NZ622165B2 - Sustained-release lipid pre-concentrate of pharmacologically active substance and pharmaceutical composition comprising the same - Google Patents

Abstract

Disclosed herein is a sustained release lipid pre-concentrate, comprising: a) a sorbitan unsaturated fatty acid ester having a polar head with at least two or more -OH (hydroxyl) groups represented by the formula I, where R1 is OH, R2 is OH or an unsaturated C4-30 alkylester and R3 is an unsaturated C4-30 alkylester; b) a phospholipid; and c) a liquid crystal hardener, free of an ionizable group, having a hydrophobic moiety of 15 to 40 carbon atoms with a triacyl group or a carbon ring structure, wherein the lipid pre-concentrate exists as a liquid phase in the absence of aqueous fluid and forms into a liquid crystal in the presence of aqueous fluid. Also disclosed is a pharmaceutical composition comprising the pre-concentrate as well as a pharmacologically active ingredient. C4-30 alkylester; b) a phospholipid; and c) a liquid crystal hardener, free of an ionizable group, having a hydrophobic moiety of 15 to 40 carbon atoms with a triacyl group or a carbon ring structure, wherein the lipid pre-concentrate exists as a liquid phase in the absence of aqueous fluid and forms into a liquid crystal in the presence of aqueous fluid. Also disclosed is a pharmaceutical composition comprising the pre-concentrate as well as a pharmacologically active ingredient.

Description

Description Title of Invention: SUSTAINED-RELEASE LIPID PRE- CONCENTRATE OF PHARMACOLOGICALLY ACTIVE SUBSTANCE AND PHARMACEUTICAL COMPOSITION COMPRISING THE SAME Technical Field The present invention relates to a sustained release lipid ncentrate of a pharma— cologically active substance and a pharmaceutical composition comprising the same.

Background Art Sustained release formulations are ed to release a single dose of a pharmaco— logically active substance at a predetermined rate in order to maintain the effective plasma concentration of the substance in blood stream for a specific period of time, with zation of the side effects caused by multiple doses.

PLGA [poly(lactic—co—glycolic acid)] is a representative of the currently used biodegradable materials which are approved for use in sustained e by the Food and Drug Administration (FDA). U. S. Patent No. 5,480,656 ed the sustained release of a pharmacologically active nce by way of the degradation of PLGA into lactic acid and glycolic acid over a specific period of time in vivo. However, the acidic ation products of PLGA induce inflammation, decreasing cell growth (K.

Athanasiou, G. G. Niederauer and C. M. l, Biomaterials, 17, 93 (1996)).

For the sustained release, PLGA solid particles of 10 ~ 100 eters in diameter, including a drug therein must be injected. The injection of the PLGA solid les is accompanied by pain or inflammation, because the solid particle of 10 ~ 100 mi— crometers in diameter should be applied through sc or im injection and is degraded over a period of up to several months in injection site. There is therefore a need for a novel sustained release formulation that supplies the effective plasma concentration of a pharmacologically active substance for a prolonged period of time with improved patient compliance. ating in the present invention, ive and thorough research of the present inventors into the sustained release formulation led to the findings that a lipid pre— concentrate comprising a) a sorbitan unsaturated fatty acid ester having a polar head with at least two or more —OH (hydroxyl) groups; b) a olipid; and c) a liquid crystal hardener, free of an ionizable group, having a hydrophobic moiety of 15 to 40 carbon atoms with a triacyl group or a carbon ring structure, exists as a liquid state in the absence of aqueous fluid and transits into a gel—like liquid crystal upon exposure to aqueous fluid, showing an excellent sustained release profile, and that the pre— concentrate is safe to the body and highly biodegradable.

A description is given of the prior arts relevant to the present ion, infra.

International Patent Publication No. WC 2005/ 1 17830 describes a pre—formulation comprising a low viscosity, non—liquid crystalline, mixture of: at least one neutral diacyl lipid and/or at least one tocopherol, at least one phospholipid, and at least one biocompatible, oxygen—containing, low viscosity organic t. International Patent Publication No. discloses pre—formulations of a low viscosity mixture containing at least one diacyl glycerol, at least one phosphatidyl choline, at least one —containing organic t, and at least one somatostatin analogue.

All these pre—formulations release the pharmacologically active materials in vivo for two weeks or longer, but the use of a diacyl lipid, a component essential for the pre— formulations, as a pharmaceutical excipient is not usable and it has to be proven to be sufficiently safe. Another difference with the present invention is that the organic ts used in the publications are found to decrease the ty of some drugs (H.

Ljusberg—Wahre, F. S. Nielse, 298, 328—332 (2005); H. Sah, Y. bahl, Journal of Controlled Release 106, 5 l—6 l (2005)).

U. S. Pat. No. 7,731,947 discloses a composition comprising: a particle formulation comprising an interferon, e, methionine, and a citrate buffer, and a suspending e comprising a solvent such as benzyl benzoate, wherein the particle formulation is dispersed in the suspending vehicle. In one Example, it is described that phos— phatidylcholine is dissolved together with vitamin E (tocopherol) in an organic t and is used to disperse the particle ation therein. However, this ition is different from the transparent and filterable solution formulation of the present invention in that the composition is used to disperse solid particles and does not allow the formation of liquid crystals.

