(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2009/0324727 A1
`Foguet Roca
`(43) Pub. Date:
`Dec. 31, 2009
`
`US 20090324727A1
`
`(54) NANOEMULSION
`
`Publication Classification
`
`(75) Inventor:
`
`Montserrat Foguet Roca,
`Leverkusen (DE)
`
`Correspondence Address:
`SUTHERLAND ASBLL & BRENNAN LLP
`999 PEACHTREE STREET, N.E.
`ATLANTA, GA 30309 (US)
`
`(73) Assignee:
`
`BOFRONTERA BIOSCIENCE
`GMBH, Leverkusen (DE)
`
`(21) Appl. No.:
`
`12/520,759
`
`(22) PCT Filed:
`
`Dec. 21, 2007
`
`(86). PCT No.:
`
`PCT/EP07/11404
`
`S371 (c)(1),
`(2), (4) Date:
`
`Jun. 22, 2009
`
`(30)
`
`Foreign Application Priority Data
`
`Dec. 22, 2006 (EP) .................................. O6O26698.8
`
`(2006.01)
`(51) Eiki/4
`(2006.01)
`A6II 47/10
`(2006.01)
`A 6LX 3L/95
`(2006.01)
`A6IP 7/02
`(52) U.S. Cl. .......................... 424/489: 514/772; 514/561
`(57)
`ABSTRACT
`The present invention relates to a nanoemulsion comprising
`at least one aqueous component and a carrier, wherein the
`carrier comprises at least one lipophilic component, at least
`one surfactant and at least one alcohol, characterised in that at
`least one alcohol has at least three carbon atoms. The present
`invention further relates to a composition comprising said
`nanoemulsion and an active agent. In particular, the compo
`sition is present as a gel and the active agent is 5-aminole
`Vulinic acid, a derivative, precursor and/or metabolite thereof.
`The invention further relates to the preparation of said
`nanoemulsion and/or composition and to their use for the
`treatment of dermatological diseases, virus-associated dis
`eases as well as diseases associated with cell proliferation, in
`particular, tumor diseases and/or psoriasis. The present inven
`tion is further directed to the use of said nanoemulsion in
`cosmetics.
`
`

`

`Patent Application Publication
`
`Dec. 31, 2009 Sheet 1 of 6
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`US 2009/0324727 A1
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`Fig. 1
`
`GAUSSIAN SUMMARY:
`Mear. Olafest
`32,2 nin
`Sind, Deviation
`a 78.8 nm (58.3%)
`Norn. Sfind. Oew, so,583
`(Coeff, of Warr)
`
`as 0.340
`Variance (Pl.)
`3,022
`C Squared
`0.000 %
`Baseline Adi
`Z-Avg. Diff. Coeff. E .44E-007 cm2/s
`
`REL
`
`NTENS-WT GASSIAN DISTRIBUTION
`
`
`
`100
`
`

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`Patent Application Publication
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`Dec. 31,
`2009
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`Sheet 2 of 6
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`US 2009/0324727 A1
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`Patent Application Publication
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`Dec. 31, 2009 Sheet 3 of 6
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`US 2009/0324727 A1
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`2s inn
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`Patent Application Publication
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`Dec. 31, 2009 Sheet 4 of 6
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`US 2009/0324727 A1
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`42
`
`4
`
`3,8
`
`
`
`:
`O% BF200
`
`0,5% BF200
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`196 BF200
`
`3% BF200
`
`5% BF200
`
`

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`Patent Application Publication
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`Dec. 31, 2009 Sheet 5 of 6
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`US 2009/0324727 A1
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`
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`50000: 1
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`DO122O1CE1
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`

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`Patent Application Publication
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`Dec. 31, 2009 Sheet 6 of 6
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`US 2009/0324727 A1
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`

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`US 2009/0324727 A1
`
`Dec. 31, 2009
`
`NANOEMULSION
`
`0001. The present invention relates to a nanoemulsion
`comprising at least one aqueous component and a carrier,
`wherein the carrier comprises at least one lipophilic compo
`nent, at least one surfactant and at least one alcohol. The
`present invention further relates to a composition comprising
`said nanoemulsion and an active agent. In particular, the
`composition is present as a gel and the active agent is 5-ami
`nolevulinic acid (ALA), a derivative, precursor and/or
`metabolite thereof. The invention further relates to the prepa
`ration of said nanoemulsion and/or composition and to their
`use for the treatment of dermatological diseases, virus-asso
`ciated diseases as well as diseases associated with cell pro
`liferation, in particular, tumor diseases and/or psoriasis. The
`present invention is further directed to the use of said
`nanoemulsion in cosmetics.
