2-2 cohort vitamin E was associated with a significantly higher RCT as compared to placebo (12.1±0.81% vitamin E vs. 1 1.1±0.64% placebo, n=28; P=0.05). However, in the Hp 2-1 cohort vitamin E was associated with a significantly lower RCT as compared to placebo (13.9±1.1 % vitamin E vs. 15.4±1.0% placebo, n=31 ; P=0.04) (Fig. 4).
There was a statistically significant interaction between the Hp genotype (Hp 2-1 vs Hp 2-2) and vitamin E on RCT (p=0.006). There was no significant effect of vitamin E on LCAT, ApoAl or HDL in Hp 2-1 or Hp 2-2 participants (Table 4 and 5).
Example 8: Vitamin E reduces HDL associated lipid peroxides in Hp 2-2 but not
HP 2-1
Oxidative modification has been proposed to be the mechanism by which HDL is rendered dysfunctional and antioxidant therapy appears to restore HDL functionality (Asleh and Blum, ibid). This study was intended to determine if the interaction between vitamin E and Hp genotype on RCT might be explained by a differential effect of vitamin E on HDL oxidative modification in Hp 2-1 and Hp 2-2.
Vitamin E resulted in a 50% reduction in HDL associated lipid peroxides in Hp 2-2 (0.55±0.10 nmol Vitamin E vs. 1.07±0.19 nmol placebo; p=0.003 compared to placebo) while not having a significant effect on HDL associated lipid peroxides in Hp 2-1 (Fig. 5; p=0.95 compared to placebo).
Furthermore, a trend showing a greater than 20% decrease in nitration of ApoAl in Hp 2-2 with vitamin E was observed, while in Hp 2-1 vitamin E had no effect on this parameter (Tables 4 and 5).
To determine why vitamin E reduced HDL lipid peroxidation in Hp 2-2 but not Hp 2- 1 , the effect of vitamin E on the mass or activity of the antioxidant proteins GPx-3 and paraoxonase (known to be associated with HDL), as well as the amount of redox-active nontransferrin-bound iron (which has previously been implicated in the oxidation of HDL) was investigated (Tables 4 and 5). While the effect of vitamin E did not reach statistical significance for any of these measurements vitamin E was found to be associated with an approximately 50% increase in HDL associated glutathione peroxidase and a 25% reduction in redox active iron in Hp 2-2 while there was a 3-4 fold decrease in HDL associated glutathione peroxidase (p=0.06) and no change in redox active iron in Hp 2-1.
Structural analysis of HDL in study participants suggested a similar interaction in which vitamin E appeared to result in a favorable although non-statistically significant change in a number of HDL associated oxidative and inflammatory markers in Hp 2-2 individuals (the decrease in HDL associated lipid peroxides was statistically significant) while producing no beneficial effect on these markers in Hp 2-1 individuals. This study therefore supports the concept that there exists a pharmacogenetic interaction between the Hp genotype and vitamin E in individuals with DM (Blum et al., Atherosclerosis, 2010, ibid; Blum et al., Pharmacogenomics, 2010, ibid). Example 9: Vitamin E and CD163
The increase in redox active iron in Hp 2-2 DM has been attributed to the impaired clearance of Hp 2-2-Hb by the CD 163 Hp-Hb receptor. Surface expression of CD 163 is regulated by oxidative stress and hyperglycemia. The purpose of this study was to determine if the decrease in redox-active iron in Hp 2-2 DM individuals who received d-a-tocopheryl acetate may have been associated with an increase in CD 163 expression on PBMs.