U. S. Pat. No. 7,871,642 discloses a method of preparing a dispersion for delivering a pharmacologically active agent, comprising dispersing a homogeneous mixture of a phospholipid, a polyoxyethylene coemulsifier, triglyceride and ethanol in water, wherein the polyoxyethylene coemulsifier is ed from among polyethoxylated sorbitan fatty acid esters (polysorbate) and polyethoxylated vitamin E tives.

Polyethoxylated sorbitan fatty acid esters and polyethoxylated vitamin E derivatives, derived by conjugating the hydrophilic polymer yethylene to an fatty acid ester and vitamin E, respectively, are quite different in structure from sorbitan fatty acid ester and vitamin E. They are usually used as hilic surfactants utilizing the ty of polyoxyethylene, which is different from the component of the present invention.

U. S. Pat. No. 5,888,533 discloses a flowable composition for forming a solid biodegradable implant in situ within a body, comprising: a lymeric, water— insoluble, biodegradable material; and a biocompatible, organic solvent that at least partially lizes the non—polymeric, water—insoluble material and is miscible or dis— persible in water or body fluids, and capable of diffusing—out or leaching from the composition into body fluid upon placement within a body, whereupon the non— polymeric material coagulates or precipitates to form the solid implant. In this com— position, sterols, cholesteryl esters, fatty acids, fatty acid glycerides, sucrose fatty acid esters, sorbitan fatty acid esters, fatty alcohols, esters of fatty alcohols with fatty acids, anhydrides of fatty acids, phospholipids, n, n alcohols, and mixtures thereof are described as the non—polymeric material, and ethanol is used as the solvent.

However, differences from the present ion reside in that this composition cannot form liquid crystals and is designed to form solid implants by simple coagulation or precipitation of water—insoluble als and that a lot of the c solvent is nec— essarily used.

Disclosure of Invention Technical m It is therefore an object of the present invention to e a lipid pre—concentrate based on a sorbitan unsaturated ester having a polar head with at least two —OH (hydroxyl) groups that has significantly high safety and biodegradability and exists a liquid state advantageous for injection applications of dosage form while forming into a liquid crystal upon exposure to aqueous fluid, thus enhancing the sustained release of a drug in vivo.

It is another object of the present invention to provide a lipid pre—concentrate which can be injected without ing pain or inflammations, problems with tional ations.

It is a further object of the present invention to provide a pharmaceutical composition further comprising a pharmacologically active ingredient plus the pre—concentrate of the present ion.

Solution to Problem In accordance with an aspect f, the present invention provides lipid pre— concentrate for a sustained release, comprising a) a sorbitan unsaturated fatty acid ester having a polar head with at least two or more —OH (hydroxyl) groups; b) a phos— pholipid; and c) a liquid crystal hardener, free of an ionizable group, having a hy— drophobic moiety of 15 to 40 carbon atoms with a l group or a carbon ring structure, wherein said lipid pre—concentrate exists as a liquid phase in the absence of aqueous fluid and forms into a liquid crystal in the presence of aqueous fluid.

The sorbitan unsaturated fatty acid ester having a polar head with two or more – OH (hydroxyl) groups, useful in the present invention, is represented by the following Chemical Formula 1: [Chemical a 1] wherein R1 is OH, R2 is OH or R and R3 is R wherein R is an alkylester of 4 to carbon atoms with one or more unsaturated bonds.

The sorbitan fatty acid ester, which accounts for the formation of a liquid crystal in the present ion, is different from tional counterparts such as oleyl glycerate (OG), yl glycerate (PG), and glycerine monooleate (GMO), glycerine dioleate (GDO, a kind of diacyl glycerol) of the following al Formula 2. That is, the conventional molecules responsible for liquid crystalline phases share the common structure consisting of a polar head derived from glycerine or glyceric acid and a nonpolar tail derived from a lipid alcohol or fatty acid.

[Chemical Formula 2] However, the conventional molecules responsible for liquid crystalline phases are somewhat difficult to apply to the development of medications because of the following disadvantages. Oleyl glycerate (0G) and phytanyl glycerate (PG), although capable of readily form into liquid crystals, are rarely used as pharmaceutical eX— cipients for human medicine because of their relatively high ty. On the other hand, glycerine monooleate is useful as a pharmaceutically acceptable ent, but has weak crystallinity to form liquid crystals necessary for sustained release med— ications. ol dioleate, which is used in International Patent Publication No. WC 2005/ 1 17830 as described supra, is a diacyl lipid with glycerin functioning as a polar head. This molecule is not generally used as a pharmaceutical excipient because its safety has not yet been proven. In addition, it is significantly poor in radability.

As a result of ive and thorough research, the present inventors found that an rated fatty acid esters have advantages over conventionally used liquid crystalline molecules, glycerine or glyceric acid derivatives in that they form liquid crystals very effective for the sustained release of active ingredients, with superiority in safety and radability and are applicable to the development of medical products overcoming the problems encountered in the prior art. For use in com— ons for medicaments, materials must be guaranteed to be safe and biodegradable.

Further, radability is a very important factor for the material which is in charge of sustained release in the body. If the sustained release injection using PLGA is designed to release an active ingredient for one week, it is ideal that the PLGA is degraded in vivo one week after injection. In fact, however, PLGA remains intact for one to several months even after the function of sustained release is finished.

Therefore, the sorbitan unsaturated fatty acid ester of the t ion, which has excellent sustained release property, safety and biodegradability, is applicable for a novel liquid crystal—inducing material with great value in pharmaceutical industry.

