FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
AND
THE PATENT RULES, 2003
COMPLETE SPECIFICATION (See section 10; rule 13)
"Stable oil suspensions with enhanced bioavailability and compositions thereof
OMNIACTIVE HEALTH TECHNOLOGIES LIMITED,
An Indian Company, registered under the Indian Companies Act, 1956 having its registered
office located at Rajan House, Appasaheb Marathe Marg, Prabhadevi, Mumbai- 400025,
Maharashtra, India,
The following specification particularly describes the invention and the manner in which it is to be performed.

Field of invention:
The invention relates to stable oil suspension of lipophilic nutrients comprised of soluble granules suspended in oil medium which exhibit enhanced bioavailability. Soluble granules of the invention are comprised of lipophilic nutrient uniformly dispersed in effective amount of at least one solid hydrophilic carrier and one more food grade excipient. The invention also relates to process for preparation of soluble granules and oil suspension. Soluble, granules of the invention are prepared by uniformly dispersing lipophilic nutrient in effective amount of solid hydrophilic carrier and these exhibit enhanced bioavailability. These soluble granules are suspended in suitable oil medium to form suspension which is stable over shelf life of the composition. Stable oil suspensions can be encapsulated in soft gel capsules, filled in sachets or adsorbed onto solid carriers to form granules for beverage applications or can be filled in gelatin capsules and are useful for skin and eye care.
Background of the invention:
Lipophilic nutrients are essential for maintaining good health and prescribed by nutritionists for curing certain deficiencies in the body. Owing to their lipophilic nature, these nutrients exhibit low aqueous solubility and permeability through body membranes. Since the gastrointestinal tract is an aqueous environment, and since only dissolved molecules can be taken up by the body, these nutrients often show limited bioavailability.
Looking at the wide range of lipophilic nutrients which are essential for health, such as fatty acids, fat soluble vitamins, glycerides, carotenoids, curcumin, capsaicin, coenzyme Q 10 as well as Ubiquinol, the challenge of oral bioavailability of these nutrients is very high. In order to increase solubility of nutrients and enhance therapeutic efficacy, different techniques are employed by person skilled in the art. These can be decreasing particle size of nutrient, use of solubilizers, complexation with excipients such as Beta cyclodextrin, saccharin, milling with surfactant, solid dispersion, melt granulation and the like.
Along with increasing solubility of lipophilic nutrients, it is also desirable to formulate such nutrient into the dosage forms which are stable, owing to the inherent instability due to chemical structure, because of which these are prone to oxidative degradation. This highlights the

requirement of designing dosage forms for lipophilic nutrients which increase stability and exhibit enhanced therapeutic effectiveness, thus resulting into achieving desirable positive health benefits.
Various prior art applications are directed to increase solubility of lipophilic nutrients or formulate such nutrients into various dosage forms.
U.S. Patent 8748495 relates to a method for preparing a carotenoid oil suspension, by treating carotenoid crystals with tetrahydrofuran to remove non-soluble phospholipids in order to get oil suspension with low viscosity and high fluidity. The resulting crystals are ground and mixed with plant oils to provide a carotenoid oil suspension, which provides easy filling, for example, into capsules. Thus the application aims at providing oil suspension having low viscosity to aid in processing it into soft gel capsules.
Japanese patent application JP2012006943A teaches a stable microcapsule product with high levels of carotenoid. The microcapsules are coated with a protective coating of a sugar or polyhydric alcohol, a starch or dextrin, and optionally a protein, which release carotenoids upon ingestion of the microcapsules. The microcapsules are formed using a fluidized bed coating machine for spraying the coating material.
U.S. patent application US20140030419A1 relates to a carotenoid oil suspension and preparation method which includes the steps of mixing carotenoid with organic solvent, heating the mixture to dissolve the carotenoid sufficiently; introducing the carotenoid solution into a oil solution which is stirred at high speed by spraying to obtain carotenoid oil suspension. Such oil suspension comprises a carotenoid crystal with an average particle size of less than 5pm.
U.S. patent application US20130216512A1 discloses an aqueous suspension of a hydrophobic nutrient which is prepared by treating the nutrient with a selected dispersion aid such as triglyceride, an essential oil such as night primrose oil, fish oil, and a mixture to form a modified nutrient compound. Such compound is then combined with a dispersion agent such as lecithin,

hydrocolloid or surfactant in aqueous medium under high shear to form a stable aqueous suspension.
Another U.S. patent application US20130310302A1 relates to use of rice endosperm protein as novel protective hydrocolloid for fat-soluble nutrient ingredients. These compositions are used for the enrichment, fortification and/or coloration of food, beverages, animal feed, personal care or pharmaceutical compositions.
PCT application WO2012139895A1 teaches a process for the manufacture of a powder containing lutein and food composition containing said powder. As per the patent application, lutein is suspended in an aqueous solution/suspension of a polysaccharide and resulting suspension is milled followed by drying to obtain lutein particles having mean particle size of less than 0.6 micrometer.
U.S. patent application US20120039970A1 relates to a ready-to-use stable suspension of partially amorphous carotenoid particles and a process for production thereof, wherein the carotenoid and the edible oil are dissolved in a water-miscible organic solvent or water-immiscible organic solvent. This solution is mixed with a molecularly dispersed or colloidally dispersed solution of the hydrophilic protective colloid in a mixture of water and polyhydric alcohol. The carotenoid-comprising hydrophobic phase is produced as a nanodispersed hydrophobic phase and the organic solvent is removed thus concentrating the suspension formed.
U.S. patent 6616942 discloses a method for improved absorption of lipophilic nutrient from intestinal tract by administration in a soft gel capsule of a formulation of mixture of lipophilic nutrients in rice bran oil and thickener such as beeswax. The process comprises of heating rice bran oil and adding beeswax to the heated rice bran oil to which mixture of lipophilic nutrients is added. The resultant mixture is cooled and encapsulated in a soft gel capsule.
Although prior art applications disclose treatment of lipophilic nutrients with excipients such as surfactant, colloids, proteins, wax, oil or organic solvent vehicles, these do not address problem of low solubility of these inherently hydrophobic nutrients. Use of excessive amount of surfactants to increase solubility of actives is not allowed in nutraceutical formulations, owing to