`0002 Nanoemulsions constitute a colloidal system. Col
`loidal systems include micelles, liposomes, Virosomes, nano
`Suspensions, microemulsions and polymer Solutions.
`Nanoemulsions, based on their physical and chemical char
`acteristics, belong to the group of the microemulsions. Micro
`emulsions are aqueous dispersions of homogeneous, micro
`sized particles composed of a lipid core surrounded by
`Surfactant and co-surfactant monolayers. Nanoemulsions are
`characterized by a mean particle size (mean diameter) of less
`than 200 nm, often less than 100 nm, and a narrow monodis
`perse particle size distribution. Further, nanoemulsions are
`transparent and slightly opalescent. They are generally manu
`factured by mechanical fragmentation of an oily phase in an
`aqueous phase in the presence of a surfactant. The very Small
`size of the oily globules is often obtained by virtue of at least
`one pass through a high-pressure homogenizer or a Sonicator.
`The preparation of the nanoemulsions described here does
`not require Such high shear devices. The Small size of the
`globules and their high homogeneity confers on them cos
`metically advantageous properties which distinguish them
`from conventional emulsions: They are transparent and
`exhibit a novel texture. Further, they can carry active agents
`more efficiently and, thus, become increasingly important in
`the field of medicine and pharmacy.
`0003 Microemulsions, called nanodispersions, are
`known in the art which comprise a) a membrane-forming
`molecule, e.g. Soy lecithin, b) a co-emulgator, c) a lipophilic
`component, e.g. caprylic and/or capric triglyceride (Miglyol
`812 or Myritol 318) and, optionally, d) alcohol, in particular,
`ethanol (EP 0956 853). These nanodispersions are used in
`pharmaceutical formulations as transport vehicle for pharma
`ceutically active agents.
`0004. However, the use of ethanol in nanoemulsions suf
`fers from several disadvantages.
`0005 First, the use of ethanol as co-surfactant leads to
`larger nanoparticles than the use of alcohols with larger car
`bon chains. Larger particles cause a decrease of the contact
`Surface between skin and nanoemulsion leading to a decrease
`in the penetration rate. Second, ethanol has a relatively low
`viscosity of 1.10 cp which is not optimal for the stability of
`microemulsions. Further, due to the relatively low hydropho
`bicity of ethanol, the penetration of the nanoemulsions
`through the lipophilic physiological membranes of the skin
`may be impaired. As a result, the stability of the nanoemul
`sion as well as its bioavailability, i.e. penetration into tissues,
`is reduced. Further, ethanol is a very expensive alcohol com
`
`pared to other kinds of alcohol Such as isopropylalcohol. In
`addition, ethanol is liable to a special alcohol tax under cer
`tain conditions in several countries.
`0006. Therefore, it was an object of the present invention
`to provide a nanoemulsion which overcomes the disadvan
`tages of those mentioned in the prior art and which, in par
`ticular, exhibits optimal physical properties which may
`improve the stability as well as penetration into cells and
`tissues, while at the same time being less expensive than the
`nanoemulsions of the state of the art.
`0007. This object is achieved according to the present
`invention by providing a nanoemulsion comprisinga) at least
`one aqueous component and b) a carrier, which comprises i)
`at least one lipophilic component, ii) at least one surfactant
`and iii) at least one alcohol, wherein the at least one alcohol
`has at least three carbon atoms.
`0008. The inventors have now discovered that a
`nanoemulsion according to the invention has an optimally
`lower mean particle size with a narrow particle-size distribu
`tion. The reduction of the particle size contributes to an
`improved stability and a better penetration into cells and
`tissues of the nanoemulsion.