The fatty acid of sorbitan unsaturated fatty acid ester of the present invention may be d from ble oil (e. g., palm oil, castor oil, olive oil, peanut oil, sweet oil, corn oil, sesame oil, cottonseed oil, soybean oil, sunflower oil, safflower oil, linseed oil), animal fat and oil (e. g., milk fat, lard, tallow, etc.), whale oil and fish oil. Sorbitan unsaturated fatty acid ester of the present invention may be selected from among sorbitan monoesters, sorbitan sesquiesters, sorbitan diesters and mixtures thereof.

Sorbitan monoester is a sorbitan molecule with one fatty acid group attached thereto via an ester bond and may be selected from among sorbitan monooleate, sorbitan noleate, sorbitan monopalmitoleate, sorbitan monomyristoleate and a e thereof. Sorbitan sesquiester is a sorbitan molecule to which 1.5 fatty acid groups are attached on average via an ester bond. Representative among the sorbitan sesquiester useful in the present invention are sorbitan oleate, sorbitan sesquilinoleate, sorbitan sesquipalmitoleate, sorbitan sesquimyristoleate and a mixture thereof. an diester is a sorbitan molecule with two fatty acid groups attached thereto via an ester bond, and may be selected from an dioleate, sorbitan dilinoleate, sorbitan dipalmitoleate, sorbitan stoleate and a mixture thereof.

Phospholipids are ial for the construction of lamellar structures such as liposomes, but cannot form a non—lamellar phase structure, such as a liquid crystal, by themselves. However, phospholipids can participate in the sorbitan rated fatty acid ester—driven formation of non—lamellar phase structures, serving to stabilize the resulting liquid crystals. The phospholipid useful in the present invention contains a saturated or unsaturated alkyl ester group of 4 to 30 carbon atoms with a polar head.

The phospholipid may be selected from among phosphatidylcholine, phos— phatidylethanolamine, atidylserine, phosphatidylglycerine, phos— phatidylinositol, atidic acid, sphingomyelin, and a mixture f. Phos— pholipids are found in plants and animals such as soybean and eggs. In phospholipids, long fatty acid hydrocarbon chains which account for the hydrophobic tails include saturated fatty acid chains such as mono— and dipalmitoyl, mono— and dimyristoyl, mono— and dilauryl, and mono— and distearyl, unsaturated fatty acid chains such as mono— or dilinoleyl, mono— and dioleyl, mono— and dipalmitoleyl, mono— and stoleyl, and a e thereof.

The liquid l hardener cannot form a non—lamellar structure (liquid crystal) unlike sorbitan unsaturated fatty acid esters, nor a lamellar structure (liposome) unlike phospholipids, by itself. However, the liquid crystal hardener contributes to the WO 32207 sorbitan unsaturated fatty acid ester—driven formation of non—lamellar phase ures by increasing the curvature of the non—lamellar structures to enhance the ordered co— existence of oil and water in nano—scale. In the interests of this on, the liquid crystal hardener is required to have a highly limited polar moiety and a bulky non— polar moiety within the inside of its molecular structure.

In practice, biocompatible molecules which are injectable into the body can be selected as the liquid crystal hardener of the present invention only via experimental ‘trial and error’. As a result, liquid crystal hardeners suitable for the composition of the present invention have molecular structures which are ent from one another and thus cannot be elucidated as one molecular structure. The common structural feature deduced from all of the selected liquid crystal hardeners is that they are free of ionizable groups, such as carboxyl and amine groups, and have hydrophobic moieties of 15 to 40 carbon atoms comprising a bulky carbon ring structure or a triacyl group.

Preferred examples of the liquid crystal hardener of the present invention may be free of ble groups, such as carboxyl and amine groups, and having with at most one ester and —OH xyl) group as a polar head, and having hydrophobic moieties of to 40 carbon atoms comprising a bulky carbon ring structure or a triacyl group.

Preferred es of the liquid crystal hardener of the present invention may include, but are not limited to, triglyceride, retinyl palmitate, tocopheryl acetate, cholesterol, benzyl benzoate and a mixture.

In the composition of the t invention, the weight ratio between components of a) and b) is in a range of from 10:1 to 1:10 and preferably in a range of 5:1 to 1:5. The weight ratio of a) + b) to c) falls within the range of from 100:1 to 1:1 and preferably within the range of from 50:1 to 2: 1. Forming desired liquid crystals, the components in such weight ratios guarantee effective sustained release.

As used herein, the term “aqueous fluid” is intended to include water and body fluid such as a mucosal solution, a tear, sweat, saliva, gastrointestinal fluid, extravascular fluid, extracellular fluid, titial fluid, and plasma. When brought into contact with body surfaces, s or cavities (e.g. inside the body) whose external nments are accounted for by aqueous fluids, the composition of the t invention undergoes transition from a sol—like liquid phase to a gel—like liquid crystalline phase.

That is, the composition of the present ion is a pre—concentrate which exists as a liquid state before application to the human body and shifts into a liquid crystalline phase promising sustained release within the body.