A trend showing a greater than 50% increase in CD 163 expression in PBMs of Hp 2-2 individuals receiving d-a-tocopheryl acetate was observed, d-a-tocopheryl acetate appeared to be associated with a 50% reduction in CD 163 expression in Hp 2-1 individuals (Tables 4 and 5). Example 10: Vitamin E decreases association of the inflammatory marker C3 with HDL in Hp 2-2 but not in Hp 2-1
In addition to functioning to promote RCT and to prevent the oxidation of LDL, HDL has been described as having an anti-inflammatory function. However, pro -inflammatory biomarkers such as C3 have been associated with dysfunctional HDL in individuals with CVD. This study was aimed to determine whether vitamin E would decrease the association of C3 with HDL in Hp 2-2. The results indicate that vitamin E treatment was associated with a borderline significant decrease in C3 associated HDL in Hp 2-2 individuals (1.07±0.09 vitamin E vs. 1.34±0.20 placebo; n=25; p=0.09) but had no effect on the association of C3 with HDL in Hp 2-1 (1.08±0.07 vs. 1.08±0.07, n=30; p=0.99). This effect of vitamin E on a HDL associated inflammatory marker was not associated with an overall change in serum markers of inflammation such as CRP and adiponectin (Tables 4 and 5).
The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

1. A pharmaceutical composition comprising: a lipid component comprising d-alpha- tocopheryl acetate; and
at least one surfactant;
wherein the composition forms a translucent emulsion of oil-in-water droplets when dispersed in an aqueous solution.
2. The pharmaceutical composition of claim 1 , wherein said composition is essentially devoid of aqueous solution.
3. The pharmaceutical composition of claim 1, comprising at least 300 units of d-alpha- tocopheryl acetate.
4. The pharmaceutical composition of claim 3, comprising at least 400 units of d-alpha- tocopheryl acetate.
5. The pharmaceutical composition of claim 1 , wherein the average particle size of the oil-in-water droplets is less than 0.1 micron.
6. The pharmaceutical composition of claim 1 , wherein the lipid component further comprises at least one oil.
7. The pharmaceutical composition of claim 6 wherein said at least one oil is a Long Chain Triglyceride.
8. The pharmaceutical composition of claim 7, wherein said at least one oil is selected from the group consisting of olive oil, sesame oil, soy bean oil and corn oil.
9. The pharmaceutical composition of claim 1 , wherein the composition comprises at least 25% w/w of d-alpha-tocopheryl acetate.
10. The pharmaceutical composition of claim 9, wherein the composition comprises at least 55% w/w of d-alpha-tocopheryl acetate.
1 1. The pharmaceutical composition of claim 6, wherein the amount of d-alpha- tocopheryl acetate is at least 75% w/w of the total weight of the lipid component.
12. The pharmaceutical composition of claim 1, wherein the at least one surfactant is selected from the group consisting of: nonionic surfactants, anionic surfactants, and cationic surfactants.
13. The pharmaceutical composition of claim 12, wherein the at least one surfactant is a nonionic surfactant selected from the group consisting of: polysorbate 80, capric glycerides, lauroyl macrogol-32 glycerides, oleoyl macrogol-6 glycerides, capryol, lauroglycol and lauroyl polyoxyl-6 glycerides, ethoxylation of hydrogenated castor oil, polyethylated castor oil, sorbitane monooleate and sorbitan monolaurate.
The pharmaceutical composition of claim 1, wherein the composition is in an oral dosage form.
The pharmaceutical composition of claim 14, wherein the oral dosage form is in the form of capsules.
The pharmaceutical composition of claim 15 wherein said capsules are selected from the group consisting of l,000mg capsules, 750 mg capsules and 500 mg capsules. The pharmaceutical composition of claim 1, wherein the at least one surfactant has an HLB higher than 12.
The pharmaceutical composition of claim 1 , wherein the HLB of the composition is greater thanl2.
A method for preventing, treating or attenuating cardiovascular events and disorders in a diabetic subject having an Hp 2-2 genotype, comprising administering to the subject the composition of any one or more of claims 1-18.
The method of claim 19, wherein preventing, treating or attenuating cardiovascular events and disorders is selected from the group consisting of: improving serum stimulated cholesterol efflux, reducing HDL associated lipid peroxides, increasing CD 163 expression on peripheral blood monocytes and decreasing association of the inflammatory marker C3 with HDL.
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