its taste, odor and consumer acceptability. Further merely suspending these lipophilic nutrients into oils, waxes or organic solvents do not tackle the underlying problem of stability, solubility and therefore bioavailability. The lipophilic nutrients are poorly absorbed if merely administered either as oil suspensions or as beadlets. This is because the main reason for poor absorption is their poor solubility in water. Thus there is need of stepwise alternate formulation approaches for lipophilic nutrients so as to design dosage forms with enhanced stability, solubility and therefore bioavailability and which are convenient to administer in their stable form.
The researchers of the present invention have carried out exhaustive trials and formulated stable and bioenhanced oil suspensions by following stepwise approach of first tackling problem of inherent poor water solubility of lipophilic nutrient and then addressing stability issue, by careful selection of food-grade excipients and conventional equipments. The inventors formulated soluble granules by uniformly dispersing lipophilic nutrients in effective amount of solid hydrophilic carrier, thus enhancing solubility and bioavailability of the nutrient. These solubilized granules are further suspended in suitable oil medium, resulting into stabilization of the nutrient. Thus stable oil suspensions of the instant invention are comprised of soluble granules of lipophilic nutrients suspended in oil medium. The finding that soluble granules of lipophilic nutrients exhibit enhanced solubility and bioavailability and it is further stabilized after suspending in oil medium, is not reported anywhere in the prior art. Such oil suspensions are even found to be more permeable than the marketed comparator formulations, thus reflecting enhanced in vivo absorption phenomenon. Such oil suspensions are the delivery forms which are convenient for further processing and are not limited by particle size of granules or bulk density and therefore can be formulated in compositions like soft gel capsules, dry granules by adsorbing onto solid carrier, or filled in sachets for increasing administration convenience, patient compliance and dose accuracy.
Objectives of the present invention:
The main objective of the present invention is to provide stable oil suspensions comprising soluble granules of lipophilic nutrients, suspended in suitable oil medium.
According to one more objective, the present invention also provides the stepwise process for first addressing inherent poor water solubility property of lipophilic nutrient and then handling

stability problem of these nutrients, to formulate composition, which is convenient to administer and provides dose accuracy.
As per one more objective of the invention, solubility of lipophilic nutrient is increased by uniformly dispersing the nutrient in effective amount of solid hydrophilic carrier to result into soluble granules. These soluble granules are further suspended in suitable oil medium to enhance further bioavailability and conferring stability to lipophilic nutrient.
As per one more objective of this invention, in the first step of preparation, the soluble granules are prepared using lipophilic nutrient, effective amount of at least one hydrophilic carrier and one more food grade excipient.
According to one more objective of this invention, amount of solid hydrophilic carrier employed in soluble granules is in the ratio of lipophilic nutrients: solid hydrophilic carrier of 1:0.5 to 1:5.
One more objective of the present invention is to provide the process for preparation of soluble granules of lipophilic nutrients wherein the nutrient is dispersed in effective amount of solid hydrophilic carrier along with at least one more food grade excipient in suitable organic solvent, which is safe for human consumption and spray dried to get the solubilized product.
According to one more objective of the present invention, soluble granules prepared by dispersing lipophilic nutrient in solid hydrophilic carrier and spray drying are non-sticky, easy for further processing and non-cohesive, although these are prepared by sticky lipophilic nutrients, thus these are convenient for processing into oil suspension.
Another objective of the present invention is to describe second step of process for preparation of oil suspension, wherein soluble granules are suspended in suitable oil medium with stirring and followed by milling to get uniform, stable and bioenhanced suspension, containing 1 to 50 % by weight of lipophilic nutrient.
One more objective of the present invention is to provide oil suspensions of lipophilic nutrients which are stable and exhibit enhanced bioavailability as compared to granules, even after