`0009. The amount of component i) of the carrier, i.e. the
`lipophilic component, is preferably present in an amount of
`from 0.1% by weight to 15% by weight, more preferably from
`1% by weight to 8% by weight and most preferably from 3%
`by weight to 4% by weight, based on the total weight of the
`nanoemulsion. The amount of component ii) of the carrier,
`i.e. the surfactant or surfactants, is preferably present in an
`amount of from 1% by weight to 30% by weight, more pref
`erably from 2% by weight to 15% by weight and most pref
`erably from 4% by weight to 6% by weight, based on the total
`weight of the nanoemulsion. The amount of component iii) of
`the carrier, i.e. the alcohol, is preferably present in an amount
`of from 0.1% by weight to 10% by weight, more preferably
`from 0.5% by weight to 5% by weight and most preferably
`from 1% by weight to 2% by weight, based on the total weight
`of the nanoemulsion.
`0010. The aqueous component is preferably present in the
`nanoemulsion in an amount of from 50% by weight to 98% by
`weight, more preferably from 70% by weight to 95% by
`weight and most preferably from 88% by weight to 92% by
`weight, based on the total weight of the nanoemulsion.
`Advantageously, this specific constitution of the nanoemul
`sion leads to a nanoemulsion which is highly tolerable to the
`skin and does not result in a sticky feel during application to
`the skin, which, interalia, is due to the relatively low propor
`tion of Surfactants and lipophilic components.
`0011. In a preferred embodiment of the present invention,
`the nanoemulsion comprises as Surfactant a membrane-form
`ing Surfactant and an O/W-emulsion-forming co-surfactant.
`The weight ratio of the amount of membrane-forming Surfac
`tant: O/W-emulsion-forming co-surfactant ranges from 0.1:1
`to 10:1, preferably from 0.2:1 to 0.8:1, more preferably from
`0.4:1 to 0.6:1.
`0012 A preferred alcohol having at least 3 carbon atoms
`within the scope of the present invention is an alcohol having
`3-10 carbon atoms, preferably 3-7 carbon atoms, more pref
`erably 3-5 carbonatoms, and most preferably 3 carbonatoms.
`Particularly suitable alcohols having 5 carbon atoms are
`1-pentanol and/or 4-methyl-2-pentanol. Suitable alcohols
`having 4 carbon atoms are 1-butylalcohol, tert-butylalcohol
`(2-methyl-2-propanol) and/or sec-butylalcohol (2-butanol).
`Most preferred are alcohols having 3 carbon atoms, namely
`
`

`

`US 2009/0324727 A1
`
`Dec. 31, 2009
`
`1-propylalcohol and isopropylalcohol, wherein isopropylal
`cohol is preferred. By using a C-alcohol, in particular, iso
`propylalcohol in the inventive nanoemulsion, the present
`inventors have discovered that the nanoemulsion shows a
`reduced particle size and narrower particle-size distribution
`as well as improved Stability and an enhanced penetration into
`tissues compared to the nanoemulsions of the state of the art
`using ethanol as alcohol. This was Surprising, as isopropanol
`and ethanol are very similar in their physiological and chemi
`cal features.
`0013 When isopropanol is used as alcohol in the inventive
`nanoemulsion, the size of the emulsified particles is Smaller
`compared to the use of ethanol. Due to the resulting increased
`contact surface of the nanoemulsion with the skin, the pen
`etration properties are increased. The reduction of the particle
`size is also decisive for an improved stability of the
`nanoemulsion since it is known that the rate of particle agglu
`tination ultimately leading to phase separation increases with
`the particle size. The advantageous effects of isopropanol
`may be due to its slightly higher hydrophobicity, which may
`result in a better ability to penetrate through the lipophilic
`physiological membranes. Further, isopropanol has a higher
`viscosity (cp at 25°C. of 2.32) compared to ethanol (viscos
`ity/cp, 25°C., 1.10). Said higher viscosity ofisopropanol may
`serve for a better stabilization mechanism in nanoemulsions,
`as viscosity reduces the mobility of molecules. A further
`advantage of isopropanol compared to ethanol is that it is
`available at relatively low costs, which is about/3 of the costs
`of ethanol, and free of an additional alcohol tax.