The liquid ls formed by the composition of the present invention have a non— lamellar phase structure in which oil and water are in d mixture and arrangement without discrimination between inner and out . The ordered arrangement of oil and water renders the non—lamellar phase structure of a mesophase, which is a state of matter ediate between liquid and solid. The pre—concentrate of the present invention is different from conventional compositions that form lamellar structures, such as micelles, emulsions, microemulsions, liposomes, and lipid bilayers, which have been widely used in designing pharmaceutical formulations. Such lamellar structures are in oil in water (o/w) or water in oil (w/o) type in which there is clear dis— ation inner and out phases.

The term “liquid crystallization,” as used herein, refers to the formation of liquid crystals having a non—lamellar phase structure from the pre—concentrate upon exposure to aqueous fluid.

The lipid pre—concentrate of the present invention may be prepared at room tem— perature from a composition comprising at least one an unsaturated fatty acid ester having a polar head with at least two or more —OH (hydroxyl) groups, at least one phospholipid, and at least one liquid l hardener, if necessary, by heating or using a homogenizer.

The homogenizer may be a high—pressure homogenizer, an ultrasonic homogenizer, a bead mill homogenizer, etc.

As described above, e the lipid pre—concentrate of the present invention may be a ceutical composition which exists as a liquid phase in the absence of aqueous fluid and forms into liquid crystals in the presence of aqueous fluid in the body, it can be administered using a method selected from among injection, coating, ng, padding, oral administration, and ng. And the pre—concentrate of the present invention may be formulated into various dosage forms including injections, ointments, gels, lotions, capsules, tablets, liquids, suspensions, sprays, inhalers, eye drops, adhesives, and patches.

Particularly, when an ion route is taken, the pre—concentrate of the present invention may be stered by aneous or uscular injection or other injection routes depending on the ties of the pharmacologically active ingredient used.

The pharmacologically active ingredient applicable to the pre—concentrate of the present invention may be selected from among a protein, a peptide, a vaccine, a gene, a non—peptidic hormone, a synthetic chemical, and a combination thereof.

Examples of the protein or peptide as a pharmacologically active ingredient in the composition of the present invention include erythropoietin, growth hormones (human, pig, cow, etc.), growth hormone releasing factors, nerve growth factors, G—CSF, GM— CSF, M—CSF, blood coagulation factors, n, oxytocin, vasopressin, adrenocorti— cotropic hormone, epidermal growth factor, platelet—derived growth factor, prolactin, somatostatin, glucagon, eukin—2 (IL—2), interleukin—ll (IL—1 1), gastrin, tetragastrin, pentagastrin, urogastron, secretin, calcitonin, enkephalin, endorphin, an— WO 32207 giotensin, thyroid stimulating hormone—releasing hormone, tumor necrosis factor, tumor necrosis factor—related apoptosis inducing ligand, heparinase, bone morphogenic protein, hANP, glucagon—like e, rennin, bradykinin, bacitracin, polymyxin, in, tyrocidin, gramicidin, cyclosporine, polyethylene glycol—conjugated proteins and their synthetic analogs, monoclonal antibodies, enzymes, cytokines and a com— bination, but not limited thereto.

The non—peptidic hormones are a class of hormones which are not proteins or peptides and may be selected from among, but not limited to, terone, estradiol, progesterone, glandin, ride, dutasteride, tic analogs thereof, and combinations thereof.

Examples of the gene entrapped within the ncentrate of the present invention include plasmid DNA, siRNA, polynucleotides, oligodeoxynucleotides, anti—sense oligonucleotides, and a mixture thereof, but are not limited thereto.

The synthetic chemical may be selected from among tacrolimus, anatrozole, olanzapine, aripiprazole, risperidone, yprogesterone, naltrexone, methotrexate, pinitol, olopatadine, latanoprost, anecortave, relin pamoate, minoxidil, tibolone, solifenacin, tadalafil, varenicline, ropinirole, fentanyl, ketotifen, montelukast and a combination thereof, but are not limited thereto.

Accordingly, in accordance with another aspect thereof, the present invention provides a pharmaceutical composition comprising d) a pharmacologically active in— gredient selected from among proteins, peptides, vaccines, genes, non—peptidic hormones, synthetic chemicals, and a combination thereof, in addition to the lipid pre— concentrate of the present invention.

Descriptions about the ingredients a) to c) and the liquid crystal used in the pharma— ceutical composition may refer to those given with regards to the lipid pre—concentrate.

In addition, the description of the pharmacologically active ingredient d) of the phar— maceutical composition may be the same as that given with respect to the lipid pre— concentrate.

The pharmaceutical composition may preferably be formulated as an injection, an ointment, a gel, a lotion, a capsule, a tablet, a liquid, a suspension, a spray, an inhaler, an eye drop, an adhesive, and a patch, but not limited thereto. More ably, it may be formulated as an injection.

The content of the pharmacologically active ingredient in the ceutical com— position of the t invention varies ing on the kind thereof and the for— on to be used, and is generally within the range of from 0.0001 to 90 weight % based on the total weight of the pharmaceutical composition.

The pharmaceutical composition of the t invention may be prepared by adding a cologically active ingredient to the pre—concentrate of the t invention. If necessary, heat or a homogenizer may be used in the preparation of the ceutical composition of the present invention, but this is not a limiting factor to the present invention.

The dose of the pharmaceutical composition of the present invention s to the nown dose of the pharmacologically active ingredient ed and may vary depending on s factors including the patient’s condition, age and sex. It may be administered orally or parenterally.