suspending in hydrophobic edible oily medium. The bioavailability of such oil suspensions is many folds increased as compared to marketed comparator formulations.
Another objective of the present invention is to provide oil suspensions containing soluble granules of lipophilic nutrients such as fatty acids, fat soluble vitamins, glycerides, carotenoids, curcumin, capsaicin, coenzyme Q 10 as well as Ubiquinol, which are suspended in suitable oil medium.
As per one more objective, these oil suspensions can be formulated as soft gelatin capsule or delivered in the form of sachets, or adsorbed on solid carrier to form granules, which is convenient to administer to the patients.
Summary of the Invention:
Accordingly the present invention provides stable oil suspension with enhanced bioavailability containing soluble granules of lipophilic nutrients and suitable oil medium; wherein soluble granules are comprised of;
at least one lipophilic nutrient,
effective amount of at least one solid hydrophilic carrier and
at least one more food grade excipient.
The instant invention also provides process for preparation of oil suspensions of lipophilic nutrients which are more stable than the granules and compositions of these suspensions such as soft gelatin capsules.
The oil suspensions are comprised of soluble granules of lipophilic nutrients suspended in suitable oil medium. The soluble granules are comprised of at least one lipophilic nutrient and effective amount of at least one hydrophilic carrier along with one more food grade excipient such as antioxidant. Stable oil suspensions of the invention are comprised of at least 1% to about 50% carotenoids wherein free lutein is present in combination with zeaxanthin and/or neoxanthin and/or a-cryptoxanthin and/or P-cryptoxanthin and/or mesozeaxanthin along with other excipients and antioxidants. Such oil suspensions are resistant to oxidation and exhibit enhanced

stability and bioavailability as compared to granules as well as marketed comparator product. Oil suspensions of the invention can be formulated into various delivery systems such as soft gel capsules, filled in sachets or adsorbed onto solid carriers to form granules to administer conveniently to the patients.
Detailed Description:
The invention relates to stable and bioenhanced oil suspensions of lipophilic nutrients containing soluble granules suspended in suitable oil medium and compositions prepared by encapsulating oil suspensions in compositions such as soft gel capsules and the like. The invention also relates to method for preparation of soluble granules as well as oil suspensions containing lipophilic nutrients and other compositions thereof.
As used herein, the term "about" refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term "about" generally refers to a range of numerical values (e.g., +/-5-l0% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term "about" may include numerical values that are rounded to the nearest significant figure.
The lipophilic nutrients suitable for the instant invention are sensitive to heat or oxygenating conditions. The term "lipophilic" though refers to lipid-like, it generally covers all compounds that are poorly water soluble. Non-limiting examples are carotenoids, fat soluble vitamins, fatty acids, glycerides, capsaicin, curcumin and mixtures thereof.
Preferably the lipophilic nutrient is selected from the group such as, but not limited to, carotenoids (especially alpha-carotene, beta- carotene, 8'-apo-beta-carotenal, 8'-apo-beta-carotenoic acid esters such as the ethyl ester, canthaxanthin, astaxanthin, astaxanthin ester, betacryptoxanthin, lycopene, lutein, lutein (di) ester, zeaxanthin or crocetin, mesozeaxanthin, alpha or beta-zeacarotene or mixtures thereof), vitamins (A, D, E, K, CoQ 10) or derivatives thereof (such as their acetates, e.g. vitamin A acetate or tocopherol acetate, or their longer chain fatty acid esters, e.g. vitamin A palmitate or tocopherol palmitate), capsaicin, dihydrocapsaicin, derivatives thereof, polyunsaturated fatty acids (PUFAs) or derivatives thereof, and triglycerides rich in polyunsaturated fatty acids such as eicosapentaenoic acid (EPA), docosahexaenoic acid

(DHA) or gamma-linolenic acid (GLA), Omega 3, Omega 6 oils or derivatives thereof, ethanolic extracts of Terminalia, Salacia and or mixtures thereof. Further lipophilic nutrients suitable for the present invention are compounds which have a taste or smell which is required to be masked, e.g., bitter tasting vitamins and fish oil.
More preferably the lipophilic nutrients of the instant invention are selected from the carotenoids such as, but not limited to, beta-carotene, lutein, lycopene, astaxanthin, astaxanthin ester, zeaxanthin, neoxanthin, a-cryptoxanthin, P-cryptoxanthin and canthaxanthin. Further carotenoids are most preferably used in combination of free lutein with zeaxanthin and/or neoxanthin and/or a-cryptoxanthin and/or p-cryptoxanthin and the like or the mixtures thereof. Throughout the description the term fat soluble nutrients encompasses the foregoing definition and mixtures of said compounds.
According to one of the embodiments of the present invention, carotenoids of the present invention are obtained from Marigold flowers and employed in combination of free lutein and zeaxanthin, more preferably t-lutein and zeaxanthin. The ratio in which they are used in instant invention varies from about 4.5:1 to 5.5:1. More preferably these carotenoids are used in the ratio of5:l.
"Soluble granules" of the present invention can be defined as the solubilized systems comprised of lipophilic nutrient embedded in effective amount of solid hydrophilic carrier and at least one more food grade excipient to form granules, which are prepared by spray drying method.
"Hydrophilic carrier" of the invention is a food grade excipient, which is soluble in water and imparts its hydrophilic property, when used in effective amount, so that lipophilic nutrient is embedded well to form soluble granules.
"Effective amount" of solid hydrophilic carrier means the amount to be used such that lipophilic nutrients get embedded in this excipient and it confers upon its own hydrophilic properties to the hydrophobic nutrient, thus solubilizing it, to form soluble granules. Apart from imparting its hydrophilic properties, this solid carrier also helps to prepare non-sticky and non-cohesive granules of lipophilic nutrient, which are easy for processing into oil suspension.