`0014 Preferably, the at least one lipophilic component of
`the carrier of the inventive nanoemulsion is a lipid, a Veg
`etable oil and/oran animal oil. Suitable lipids according to the
`present invention are physiologically acceptable lipids Such
`as ceramide, mono-, di- and triacylglycerin (triglycerides), in
`particular, caprylic and/or capric triglyceride and/or a mix
`ture thereof, particularly preferably Miglyol (such as Miglyol
`812 or Myritol 318 available e.g. from Henkel). Suitable
`Vegetable and animal oils e.g. are Sunflower oil, soybean oil,
`peanut oil, rape oil, fish oil and/or cetaceum.
`0015. A suitable membrane-forming surfactant is a phos
`pholipid, a lysophospholipid, a ceramide and/or a mixture
`thereof. Preferably, the phospholipid is lecithin or cephalin
`from Soybeans or hens' eggs, more preferably the lecithin is
`Soy lecithin.
`0016 Preferably, the lecithin has a phosphatidylcholine
`content of at least 80% by weight, more preferably of at least
`90% by weight, and most preferably of at least 94% by
`weight. The inventors have found that the quality of the leci
`thin, namely its phosphatidylcholine content, plays a crucial
`role for the size of the particles of the nanoemulsion. The
`higher the phosphatidylcholine content of the lecithin, the
`smaller is the size of the particles of the nanoemulsion.
`0017. As O/W emulsion-forming co-surfactant, anionic,
`nonionic, cationic and/or amphoteric Surfactants are suitable
`as well as block copolymers. Suitable anionic Surfactants are
`Soaps, alkylbenzene Sulphonates, alkane Sulphonates, alkyl
`sulfates and/or alkyl ether sulfates. Suitable cationic surfac
`tants are quaternary ammonium compounds, preferably hav
`ing
`one
`or
`two
`hydrophobic
`groups
`(e.g.
`cetyltrimethylammonium bromide and cetyltrimethylammo
`nium chloride) and/or salts of long-chain primary amines. A
`suitable amphoteric surfactant is N-(acylamidoalkyl)betaine,
`N-alkyl-3-aminopropionate and/or amine-N-oxide. A Suit
`able block copolymer, for example, is propylene oxide. In the
`
`Scope of the present invention, a nonionic Surfactant is par
`ticularly preferred as O/W emulsion-forming co-surfactant.
`A Suitable nonionic Surfactant is selected from the group
`consisting of fatty alcohol polyglycolether, alkylphenol
`polyglycolether, alkylpolyglucoside, fatty acid glucamide,
`fatty acid polyglycolether, ethylene oxide-propylene oxide
`block polymer, polyglycerol fatty acid ester, fatty acid alca
`nolamide and (ethoxylated) sorbitan fatty acid ester (sorbi
`tan). A particularly preferred ethoxylated sorbitan fatty acid
`ester is polyoxyethylene Sorbitan monooleate, most prefer
`ably Polysorbate 80.
`0018. The aqueous component of the nanoemulsion of the
`present invention preferably comprises a weak buffer system
`with low salt content, more preferably a 5 mM to 30 mM
`phosphate buffer and most preferably 10 mM phosphate
`buffer. The pH value of the phosphate buffer preferably
`ranges from pH 4 to pH 8, more preferably from pH 5 to pH
`7, and is most preferably from pH 5.5 to pH 6.5. The water
`used for preparing the phosphate buffer is preferably sterile
`deionized water and/or water for injections, more preferably
`water for injections.
`0019. The mean diameter of the emulsified particles in the
`nanoemulsion (nanosomes) is 5 nm to 500 nm, preferably 10
`nm to 200 nm, more preferably less than 100 nm, particular,
`up to 90 nm, preferably up to 70 nm, still more preferably 10
`nm to 50 nm, and most preferably 15 nm to 35 nm. The size
`distribution of the nanoparticles is preferably monodisperse
`and follows Gaussian distribution. The diameter of the emul
`sified particles of the invention is determined by means of the
`particle size distribution, which is measured by the method of
`dynamic light scattering (DLS) (also called photon correla
`tion spectroscopy (PCS)). The statistical analysis of the dis
`tribution of the particles is performed by a method called
`particle number-weighted distribution in accordance with the
`present invention.