In accordance with a further aspect thereof, the present invention contemplates a method of maintaining ceutical cy through the sustained release of a phar— macologically active ingredient by administering the pharmaceutical composition of the present invention to a mammal including a human, and the use of the pharma— ceutical composition for the sustained release of a pharmacologically active ingredient.

Advantageous Effects of Invention As described hitherto, the lipid pre—concentrate of the present invention, based on a sorbitan unsaturated fatty acid ester, is highly safe and biodegradable and exists as a liquid phase in the absence of aqueous fluid but rapidly changes into liquid crystals upon exposure to aqueous fluid within the body. When formulated with a pharmaco— logically active ingredient, therefore, the pre—concentrate in a liquid phase improves t compliance and ts excellent sustained release without side s such as pain and inflammation, compared to conventional sustained e formulations in solid particle phases.

Brief Description of Drawings The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in con— junction with the accompanying drawings, in which: shows in vivo biodegradability of the compositions of es 4 and 5 and Comparative Examples 1 to 3. shows in vitro drug release behaviors of the composition of Example 14; is a pharmacokinetic e showing the in vivo drug release behavior of the compositions of Example 16 and Comparative Example 5; shows phase changes of the compositions of Examples 4 and Comparative Example 4 upon exposure to aqueous fluid; and shows the liquid crystalline structures of the composition of Example 4 in Cryo TEM microphotographs.

Mode for the ion The following non—limiting es serve to illustrate selected embodiments of the invention. It will be appreciated that variations in proportions and alternatives in elements of the components shown will be apparent to those skilled in the art and are within the scope of embodiments of the present invention.

The additives and excipients used in the present ion satisfied the requirements of the Korean copoeia and were purchased from h, Lipoid, and Croda.

F—lf—lf—lf—l NON WWI—*0 I—JI—JI—JI—J EXAMPLES 1 TO ll: Preparation of Lipid Pre—Concentrates an unsaturated fatty acid esters having a polar head with at least two —OH groups, phospholipids and liquid crystal hardeners were mixed at the weight ratios shown in Table 1 below, optionally in a solvent. In Examples 1 to 4, the ingredients were mixed in a water bath maintained at 25~45°C using a homogenizer (PowerGen mode1125 . Fisher) for about 10 min at 3,000 rpm. The ingredients of Examples 5 and 6 were stirred for 3 hours in a water bath maintained at 30~50°C. In Examples 7 to ll, the ingredients were mixed in a water bath maintained at 45~75°C using a ho— mogenizer (PowerGen mode1125 . Fisher) for about 20 min at 3,000 rpm. Thereafter, the resulting lipid solutions were left at room temperature to make a thermal equilibrium at 25°C before being loaded into 1 cc disposable syringes. Lipid pre— trates afforded by the above method are injected into water (2 g of distilled water) and formed into a liquid crystal phase.

[TABLE I] . Example No. (Unit: mg) ‘ Ingredient . a LL ‘ L7 l . l 2 3 4 5 ‘ i 6 7 ‘ 8 9 10 i ll ’ setbitéhfiahéfiéééé _- 210; ’ 7 50 60 60 ; 4065 l ‘ ‘ 7 I Sorbitan sesquioleate ; ‘g 40 50 6O 6O 65 'Efioggfi'a’t’ia’giabiine 55 j 35 48 55 30 .

If“ 472.5]; " 775i Phosphatidylethanolaminc L42 (5‘ 25 Phosphatidylserine , 32 . 5‘ ; €32 . 5 l Triglycéfiae 3 ; 5 7.5 : 5 7.5; ‘ { W 7 Retinyi- palmitate 7 E; 7 .5 1 ‘ if 7 ”7 limgczfii'tg .1 5 i 10 l 1 ‘ I m I 7‘ Benzy; benzoate 7 i i WV Ch0:_es:erol i "‘5’""“"""“"T'"‘ ”""""i"”"""54§ ‘kmviwiiiii V V 5 5 T "Tram I; wa'Ler phase .LCILC L6 L6 L6 LC‘ Lc* L2? LC* 1 Lc* Lc* ‘ ; *LC: liquid crystal‘ EXAMPLES 12 TO 21: Preparation of Pharmaceutical Compositions Containing Pharmacologically active Ingredients Sorbitan unsaturated fatty acid esters having a polar head with at least two —OH groups, olipids and liquid crystal hardeners were mixed at the weight ratios shown in Table 2 below.

In Examples 12 to 15, the ingredients were mixed in a water bath maintained at ~60°C using a homogenizer (PowerGen modell25. ) for about 10 min at 3,000 rpm. In Examples 16 to 21, the ingredients were mixed in a water bath maintained at ~50°C using a homogenizer (PowerGen modell25. Fisher) for about 5 min at 3,000 rpm. The resulting lipid solutions were left at room temperature to make a thermal equilibrium at 25°C, followed by adding pharmacologically active ingredients thereto.

As the pharmacologically active ingredients, the gene drugs siRNA (Bioneer) and fluo— rescence—conjugated siRNA (InVitrogen, Block—iT Fluorescent oligo), the peptide drug exenatide (Teva), and the synthetic drug tamsulosin (Lekpharmaceuticals) were used.

Subsequently, the ingredients were homogenized using a homogenizer at 3,000 rpm for about 5 min to afford a pharmaceutical composition in a solution phase. In the case of the gene drugs , fluorescence—conjugated siRNA), they were mixed in the amounts shown in Table 2, together with a solution of chitosan in led water, to form xes before application to the lipid solutions.