Thus soluble granules are the non- sticky and non-cohesive systems, wherein lipophilic nutrients are conferred hydrophilic properties to enhance solubility and release of active by using effective amount of solid hydrophilic carrier, thus it is first step in formulation of oil suspensions. Although the granules are prepared from sticky starting material such as lipophilic nutrient, these are non-cohesive and aid in further processing.
An "oil", as used herein means any neutral, nonpolar chemical substance that is a viscous liquid at ambient temperatures and is both hydrophobic (immiscible with water) and lipophilic (miscible with other oils), the oils used in the present invention are food -grade or edible oils and may be selected from animal, vegetable or synthetic source.
According to one embodiment of the instant invention, granules of lipophilic nutrients are prepared as first step of formulation by using effective amount of solid hydrophilic carrier. Resulting soluble granules exhibit desired solubility and active release properties, owing to embedding of lipophilic nutrient in solid hydrophilic carrier, thus conferring its properties to resulting granules and increasing the solubility. As a second formulation step, when these granules are further suspended in suitable liquid medium such as oil, it was observed that such oil suspension exhibits enhanced stability and solubility as compared to granules as well as marketed product. Thus oil suspensions of the present invention are comprised of soluble granules of lipophilic nutrients, suspended in oil medium and stabilized from inherent oxidation by employing one more food grade excipient such as antioxidant during preparation of granules.
As the granules are suspended in oil, these oil suspensions do not have any limitation of solid dosage forms like bulk density and particle size and thus are convenient to be formulated into soft gel capsules, or filled in sachets or transformed into granules by adsorbing on suitable solid excipient, for ease of administration to consumers. It was also observed that resulting compositions exhibit improved stability and desired dissolution profile.
In accordance with one more embodiment of the present invention, a stable system is provided which acts as an immediate release formulation in the form of oil suspensions of lipophilic

nutrients and its subsequent compositions, which are convenient to administer to consumers of different age groups and results in patient compliance and therapeutic efficacy.
In one of the embodiments of the invention, granules are prepared by dispersing lipophilic nutrient and effective amount of solid hydrophilic carrier in common organic solvent, which is acceptable for human consumption and spray drying the dispersion to get granules of lipohilic nutrients. Thus granules are preferably comprised of carotenoids, which are combination of free lutein and zeaxanthin, embedded in effective amount of solid hydrophilic carrier and at least one more food grade excipient.
In one more embodiment, the solid hydrophilic carrier employed in preparation of granules is selected from the group such as, but not limited to, cellulose derivatives, polyacrylates, polyethylene glycols, povidones, starch and starch derivatives, gums, sugars, and the like.
The solid hydrophilic carrier of the invention may be selected from the group such as cellulose and cellulose derivatives, but not limited to, alkyl cellulose (methyl cellulose), a hydroxyalkyl cellulose (e.g., hydroxymethyl cellulose, hydroxypropyl cellulose), carboxyalkyl cellulose (e.g., carboxymethyl cellulose and alkali metal salts thereof, such as sodium salts), a carboxyalkylalkyl cellulose (e.g., carboxymethylethyl cellulose), a carboxyalkyl cellulose ester (e.g., carboxymethyl cellulose butyrate, carboxymethyl cellulose propionate, carboxymethyl cellulose acetate butyrate, and carboxymethyl cellulose acetate propionate), and the like.
According to one more embodiment of the present invention, the solid hydrophilic carrier may also be selected from the group such as polyacrylates, but not limited to polymethacrylate, a methacrylate copolymer (e.g., a methacrylic acid-methyl methacrylate copolymer, dimethylaminoethyl methacrylate-butyl methacrylate-methyl methacrylate copolymer, and a diethylaminoethyl methacrylic acid-methyl methacrylate copolymer), and an ethacrylate copolymer (e.g. methacrylic acid ethacrylate copolymer), and the like.
According to still one more embodiment of the present invention, the hydrophilic carrier may be selected from the group of povidones, but not limited to polyvinyl pyrrolidone (e.g., Povidone),

polyvinyl acetate ester (e.g., polyvinyl acetate phthalate (PVAP)), and a polyethylene glycol polyvinylacetate copolymer (e.g. polyethylene glycol-polyvinylcaprolactam-polyvinylacetate copolymer), and the like.
According to one more embodiment of the present invention, the solid hydrophilic carrier may also be selected from the group such as polyethylene glycols, but not limited to, polyalkylene oxide (e.g., polyethylene glycols, such as PEG 300, PEG 400, PEG 4000, and PEG 8000, and polypropylene glycols), a copolymer of ethylene oxide and propylene oxide (e.g., ethoxylated propoxylated block copolymers, and a polyethoxylated glyceryl ester (e.g., polyoxyl 35 castor oil and polyoxyl 40 castor oil having 40-45 moles of ethylene oxide), and the like.
According to one more embodiment of this invention, the hydrophilic carrier may be selected from the group such as starch and starch derivatives, but not limited to, dextrins, acid-treated starch alkaline-treated starch, bleached starch, oxidized starch derivatives, enzyme-treated monostarch phosphate, distarch phosphate, phosphated distarch phosphate, acetylated distarch phosphate, starch acetate, acetylated distarch adipate, hydroxypropyl starch, hydroxypropyl distarch phosphate, hydroxypropyl distarch glycerol, starch sodium octenyl succinate, acetylated oxidized starch and the like.
As per one more embodiment of this invention, the hydrophilic carrier may be selected from the group such as gums, but not limited to, pectin, alginate, carrageenan, agar. Gum arabic, Gum tragacanth, Gum karaya, Gum ghatti, Gum guar, Locust bean gum, Tara gum, Xanthan gum, Gellan gum, Welan gum and the like.
As per one more embodiment of the present invention, the hydrophilic carrier may be selected from the group such as sugars and alcohols, but not limited to glycerol, sorbitol, glucose syrup, corn steep liquor, mannitol, sucrose, glucose, sodium chloride, polyvinyl alcohol, and mixtures thereof and the like.