`0020. A further subject of the present invention is a pro
`cess for the preparation of the nanoemulsion according to the
`invention, comprising the following steps: a) providing an
`aqueous component, b) providing a carrier comprising at least
`one lipophilic component, at least one surfactant and at least
`one alcohol, wherein the at least one alcohol has at least three
`carbon atoms, and c) mixing the aqueous component of step
`a) with the carrier of stepb). By preparing the nanoemulsion,
`the components of the carrier are provided in the aqueous
`component and the mixture is converted into a nanoemulsion
`by intensive or gentle homogenization. Homogenization may
`be carried out, for example, by commercially available
`homogenizers. After the preparation of the nanoemulsion,
`further additives and excipients may be added, the presence of
`which is not appropriate during the homogenization.
`0021. The process for the preparation of a nanoemulsion
`preferably is performed under aseptic conditions, e.g. using a
`laminar flow hood.
`0022. The inventors found out that, besides the composi
`tion, some steps of the process of preparation are decisive for
`the final size and particle-size distribution of the emulsified
`particles in the nanoemulsion. Particularly, temperature and
`homogenisation conditions during step c play a major role.
`Step c is carried out at a temperature between 50 to 60°C. All
`ingredients are previously heated up to this temperature. The
`container and the mixer should also be optimized to achieve a
`very fast homogenous mixture of the components (within
`seconds) avoiding formation of foam. See Example 1 for an
`illustration of Such procedure. Alternatively, the homogeni
`
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`US 2009/0324727 A1
`
`Dec. 31, 2009
`
`Zation does not require high shear devices such as Sonicators
`or high-pressure homogenizers.
`0023. A further aspect of the present invention is a phar
`maceutical and/or cosmetic composition comprising the
`inventive nanoemulsion. The inventive nanoemulsion is Suit
`able in the field of cosmetics, e.g. as anti-aging agent, as it
`provides an elegant, translucent and transparent vehicle that
`can be used for a variety of different product types. The
`nanoemulsion provides a system in very delicate balance,
`which has relatively low levels of emulsifiers that are consid
`ered as irritating. The inventive nanoemulsion retains the
`desirable transparency or translucency, it is able to accom
`modate a reasonable amount of additives such as fragrances
`or moisturizers, while remaining stable, mild and gentle on
`the skin of the user due to the low level of emulsifier. For the
`intended use of the final product in the case of cosmetics, it is
`possible to add non-therapeutic or non-active agents such as
`emollients, flavors, colorants, fragrances, gellants, thicken
`ers, Sunscreens and the like, which enhance the ultimate use
`of a product, particularly for topical cosmetic purposes, and
`provided care is taken to avoid choosing components that will
`interfere with the clarity of the product. The final product can
`take the form of a milk, cream, lotion, gel, serum or liquid
`spray, among others. In the case of Sunscreens the nanoemul
`sions of the invention may incorporate one or more Sunscreen
`agents such as benzophenones, avobenzones, cinnamates,
`salicylates and the like.
`0024. The nanoemulsions may also be employed in phar
`maceutical compositions, in particular, they can be used for
`the manufacture of a topical medicament for the treatment of
`dermatological diseases such as neurodermatitis, psoriasis,
`keratosis, in particular, actinic keratosis, and diseases associ
`ated with cell proliferation such as tumor diseases. Prefer
`ably, the tumor disease is selected from the group consisting
`of basal cell carcinoma, squamous cell carcinoma, Morbus
`Bowen, Vulvar intraepithelial neoplasia (VIN) or a nodular or
`Subcutaneous cancer disease. Further, the nanoemulsion is
`Suitable for the treatment of virus-associated diseases caused
`by a human papillomavirus Such as Condylomata acuminata.
`By incorporating the lipophilic component of the nanoemul
`Sion, the transepidermal loss of water can be influenced
`advantageously, i.e. the barrier function of the skin can be
`enhanced and, thus, dermatological diseases Such as neuro
`dermatitis can be affected advantageously.