[TABLE 2] 2012/006855 Example No. (Unit: mg) ient 1 g 12 13 14 15 16 1/. g he.“ ,__ mart i 3 0.02 0.02 siRNA/Chitosan E z : /0.4 ; /o.4 1 l , , 111111 7 11111 Fluorescence— Conjugated ‘ siRNA/Chitosan rfiXenatide 0.13 0.13 , ",7" i—r Sorbitan 4; K0 4:. 0 p p monooleate fiSorbitan 1 59 ; . 59 54 sesqu1oleate. ; Phosphatidyl— l 46 46 46 choline Phosphatidyl— l 36 36 36 ethanolamlne. ‘ i Tocopheryl acetate g fienzyl benioate 1 5 1 10 _ ‘ Exanple No. (unitt mg) Ingredient ,fima yyyyyyy447444444, 1 18 19 20 21 Dutasteride 0.5 o 5 Wiéfisulosin 0.2 0.2 Sorbitan monooleate 49 45 Sorbitan sesquioLeate 59 35 Phosphatidylcho;ine 46 40W Phoaphatidylethanolamine“ 36 3 Tocopheryl acetate 5 l5 ‘ Retinylipalmitaterfl 5 COMPARATIVE EXAMPLES 1 TO 4 In Comparative Examples 1 t0 3, dioleyl ide, a Class of diacyl glycerides, was used in the amounts shown in Table 3, together with phosphatidylcholine, erol and/0r l, followed by homogenization for about 10 min at 3,000 rpm in a ho— m0genizer (PowerGen mode1125 . Fisher).

In Comparative Example 4, polyoxyethylene sorbitan monooleate, phos— phatidylcholine and tocopheryl acetate were used in the amounts shown in Table 3, followed by homogenization for about 30 min for 3,000 rpm in a homogenizer. Here, polyoxyethylene an monooleate has a polyoxyethylene group substituted for an — OH group on the sorbitan polar head and is different from sorbitan monooleate, used in the present ion. Polyoxyethylene an monooleate is generally used as a hy— drophilic surfactant or emulsifier due to the bulky polyoxyethylene moiety.

[TABLE 3] _ Comparative Example No. (unit: mg) ; Ingredient 1 l 2 l 3 4 GlyceinWdioleate 65 55W ”:“WSWXBV —W Polyoxyethylene sorbitan _ _ ’ _ ‘ 50 monooleatc l 1 WWTOCOpherOl WWAZAW - 7.5 — l Tocopheryl acetate l i —- — — 10 Phosphatidylcholine 35 35 W7§0VWWWWW§0WWW VVWWEthanol ““91 10 t io — COMPARATIVE EXAMPLE 5 To 1 mL of physiological saline was added 20 ug of exenatide, followed by homoge— nization at room temperature. f—lf—lf—l 000000 WNH 7—1 00 J;I—JI—JI—JI—J EXPERIMENTAL EXAMPLE 1: Comparison of In Vitro Safety The safety of the itions of the present ion was ed in vitro by executing an extraction colony assay cytotoxicity test as follows. In 18 mL of Eagle’s Minimal ial Media (EMEM) supplemented with 10% fetal bovine serum, 2 g of each of the itions of Examples 1, 4 and Comparative Examples 1 and 2 was extracted. L929 cells (mouse fibroblast, an Type Culture Collection) were seeded at a density of 1x102 cells/well into 6—well plates and incubated for 24 hours at 37°C in 5% CO2 humidified incubator. The extracts were diluted in EMEM (0, 5, 25, 50%) and then placed in an amount of 2 mL/well in contact with the stabilized L929 cells. After incubation for 7 days at 37°C in a 5% CO2 humidified incubator, the cells were fixed with a 10% formalin solution and stained with a Giemsa solution to count colonies. The results are ized in Table 4, below.

[TABLE 4] Extract Relative colony formation rates(%)* Medium(V/V)%** 3x. 1 3x. 4 c. Ex. 1 c. 3x. 2 0 % Medium 1 100.0 100.0 100.0 100.0 o ) % Medium 100.0 96.6 71.4 72.2 % Medium 66.7 72.4 23.8 27.87" "5704 % Médifimfi 4 7 11.1W 7 17:27 WWW-'6‘TOWW 7 7 * Relative colony formation rates (%) = No. of Colonies on Test Medium / No. of Colonies on 0% Medium x 100 (%) ** Extract Medium % 2 Extract Medium /(Diluted Medium + t Medium) x 100(%) As can be seen in Table 4, the groups administered with the compositions of Examples 1 and 4 showed significantly high cell growth rates on all diluted mediums (5%, 25% and 50%), compared to those administered with the compositions of Com— parative Examples 1 and 2, indicating that the itions (lipid pre—concentrates) of the present invention are far safer than the conventional compositions (disclosed in In— ternational Patent Publication No. ).

MENTAL EXAMPLE 2: Comparison of In Vivo Biodegradability The compositions of the present invention were evaluated for in vivo biodegradability in the following experiments. Each of the compositions of Examples 4 and 5 was subcutaneously injected at a dose of 400 mg into the back of SD rats and monitored for a predetermined period of time. For comparison, the compositions of Comparative Examples 1 to 3 were tested in the same manner. The injection sites were raphed two weeks after injection and are shown in As can be seen in the compositions of Examples 4 and 5 were observed to be mostly biodegraded almost without producing a feeling of irritation whereas the com— positions of Comparative Examples 1 to 3 remained one to two third their original volume.