As per preferred embodiment of the instant invention, the hydrophilic carrier excipient may be polyvinyl pyrrolidone (PVP). Various grades of polyvinyl pyrrolidone can be employed within scope of this invention.
As per one more embodiment of the invention, hydrophilic carrier is used in the effective amount in the present invention. The amount of hydrophilic carrier incorporated into soluble granules is such that it is effective to impart its hydrophilic properties to the matrix of lipophilic nutrient, thus increasing their solubility. In certain preferred embodiments of the present invention, the hydrophilic carrier is included in the granules so that ratio of carotenoid to such carrier is 1:0.5 to 1:5.
As per one more important embodiment of the present invention, the amount of carotenoid, used in the granules varies from about 1% to 50%, more preferably about 2 to 25%. Preferably these granules contain carotenoids, which is present in the form of combination of free lutein and zeaxanthin.
According to further embodiment of the invention, size of the granules is 0.5 to 30 microns, as analyzed by Malvern instrument, after suspending into oil suspension, by following process of stirring and milling of suspension for homogeneous dispersion of granules in oil medium.
Stable oil suspensions of the instant invention are formed of granules of carotenoids, suspended in suitable oil, wherein granules are comprised of lipophilic nutrient, effective amount of solid hydrophilic carrier and at least one more food grade excipient.
Granules of the present invention typically may also include other food grade excipients such as, but not limited to, diluents, antioxidant, surfactant, binders, solvents, and the like. As is well known to those skilled in the art, pharmaceutical excipients are routinely incorporated into solid dosage forms. This is done to ease the manufacturing process as well as to improve the performance of the dosage form.

As per one embodiment of the instant invention, examples of suitable diluents include, but are not limited to, starch, dicalcium phosphate, microcrystalline cellulose, lactose monohydrate, dextrate hydrated, colloidal grade carboxymethyl cellulose sodium, carboxymethyl cellulose calcium and other cellulose containing polymers and their derivatives or the like and mixtures thereof. Preferably, suitable diluent is selected from conventional marketed grades of microcrystalline cellulose, including, but not limited to Avicel®PH 101, Avicel®PH 102, Avicel®PH 103, Avicel®PH 105, Avicel®PH 112, Avicel®PH 113, Avicel® PH 300, AviceI®PH 212, Avicel®PH 301, Avicel®PH 302, and the like or mixtures thereof.
As per one more embodiment of the instant invention, the antioxidant is selected from the commonly used excipients including, but not limited to a-Tocopherol, |3-Tocopherol, y-Tocopherol, mix Tocopherol, citric acid, Rosemary extract, ascorbyl palmitate, sodium ascorbate or the like and the combinations thereof.
Suitable surfactants include, but are not limited to, anionic and non-ionic surfactants or a mixture
thereof. The non-ionic surfactants employed in the composition may include, but are not limited
to, ethoxylated fatty acid ester, ethoxylated fatty acid ethers, ethoxylated sorbitan ethers,
ethoxylated alkyl-phenols, glycerol esters, glycerol sugar esters, polyoxyethylene glycerol
monolaurate, polyoxyethylene glycerol monostearate, polyoxyethylene-20-cetyl stearate,
polyoxyethylene-25-cetyl stearate, polyoxyethylene(25)-oxypropylene monostearate,
polyoxyethylene-20-sorbitan monopalmitate, poly-oxyethylene-16-tert-octylphenol,
polyoxyethylene-20-cetyl ether, polyethylene glycol(1000)monocetyl ether, ethoxylated castor oil, polyoxyethylene sorbitol-lanolin derivatives, polyoxyethylene(25)propyiene glycol stearate, polyoxyethylenesorbitol esters, polyoxyethylene-20-sorbitan monopalmitate, polyoxyethylene-16-tert-octylphenol, polyoxyethylene-20-cetyl ether, glycyeryl undecylenate and Polysorbate 60, capmul (medium chain glyceride), peceol(glyceryl monooleate), glyceryl laurate and glyceryl caprylate (Capmul MCM), PEG sorbitan fatty acid esters like PEG-20 sorbitan monolaurate (Tween 20), PEG-20 sorbitan monostearate (Tween 60), PEG-20 sorbitan monooleate (Tween 80), sorbitan fatty acid esters like sorbitan monolaurate (Span 20), glyceryl stearate (Cithrol GMS) or the like and mixtures thereof. Suitable anionic surfactants include, but are not limited to, fatty alcohol sulfates, alpha olefin sulfonates, sulfosuccinates, phosphate esters, carboxylates,