`0025. A further aspect of the present invention relates to a
`composition comprising the inventive nanoemulsion and an
`active agent. In this respect, the composition is particularly
`useful as a pharmaceutical and/or cosmetic composition, e.g.
`for application to the skin or hair. The nanoemulsions of the
`invention comprised in said compositions provide a very
`efficient delivery system for a wide variety of active agents.
`Examples of active agents that may be useful include agents
`for the eradication of age spots, keratoses and wrinkles, anal
`gesics, anesthetics, anti-acne agents, antibacterials, anti
`yeast agents, antifungal agents, antiviral agents, anti-dandruff
`agents, antidermatitis agents, antipruritic agents, anti-emet
`ics, antimotion sickness agents, anti-inflammatory agents,
`anti-hyperkeratolytic agents, anti-dry skin agents, antiperspi
`rants, antipsoriatic agents, antiseborrheic agents, hair condi
`tioners and hair treatment agents, anti-aging agents, anti
`wrinkle agents, antiasthmatic agents and bronchodilators,
`Sunscreen agents, anti-histamine agents, skin lightening
`agents, depigmenting agents, vitamins, corticosteroids, hor
`mones, retinoids such as retinoic acid and retinol, topical
`
`cardiovascular agents, clotrimazole, ketoconazole, micona
`Zole, griseofulvin, hydroxy Zine, diphenhydramine, pramox
`ine, lidocaine, procaine, mepivacaine, monobenzone, eryth
`romycin,
`tetracycline,
`clindamycin,
`kanamycin,
`meclocyline, hydroquinone, minocycline, naproxen, ibupro
`fen, theophylin, cromolyn, albuterol, topical steroids Such as
`hydrocortisone, hydrocortisone 21-acetate, hydrocortisone
`17-valerate, and hydrocortisone 17-butyrate, betamethasone
`Valerate, betamethasone diproprionate, triamcinolone
`acetonide, fluocinonide, clobetasol proprionate, benzoyl per
`oxide, crotamiton, propranolol, promethazine, Vitamin A
`palmitate, vitamin E acetate and mixtures thereof.
`0026. In a preferred embodiment of the present invention
`the active agent is selected from the group consisting of
`5-aminolevulinic acid, a derivative, precursor and/or metabo
`lite thereof. “Derivative' is to be understood as being, in
`particular, an estherification of the amino group of 5-aminole
`Vulinic acid Substituted with one or two alkyl groups, particu
`larly preferably with one methyl group. The most preferred
`derivate is methylaminolevulinic acid. The derivatization
`may be understood also as Salt complexes and addition com
`pounds as well as alkylated compounds. “Precursor and
`"metabolite' are to be understood as substances converted in
`a cell into protoporphyrin IX. The active agent 5-aminole
`Vulinic acid or a derivative thereof is especially preferred.
`0027 5-Aminolevulinic acid is used as a prodrug in the
`field of photodynamic therapy. Photodynamic therapy is a
`promising method for the treatment of different pre-malig
`nant and malignant diseases which are associated with cell
`proliferation (Taylor E L and Brown S B, 2002, Journal of
`Dermatological treatment, 13, Suppl. 1, S3-11 and Peng Q. et
`al., 1997, Cancer, 79, S2282-2308). The principle of photo
`dynamic therapy is based on the introduction of a so-called
`photosensitizing agent into the lesioned tissue and Subse
`quent radiation with light of appropriate wavelength in order
`to transform the agent into a cytotoxically active agent which,
`in turn, causes the destruction of the cell. The selectivity of
`this method is due to the enhanced concentration of the sen
`sitizing agent in fast-proliferating or lesioned cells in com
`parison to normal tissue. Furthermore, irradiation of the pho
`tosensitizer gives rise to a characteristic fluorescence
`radiation which can be used for diagnostic purposes, for
`example, for detecting proliferating cells.