Therefore, the compositions of Examples 4 and 5 exhibited significantly high biodegradability, compared to the compositions of Comparative es 1 to 3 national Patent Publication No. WO 2005/ 1 17830).

For reference, the conventional material PLGA [poly(lactic—co—glycolic acid)], which has been widely used for sustained release, is known to remain undegraded in vivo even after two or three months. ingly, the lipid pre—concentrates of the present invention overcome the problem that even after it releases drugs completely, the conventional carrier system s within the body due to its low biodegradability.

MENTAL EXAMPLE 3: In Vitro Test for ned Release Drug release behaviors from the compositions of the present ion were examined in vitro in the following test. Prostate cancer cells (Prostate cancer—3, the Korean Cell Line Bank) were seeded at a density of 5x104 cells/well into transwell plates and incubated for 2 days at 37°C in a 5 % CO2 humidified incubator. The com— position of Example 14 was added in an amount of 100 mg to a transwell insert containing 3 mL of RPMI 1640 supplemented with 10% fetal bovine serum. Fluo— rescence emitted from the composition of Example 14 was measured using a fluo— rescence microscope (Eclipse Ti—S, Nikon) while the insert was applied every 24 hours for seven days to the transwell plates. The results are shown in The left photographs of were taken using differential interference contrast (DIC) microscopy while the right photographs show the intracellular uptake of fluo— rescence—conjugated siRNA. As is understood from the data of the composition of the present invention constantly released the pharmacologically active ingredient for at least 7 days.

MENTAL EXAMPLE 4: In Vivo Test for Sustained Release Drug release behaviors from the itions of the present invention were examined in vivo in the following test. The composition of Example 16 was subcu— taneously injected into 6 SD rats (male), 9 weeks old, with an average body weight of 300 g, at such a dose as to correspond to 140 ug/kg of exenatide. ide concentrations in plasma samples taken from the SD rats were monitored for 14 days using a commercially available kit (immunoassay kit, Bachem) to draw a PK profile (pharmacokinetic e) as shown in For comparison, the com— position of Comparative Example 5 was administered at a dose corresponding to 10 ug/kg of ide (herein, the reason why the dose of ide of Example 16 was 14 times as large as that of Comparative Example 5, is that the ek dose (7 days) of the sustained release formulation corresponds to 14 times as large a dose as the general injection because of the use of twice a day).

As shown in the composition of Example 16 increased the in vivo half—life of the biologically active ingredient by about 25 fold, compared to the ition of Comparative Example 1, which is a general injection, proving its excellent sustained release effect (in means of measurements taken of 6 rats are d).

EXPERIMENTAL EXAMPLE 5: In Vivo Test for Pharmacological Effect The pharmacological effect of the composition of the present ion was evaluated in the following test. The ition of Example 16 ning exenatide (anti—diabetic), which can induce a weight loss, was subcutaneously injected into 6 SD rats (male), 9 weeks old, with an average body weight of 300 g, at such a dose as to correspond to 140 ug/kg of exenatide. Average weights were calculated on day 0 and 14 and the results are given in Table 5, below.

[TABLE 5] V‘li’lljggample 16 (g) logical Sallriefi (If) ; ha"; 0 303 308 ]""" Day 14 356 379 ‘ Wt ChaEJETé; ) 4. 75 100 * Wei ght Change % = weight Change of grouP stered with the comPosition of e 16 (g) / weight change of group administered with physiological saline (g) x 100) As shown in Table 5, the group administered with the composition of Example 16 experienced about 25% weight loss for two weeks, compared to the weight of the group administered with physiological saline. Therefore, the sustained release com— position of the present invention ensures long—lasting pharmacological efficacy in vivo as well as significantly increased half—life of the biologically active ingredient through in vivo test for ned release (EXPERIMENTAL EXAMPLE 4).

EXPERIMENTAL EXAMPLE 6: Formation of Liquid Crystal in Aqueous Fluid The composition of the present ion was evaluated for ability to form liquid crystal in an aqueous phase in the following test. After being loaded into syringes, itions of Example 4 and ative Example 4 were dropped into 2 g of PBS (pH 7.4) and the results are shown in The composition of Example 4 based on the sorbitan unsaturated fatty acid ester having a polar head with at least two —OH (hydroxyl) groups (sorbitan monooleate) existed as a liquid phase in the absence of aqueous fluid, but formed liquid crystals upon exposure to aqueous fluid. On the other hand, the composition of Comparative Example 4 based on polyoxyethylene sorbitan unsaturated fatty acid ester (polyoxyethylene sorbitan monooleate) existed as a liquid phase and dispersed in PBS, but did not forms into a liquid crystal even after exposure to aqueous fluid. Con— sequently, only the composition of the present invention rapidly forms into liquid crystals contributing to sustained release effect in the presence of aqueous fluid, such as in the environment within the body.

Within the liquid crystals, there are a great number of bicontinuous water channels of nano size (below 20 nm) that resemble the Moebius strip. The water ls are surrounded with bicontinuous lipid layers. Thus, once a lipid composition forms into a liquid crystal in a semi—solid phase, a cologically active substance can be ed from the liquid crystal structure only after it has passed through numerous water channels and lipid layers, which enhances sustained release effect of a pharma— cologically active substance. Therefore, the composition of the present invention can be d to sustained release drug formulations.