sarcosinates, alkyl benzene sulfonates, alkyl sulfonates, olefin sulfonates, alkyl ethersulfonates, glycerol ethersulfonates, alpha-methyl estersulfonates, sulfonic fatty acids, alkyl sulfates, fatty alcohol ethersulfates, glycerol ethersulfates, mixed hydroxy ethersulfates, monoglyceride(ether)sulfates, fatty acid amide(ether)sulfates, sulfosuccinates, sulfosuccinamates, sulfotriglycerides, alkyl oligoglycoside sulfates, alkyl(ether)phosphates or the like and mixtures thereof.
In one more embodiment of the present invention, there is provided a process for preparation of soluble granules of lipophilic nutrient; wherein the nutrient is dispersed in suitable polar or non polar solvent or a mixture of polar and non-polar solvent. The solid hydrophilic carrier is also dissolved in a suitable polar solvent to form clear solution and mixed with at least one more food grade excipient. The dispersion of the lipophilic nutrient is then mixed well with the solution of hydrophilic carrier and excipient and subjected to spray drying to remove solvent to obtain soluble granules of lipophilic nutrient.
In another preferred embodiment the solvent employed in process for preparation of soluble granules may be selected from the group such as, but not limited to, acetone, hexane, ethyl acetate, isopropyl alcohol, ethanol, dichloromethane, methanol, and a mixture thereof, more preferably from acetone, ethanol, dichloromethane, isopropyl alcohol, and more preferably dichloromethane and isopropyl alcohol.
In a preferred embodiment the non-polar solvents which may be used for preparing the dispersion of lipophilic nutrient include, but not limited to, methylene chloride, chloroform, petroleum ether (low boiling), petroleum ether (high boiling) and the like or the mixtures thereof.
In another preferred embodiment, the polar solvents, which may be used for preparing the solution of solid hydrophilic carrier, include, but is not limited to, isopropyl alcohol, acetone, methanol, ethanol, acetonitrile or mixtures thereof.
The non-polar solvent and polar solvent can be used in varying ratios. For instance, the non-polar and polar solvents can comprise a mixture of methylene chloride and isopropyl alcohol at a ratio

of about 1:1 to about 0.1:1. The non-polar and polar solvents can also comprise a mixture of methylene chloride and isopropyl alcohol at a ratio of about 0.2:1 to about 2:1.
Soluble granules of the present invention are suspended in suitable liquid medium such as oil in the ratio of about 1:1 to 1:3 to result into suspension in which granules are dispersed well. Oils employed for this purpose are edible or food -grade category and may be selected from animal, vegetable or synthetic source and are rich sources of medium chain triglycerides.
Oils of the instant invention are selected from the group, such as, but not limited to sunflower oil, safflower oil, coconut oil, corn oil, cotton seed oil, canola oil, olive oil, palm oil, peanut oil, sesame oil, soybean oil, grape seed oil, pumpkin seed oil, argan oil, rice bran oil and other vegetable oils, as well as animal-based oils like butter and lard and the like. More preferably the oil employed to suspend granules of the present invention is selected from soybean, safflower or sunflower oil.
As per one more preferred embodiment of the present invention, granules of lipophilic nutrient are suspended in suitable oil by using homogenizer followed by milling to get oil suspension containing granules of lipophilic nutrient.
According to one embodiment of the present invention, oil suspension of lipophilic nutrient can be incorporated into suitable dosage form such as soft gel capsules by encapsulation or packed in the sachets for convenience of administration or can be adsorbed on solid hydrophilic carrier to form granules, which can be employed in beverages or filled in hard gelatin capsules.
As per one more embodiment of the invention, oil suspensions are evaluated for stability, release of active and absorption in comparison with granules as well as composition such as soft gel capsules. Release of active is studied by dissolution study in relevant dissolution medium. Absorption of active from oil suspension is evaluated by intestinal permeability studies using everted rat intestinal sac method and compared with marketed comparator formulation.

While the present invention has been described in terms of its specific illustrative embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention. The details of the invention, its objects and advantages are explained hereunder in greater detail in relation to non-limiting exemplary illustrations.
Examples:
Example 1: Preparation of Carotenoid Crystals
The preparation of carotenoid concentrate is described in Indian Patent Application No. 622/MAS/2002 (U. S. Patent 6,737,535), the disclosures of which are incorporated by reference herein, and is summarized as follows.
Commercial grade marigold extract (57.98 g) containing 11.54% free lutein-zeaxanthin content (by spectrophotometric method) was mixed with potassium isopropyl alcoholate (prepared by dissolving 15 g potassium hydroxide in 175 ml isopropanol.) The saponification mixture was heated and maintained at 70°C for a period of 3 hours. The degree of hydrolysis was monitored by HPLC during the saponification stage. Isopropanol was distilled off under reduced pressure and the solids obtained were stirred with 230 ml of water at room temperature. The mixture was taken into a reparatory funnel and extracted with equal volume of ethyl acetate (3 times). Ethyl acetate layer was collected and washed with distilled water for removing the excess alkali, soapy materials and other water-soluble impurities. The ethyl acetate layer was distilled off under reduced pressure to get saponified crude extract (25.01 g). This resultant crude extract (25.01 g) was subjected to purification by stirring with 100 ml of hexane/acetone mixture (80:20) at room temperature for 30 minutes, followed by filtration. The precipitate of carotenoid crystals obtained was washed with methanol. The resulting orange crystals were vacuum dried at ambient temperature for 72 hrs. The yield of the crystals was 3.41% (1.98 g). Carotenoid content was 86.23% by weight (as determined by UV/Vis spectrophotometry) out of which the contents of trans-lutein, zeaxanthin, and other carotenoids were 91.43%, 6.40% and 2.17% respectively as determined by HPLC analysis.