`0028 5-Aminolevulinic acid is an endogenous substance
`which is synthesized from glycine and Succinyl-CoA within
`the cells. Within the scope of heme biosynthesis, the proto
`porphyrin IX, which is photoactive to a high degree, is formed
`from 5-aminolevulinic acid (5-ALA) and is then converted
`into heme. This control mechanism is circumvented by exog
`enously administering synthetically prepared 5-aminole
`Vulinic acid, thereby giving rise to an increased production of
`protoporphyrin IX. Since the degradation of protoporphyrin
`IX is further inhibited by the natural control mechanism, this
`compound becomes concentrated in the cells. When irradi
`ated with light, protoporphyrin IX is able to undergo a pho
`tochemical oxidation reaction and consequently acts as a
`photosensitizer for the photodynamic therapy.
`0029 Systemic applications of 5-aminolevulinic acid are
`associated with a number of side effects which can be cir
`cumvented with topical application of the drug. A number of
`investigations using topically applicable 5-aminolevulinic
`acid compositions are known from the prior art. While these
`investigations have the feature in common that the 5-aminole
`Vulinic acid employed is in the form of an oil-in-water emul
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`

`US 2009/0324727 A1
`
`Dec. 31, 2009
`
`Sion, differences exist with regard to other parameters such as
`period of penetration, period of treatment, type of light
`employed and the dose of light employed.
`0030) B. Thiele et al. (H+G, Vol. 69, No. 3, pp 161-164
`(1994)) describe investigations which involve using 20%
`8-aminolevulinic acid in the form of an oil-in-water emul
`sion, with a penetration period of from 5 to 6 h, and subse
`quently irradiating with an argon ion-pumped dye laser
`(emission peak 630 nm) giving a cumulative total dose of
`from 50 to 100 J/cm.
`0031 Wolf et al. (Journal of the American Academy of
`Dermatology, Vol. 28, pp. 17-21, 1993) describe investiga
`tions which involve using 20% 5-aminolevulinic acid in the
`form of an oil-in-water emulsion, with a penetration period of
`4, 6 or 8 h, and irradiating with unfiltered light or red light,
`giving a light dose of from 30 J/cm to 100 J/cm.
`0032. Although the investigations disclosed in the prior art
`clearly demonstrate the promising potential of photodynamic
`therapy using 5-aminolevulinic acid, oil-in-water emulsions
`known so far Suffer from a number of disadvantages.
`0033 For example, M. Novo Rodriguez et al. (SPIE, Vol.
`2371, pp. 204-209) showed that, in the high concentrations
`required for a clinical application, aminolevulinic acid is
`unstable in aqueous solutions in a neutral to basic pH range.
`During the investigation period of 25 h, satisfactory results
`are only obtained at a concentration of 3% and at a pH of 5,
`which are specified as the optimal conditions for aqueous
`solutions of 5-aminolevulinic acid. For clinical use, however,
`it will in general be necessary to provide also compositions in
`a higher concentration range; furthermore, to be used com
`mercially, the 5-ALA solutions have to be stable for a period
`which is in the dimension of weeks or months.
`0034 V. von Arx et al. (J. Pharm. Pharmacol. 49: 652-656,
`1997) describe investigations relating to the topical applica
`tion of 5-aminolevulinic acid in a variety of gels. This publi
`cation states that the best formulation for maintaining the
`stability of 5-aminolevulinic acid is a combination with
`Novion AA-1, a polyacrylic acid, at a pH-6.
`0035 Hürlimann et al. (Dermatology, Vol. 197, No. 3,
`1998, pp. 248-254) disclose nanocolloid lotions containing
`5-aminolevulinic acid as well as the use thereof in photody
`namic therapy, without further specifying the emulsion.
`0036 WO 00/28971 describes compositions comprising a
`nanoemulsion and 5-aminolevulinic acid, wherein the
`nanoemulsion consists of egg lecithin (83% phosphatidyl
`choline), Miglyol 812 (triglyceride) and polysorbate 80 in 20
`mM phosphate buffer (see Example 1 of WO 00/28971), but
`no alcohol is used as solvent. Nanocolloid formulations con
`taining egg lecithin as an emulsifier, however, Suffer from the
`disadvantage that they are markedly stronger colored than
`5-aminolevulinic acid nanoemulsions containing Soy lecithin
`as an emulsifier. The color change in the formulation corre
`lates with the formation of a degradation product of the active
`agent 5-aminolevulinic acid. It is to be concluded therefrom
`that nanoemulsion formulations containing egg lecithin
`involve considerably reduced stabilization of 5-aminole
`Vulinic acid compared to formulations containing Soy lecithin
`as an emulsifier.