EXPERIMENTAL EXAMPLE 7: Determination of Inner Structure of Liquid Crystal Using Cryo TEM Inner Structure of the liquid crystals of the composition of the present invention were examined in the following experiment. The composition of Example 4 in a liquid phase was dropped to 2 g of water to produce a liquid crystalline structure. Using a ho— zer, the liquid crystals in the aqueous phase were sufficiently sed and maintained in an equilibrium state at room temperature until analysis. The diluted liquid crystals were ed onto a grid and , followed by examining the structure in a cryo Transmission Electron Microscope (Cryo TecaiF20G2, FEI). The results are shown in As shown in photographs of the liquid crystals were observed to have lline ures such as cubic phases or hexagonal phases. As a rule, lamellar structures, such as micelles, emulsions, microemulsions, liposomes, etc., typically exist in complete cal states, whereas non—lamellar structures according to the com— position of the present invention assume ordered forms with certain angles, which are quite different from sphere forms.

Although the invention has been illustrated and described with respect to one or more implementations, equivalent alterations and modifications will occur to others skilled in the art upon the reading and understanding of this specification and the d drawings. In addition, while a particular e of the invention may have been disclosed with t to only one of several implementations, such feature may be combined with one or more other features of the other implementations as may be desired and advantageous for any given or particular application.

Claims (1)

1. CLAIMS 【Claim 1】 A sustained e lipid pre-concentrate, comprising: a) a sorbitan unsaturated fatty acid ester having two or more –OH (hydroxyl) groups on a polar head and having the structure of Formula I: wherein: R1 is OH; R2 is OH or an alkylester of 4 to 30 carbon atoms with one or more unsaturated bonds; and R3 is an alkylester of 4 to 30 carbon atoms with one or more unsaturated bonds; b) a phospholipid; and c) a liquid crystal hardener, free of an ionizable group, having a hydrophobic moiety of 15 to 40 carbon atoms with a triacyl group or a carbon ring structure, wherein the lipid pre-concentrate exists as a liquid state in the absence of an aqueous fluid and transforms from the liquid state into a liquid crystal of a gel state in the presence of an aqueous fluid. 【Claim 2】 The sustained release lipid pre-concentrate of claim 1, wherein the sorbitan unsaturated fatty acid ester is selected from the group consisting of sorbitan eate, sorbitan noleate, an monopalmitoleate, sorbitan monomyristoleate, sorbitan sesquioleate, sorbitan sesquilinoleate, sorbitan sesquipalmitoleate, sorbitan sesquimyristoleate, sorbitan dioleate, sorbitan dilinoleate, sorbitan itoleate, sorbitan dimyristoleate and a e thereof. 【Claim 3】 The sustained release lipid pre-concentrate of claim 1, wherein the sorbitan unsaturated fatty acid ester is selected from the group consisting of an monooleate, an monolinoleate, sorbitan monopalmitoleate, sorbitan monomyristoleate and a mixture thereof. 【Claim 4】 The sustained release lipid pre-concentrate of any one of claims 1 to 3, wherein the phospholipid contains a ted or unsaturated alkyl ester group of 4 to 30 carbon atoms and is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerine, atidylinositol, phosphatidic acid, sphingomyelin and a mixture thereof. 【Claim 5】 The sustained release lipid pre-concentrate of any one of claims 1 to 4, wherein the liquid crystal hardener is selected from the group consisting of triglyceride, retinyl palmitate, tocopheryl acetate, cholesterol, benzyl benzoate and a mixture thereof. 【Claim 6】 The ned release lipid pre-concentrate of any one of claims 1 to 4, wherein the liquid crystal hardener is selected from the group consisting of ceride, retinyl palmitate, tocopheryl acetate, terol and a mixture thereof. 【Claim 7】 The sustained release lipid pre-concentrate of any one of claims 1 to 4, wherein the liquid crystal hardener is tocopheryl e. 【Claim 8】 The sustained release lipid pre-concentrate of any one of claims 1 to 7, wherein the weight ratio of component of a) to component b) is 10:1 to 1:10. 【Claim 9】 The sustained release lipid pre-concentrate of any one of claims 1 to 7, wherein a weight ratio of a sum of the components of a) and b) to the component of c) is 100:1 to 1:1. 【Claim 10】 A pharmaceutical composition comprising d) a pharmacologically active ingredient selected from the group consisting of a n, a peptide, a vaccine, a gene, a non-peptidic hormone, a synthetic chemical drug, and a combination f, plus the sustained release lipid pre-concentrate of any one of claims 1 to 7. 【Claim 11】 The pharmaceutical composition of claim 10, wherein a weight ratio of the component of a) to the component of b) is 10:1 to 1:10. 【Claim 12】 The pharmaceutical composition of claim 10, wherein a weight ratio of a sum of the components of a) and b) to the component of c) is 100:1 to 1:1. 【Claim 13】 The sustained release lipid pre-concentrate of any one of claims 1 to 7 or the pharmaceutical ition of claim 10, being in a ation, said ation being selected from the group consisting of an injection, an ointment, a gel, a lotion, a capsule, a tablet, a liquid, a suspension, a spray, an inhaler, an eye drop, an adhesive, and a patch. Chong Kun Dang Pharmaceutical Corp. By the patent attorneys for the applicant CULLENS