Example 2: Preparation of oil suspension from granules of Carotenoids
Table 1: Composition of carotenoid oil suspension

Ingredients Formula I Formula II Formula III Formula IV Formula V
Carotenoids (Free lutein and zeaxanthin concentrate) 12.50 12.50 12.50 12.50 12.50
Mixed Tocopherol 0.92 0.92 0.92 0.92 0.92
Tween 80 2.63 2.63 2.63 2.63 2.63
Sodium Ascorbate 1.71 1.71 1.71 ■ 1.71 1.71
Povidone K30 15.58 15.58 15.58 - -
Modified starch - - - 15.80 -
Maltodextrin - - - - 15.80
Isopropyl Alcohol 6.00 6.00 6.00 6.00 6.00
Methylene Dichloride 294.00 294.00 294.00 294.00 294.00
Soybean Oil 66.66 - - 66.66 66.66
Sunflower Oil - 66.66 - - -
Safflower Oil - - 66.66 - -
Total 100 100 100 100 100
Process for preparation of Formula I to Formula V
Step 1: Preparation of soluble granules of carotenoids
Isopropyl alcohol was mixed with methylene dichloride in to suitable vessel and carotenoid concentrate was added to this solvent system under stirring. Required amount of polyvinyl pyrrolidone (PVP K30) was added in above dispersion with stirring. Mixed tocopherol and tween 80 were added sequentially to above mixture. Weighed amount of sodium ascorbate was sifted through 100 mesh and added to the system with continuous stirring. Resulting dispersion was milled through colloid mill for 15 to 30 minutes and passed through 80 mesh. This was subjected to spray drying by adjusting suitable parameters to get granules of carotenoids.

Step 2: Preparation of oil suspension of carotenoid granules
Weighed amount of suitable oil (Soybean oil/Safflower oil/Sunflower oil) were transferred to mixing vessel and granules of carotenoids were added to this liquid medium under stirring till it got completely dispersed. The resulting oil suspension of carotenoids was further stirred for 60 minutes and passed though colloid mill twice followed by filtration of resulting oil suspension.
Example 3: Stability Study of Carotenoid granules and Oil suspension
Carotenoid granules and oil suspension of Formula I were subjected to accelerated stability study at 40°C ± 2°C/75% RH ± 5% RH for the period of 6 months. Product was packed in sealed aluminium bottle and incubated in stability chambers. The granules and oil suspension were analyzed for lutein and zeaxanthin content at definite time interval. The result is tabulated in table no. 2 and 3 respectively.
Table 2: Lutein and Zeaxanthin content in carotenoid granules

Example Initial (%) 1 Month 2 Month 3 Month 6 Month
L Z L Z L Z L Z L Z
Formula I 22.62 5.00 20.60 4.80 20.14 4.58 20.5 4.56 19.56 4.54
L- Lutein , Z- Zeaxanthin
Table 3: Lutein and Zeaxanthin content in oil suspension during stability study

Example Initial (%) 1 Month 2 Month 3 Month 6 Month
L Z L Z L Z L Z L Z
Formula I 7.80 1.75 7.72 1.72 7.47 1.74 8.62 1.75 7.4 1.64
L- Lutein, Z- Zeaxanthin
It was found that as shown in table 2, carotenoid soluble granules exhibit certain drop (about 12 to 15%) in stability with respect to Lutein content over a period of 6 months. But when these granules are suspended in suitable oil medium, the resulting oil suspension is stabilized and the percentage drop in lutein content is significantly lowered to around 5%, as evident from table 3. Thus oil suspensions of granules exhibit enhanced stability and can be stored over a period of shelf life, without affecting active content.

Example 4: Preparation of carotenoid soft gel capsules from oil suspension
Carotenoid oil suspension was encapsulated in soft gel using suitable oval die, employing spread box temperature of 55°C and segment temperature 39 °C and ribbon thickness was adjusted as 0.85 mm. Soft gel capsules were dried at 23 °C with RH 18% for 48 hrs.
The capsules were examined physically for integrity and leakage. It was found that there was no leakage, fracture or discoloration during sealing and storage. Thus the capsules were stable during shelf life at storage conditions.
Example 5: Dissolution data of carotenoid granules, oil suspension and soft gel capsules
Carotenoid granules, oil suspension and soft gel capsules were subjected to dissolution study employing USP type II (Paddle) apparatus and using 1000 ml of pH 6.8 buffer with 2.0% SLS as dissolution medium at 100 rpm. Along with this, marketed soft gel capsules were also subjected to dissolution and mean % release (average) data of lutein release is shown in table no. 3.
Table 4: Avearge Lutein release from granules, oil suspension and soft gel capsules of carotenoids