`0037 Another disadvantage of the known oil-in-water
`emulsions in combination with 5-aminolevulinic acid is that
`the penetration depth of the photosensitizer into the damaged
`tissue is not optimal. As a result, the diseased tissue is ame
`nable to photodynamic therapy only in its Superior layers
`
`although the penetration depth of the light used for activating
`the photosensitizer would also allow treatment of more
`deeply lying layers.
`0038. Therefore, it was a further object of the present
`invention to provide compositions comprising 5-aminole
`Vulinic acid, which, at least partially, overcome the known
`disadvantages of the state of the art.
`0039. According to the present invention this object is
`achieved by providing a composition comprising the inven
`tive nanoemulsion and 5-aminolevulinic acid, a derivative,
`precursor and/or metabolite thereofas active agent. In these
`compositions, the above-mentioned advantageous properties
`with respect to stability, penetration into tissues as well as
`lower costs of the inventive nanoemulsions can be utilized to
`transport a 5-aminolevulinic acid to target sites of tissues.
`0040. A specific interaction has been found between ami
`nolevulinic acid and the nanoparticles of the emulsion. In
`particular, 5-aminolevulinic acid, a derivative, precursor and/
`or metabolite thereof, is attached at the outside of the mono
`layer of the nanoparticles of the nanoemulsion. Thus, ami
`nolevulinic acid is transported by the particles while it is not
`contained within the core of the particles. Rather, aminole
`Vulinic acid is located outside the core of the particles due to
`interactions between aminolevulinic acid and the outer
`monolayer. In conventional nanoemulsions, in contrast
`thereto, the drug is contained inside the lipid core of the
`particles of nanoemulsions.
`0041) Surprisingly, it has been found that the stability of
`5-aminolevulinic acid can be considerably increased when
`the 5-aminolevulinic acid is formulated with the inventive
`nanoemulsion comprising at least one alcohol which has at
`least three carbon atoms. While the reasons for this are not
`known, it appears that a microenvironment created by nano
`somes has a particularly favorable effect on the stability of
`5-aminolevulinic acid.
`0042. It also turned out, surprisingly, that an improved cell
`and tissue penetration can be achieved with the nanoemul
`sions according to the invention, resulting in more deeply
`lying diseases and/or diseases with higher layer thicknesses
`also becoming accessible to treatment. The greater penetra
`tion depths were Surprising especially because it had previ
`ously been assumed that, due to its Small size, 5-aminole
`Vulinic acid would in any case be readily able to penetrate
`through a damaged epidermis which is present, for example,
`in tissue associated with inflammations, precancerous stages
`and tumors.
`0043 A third surprising advantage is that, when formu
`lated with nanosomes according to the invention, 5-aminole
`Vulinic acid is evidently taken up very efficiently from the
`cells. This firstly improves targeting; secondly, it means that
`the penetration period, i.e. the time between applying the
`composition and irradiating the diseased tissue with light, can
`be reduced, which means a distinct relief for the patient. As
`can be gathered from Example 4, FIG. 5, 5-aminolevulinic
`acid uptake into cultured cells and conversion of aminole
`Vulinic acid to PpIX within the cells is enhanced with increas
`ing concentrations of the inventive nanoemulsion with con
`stant concentration of aminolevulinic acid.
`0044 Storage stability, too, can be improved by means of
`the nanoemulsions of the invention which comprise an alco
`hol having at least 3 carbon atoms as a solvent in the carrier.
`0045. According to the invention the composition prefer
`ably comprises an active agent which is selected from 5-ami
`nolevulinic acid, or a derivative, a precursor and/or a metabo
`
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`US 2009/0324727 A1
`
`Dec. 31, 2009
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`lite thereof. “Derivative' is to be understood as being, in
`particular, an estherification of the am