Time (min Granules Formula I Oil Suspension Formula I Soft gel capsules Formula I Marketed Soft gel capsule
15 81.42 52.80 42.84 14.53
30 85.36 67.78 75.44 62.74
45 88.48 84.60 92.90 90.36
60 91.59 94.02 99.89 99.88
90 97.81 108.28 109.50 99.54
The dissolution study indicates that soluble granules exhibit fast release of lutein at initial time point, but lutein release from granules, oil suspension and soft gel capsules of the invention was comparable after 45 minutes. In spite of suspending the granules in oil medium and encapsulating into soft gel, lutein was released efficiently after 30 minutes. Release of lutein from marketed soft gel capsules was slower at initial time point as compared to oil suspension and soft gel capsules of the invention. Thus granules suspended in suitable oil and encapsulated

in soft gel capsules exhibit desirable release rate of lipophilic nutrient as compared to release from granules.
Example 6: Evaluation of Intestinal Permeability of Lutein by Everted Rat Intestinal Sac Method
Rat everted intestinal sac was prepared by opening rat abdomen under anaesthesia and selecting middle small intestine by flushing with Kreb's buffer solution and everting the prepared sac by pushing the rod through whole length of intestine. Total surface area of the everted sac of the small intestine was recorded and the sac was filled with Kreb's solution and placed in a beaker containing specific concentration of test item. The sacs were incubated up to 60 minutes and sample was withdrawn from serosal side of the sac and processed for HPLC analysis. Concentrations were measured and values were used to calculate the apparent permeability. The experiment was carried out in duplicates for every sample and mean apparent permeability was calculated from permeability study result and recorded in following table no. 6. The direction of permeability was from intestinal mucosa to serosal side. The samples evaluated for permeability by this method were as follows:
Sample I: Soluble Granules of lipophilic nutrient Sample II: Oil suspension of granules in safflower oil base (Formula III) Sample III: Oil suspension of granules in Soybean oil base (Formula I) Sample IV: Marketed comparator lutein oil suspension product
Table No. 6: Apparent Permeability of Lutein from test samples

Sample Mean, Apparent Permeability
(10"06cm/sec)
Sample I 0.02
Sample II 0.195
Sample III 0.443
Sample IV 0.009

Evaluation of intestinal permeability study by everted sac method indicates that apparent permeability exhibited by oil suspension comprising granules of lipophilic nutrients in Soybean oil medium is significantly more than granules as well as oil suspension comprising granules of lipophilic nutrients in Safflower oil medium. Apparent permeability for marketed product suspension was considerably lower than oil suspension of the instant invention. As the permeability is directly related to absorption through body cells, high apparent permeability indicates that oil suspension in soybean oil medium exhibits enhanced bioavailability as compared to soluble granules and thus are useful and convenient to administer to the patients of all age groups.

Claims: We claim,
1. Stable oil suspension with enhanced bioavailability comprising,
at least one lipophilic nutrient,
effective amount of at least one solid hydrophilic carrier and
at least one more food grade excipient;
wherein the said lipophilic nutrient is uniformly dispersed in solid hydrophilic carrier to
form soluble granules and then suspended in an oil, which suspension can be formulated
into compositions such as soft gel capsules, sachets and the like.
2. Stable oil suspension of claim 1, wherein lipophilic nutrient is selected from carotenoids, vitamins, omega fatty acids, glycerides, capsaicin, curcumin, extracts of Salacia, Terminalia, Co-Enzyme Q-10, Ubiquinol and mixtures thereof,
3. Stable oil suspension of claim 2, wherein lipophilic carotenoid nutrient is selected from the group consisting of lutein, lutein esters, alpha carotene, beta-carotene, zeaxanthin, mesozeaxanthin, betacryptoxanthin, zeaxanthin esters, astaxanlhin, lycopene and mixtures thereof.
4. Stable oil suspension of claim 1, wherein solid hydrophilic carrier is selected from cellulose derivatives, polyacrylates, polyethylene glycols, povidones, starch and starch derivatives, gums, sugars, and the mixtures thereof.
5. Stable oil suspension of claim 4, wherein the solid hydrophilic carrier is selected from the group of povidones such as polyvinyl pyrrolidone, polyvinyl acetate ester, and a polyethylene glycol polyvinylacetate copolymer and the mixtures thereof.
6. Stable oil suspension of claim 5, wherein the ratio of lipophilic nutrient to hydrophilic carrier is 1:0.5 to 1:5.

.7. Stable oil suspensions of claim 1, wherein suitable oil is selected from sunflower oil, safflower oil, coconut oil, corn oil, cotton seed oil, canola oil, olive oil, palm oil, peanut oil, sesame oil, soybean oil and the mixtures thereof.
8. Stable oil suspensions of claim 1, wherein at least one more food grade excipient is selected from diluents, antioxidant, surfactant, binders, solvents, and the mixtures thereof.
9. The process for preparation of stable oil suspensions with enhanced bioavailability, which is comprised of,
i) dispersing the lipophilic nutrient in suitable polar or non polar solvent or a mixture of
polar and non-polar solvent
ii) dissolving the solid hydrophilic carrier in a suitable polar solvent to form clear
solution and mixing with at least one more food grade excipient
iii) mixing dispersion of the lipophilic nutrient with solution of solid hydrophilic carrier
and excipient and
iv) subjecting to spray drying to remove solvent to obtain soluble granules of lipophilic
nutrient.
v) suspending the granules of lipophilic nutrients in suitable oil with stirring followed by
milling.
10. Stable oil suspension prepared by process of claim 9, which is comprised of 1 to 50% by
weight of lipophilic nutrient.