Title of Invention


Abstract WE CLAIMS 1. A leave on topical composition comprising: (a) petroselinic acid and/or derivatives thereof; (b). a r.etinoi.d.. and/or. a. LRAX/ABAT inhibitor;, and. (c) a dermatologically acceptable vehicle.
Full Text F0RM2
(39 of 1970)
(See section 10; rule 13)
Title of the invention
HINDUSTAN LEVER LIMITED, a company incorporated under the Indian Companies Act, 1913 having its registered office at Hindustan Lever House, 165/155, Backbay Reclamation, Mumbai-400 020, State of Maharashtra, India

The following specification describes the nature of this invention (and the manner in which it is to be performed)


This invention relates to topical compositions for application to., human skin and to their use in improving .the condition and appearance of skin.
Skin is subject to deterioration through dermatological disorders, environmental abuse (wind, air conditioning, and central heating) or through the normal aging process (chronoageing) which may be accelerated by exposure of skin to sun (photoageing). In recent years the demand for cosmetic compositions and cosmetic methods for improving the appearance and condition of skin has grown enormously.
Consumers are increasingly'seeking "anti-ageing" cosmetic products that treat :.or delay the visible signs of. chronoageing'and; photoageing; skin ;.such as wrinkles, lines, sagging, hyperpigmentation and age spots.
Consumers also frequently seek other benefits from cosmetic products in addition to anti-ageing. The concept of "sensitive skin" has also raised the consumer demand for cosmetic products that improve the appearance and condition of sensitive, dry and/or flaky skin and to soothe red,

and/or irritated skin. Consumers also desire cosmetic products that have an oil/sebum control effect-. Many people are concerned with the degree of pigmentation of their skin. For example, people with age spots or freckles may wish such pigmented spots to be less pronounced. Others may wish to reduce the skin darkening, caused by-exposure, to. sunlight or to lighten their natural skin colour. To meet this need many attempts have been made to develop products that reduce- the,pigment,.production,-in. the melanocytes. However, the substances thus far identified tend to have undesirable side effects, e.g. skin irritation.
Consequently such substances are not suitable for cosmetic use or the.y can only be applied at a concentration at which
| their skin lightening effect is less than desired. Using a combination of different skin lightening substances may be considered to reduce adverse side effects but there is a substantial risk that by using such a combination the skin lightening is reduced as well due tc competition effects
Therefore there is -a need for improvement in the effectiveness.'of cosmetic skin lightening products : particularly, such; that -they do not irritate1-the "-sklnv •'
The use of fatty acids, including petroselinic acid, in cosmetic formulations for treating the hair is known. EP-A-116439) describes hair tonics which include fatty acids (such...as.,..petr.oseliai.c.acid) for alleviating dandruff and itch and for stimulating hair growth.

EP-A 709084 describes the' use of coriander seed oil, which is rich in petroselinic acid triglycerides, in a cosmetic composition for moisturising dry skin conditions. Retinol (vitamin A) is an endogenous compound that occurs naturally in the human body and is essential for normal epithelial cell differentiation. Natural and synthetic vitamin A. derivatives, (retinoids)! have been used extensively in the ^treatment of a variety of .skin disorders arid have been used as akin..repair,. or renewal, agentt.S. , Retional. acid for example, has been employed to treat a variety of skin conditions, e.g., acne, wrinkles, psoriasis, age spots and discoloration. See e.g., Vahlquist, A. et al., J. Invest. Dermatol., Vol. 94, Holland D. B. and Cunliffe, W. J. (1990), pp. 496-498; Ellis, C. N. et al., "Pharmacology of Retinols in Skin", Vasel, Karger, Vol. 3, (1989), pp. 249-252; Lowe, N. J. et al., "Pharmacology of Retinols in Skin", Vol. 3, (1989), pp. 240-248, PCT Patent Application No. WO 93/19743.
There .continues to be a:need .however, for alternative effectiv:e.:Cosmetic compositions for topical application to skin for. treating/delaying; the Ivisibile: sings of aging and . photodamaged skin such as wrinkles, lines, sagging, hyperpigmentation and age spots.
We have now found that effective treatment and prevention of normal, (but cosmetically undesirable), skin conditions, due to chronoageing or photoageing, such as wrinkles, lines, sagging, hyperpigmentation and age spots, may be obtained through the application of cosmetic compositions to the skin

which comprise a specific fatty acid - petroselinic acid and/or derivatives thereof, in combination with a retinoid and/or an. inhibitor of the enzyme acyl CoA retinol transferase (ARAT) or the enzyme lecithin retinol acyl transferase (LRAT) (hereinafter referred to as LRAT/ARAT inhibitors). We have also found that the use of such cosmetic compositions advantageously provides further skin, care benefits' in addition to anti-ageing such as soothing sensitive and:/or irritated; s.Jcia,, controlling .oil/sebum, secretion and for lightening the skin.
The arc discussed above does not disclose the specific synergistic combination of petroselinic acid with retinoids/LRAT/ARAT inhibitors nor the use of such a specific combination for treating wrinkles sensitive skin, dry skin, controlling oil/sebum secretion, or lightening skin.
According' to a. first aspect oft the present invention there is'provided :a topical icbmpo;s;itioh;:.comprisi:ng:i
(a) (b) (c)
petroselinic acid and/or derivatives thereof; a retinoid and/or an LRAT7ARAT inhibitor; and a dermatologically acceptable vehicle.
According to a second aspect of the present invention "here is provided a cosmetic method of providing at least one skir. care benefit selected from: treating/preventing wrinkling,

agging, dry, aged and/or photoda'maged skin; boosting ollagen deposition in skin, boosting decorin production in kin, enhancing tissue repair; soothing irritated, red nd/or sensitive skin; improving skin texture, smoothness nd/or firmness; lightening skin; controlling oil/sebum ecretion, the method comprising applying to the skin a opical composition as described above.
h:e -present, invention also,' encompasses the use of the. nventive compositions for providing at least one skin care enefit selected from treating/preventing wrinkling, agging, aged and/or photodamaged skin; boosting collagen eposition in skin, boosting decorin production in skin, nhancing tissue repair; soothing irritated, red and/or ensitive skin; improving skin texture, smoothness and/or irmness; lightening skin; controlling oil/sebum secretion.
ccording to a still further aspect of the present invention her;e .is: provided the use off petroselinic acid and;V erivatives' thereof'in combrnatio'n with' a 'retinoid and/or." a. RAT/ARAT inhibitor in a cosmetic topical composition • for1 roviding' at : least one cosmetic.skin care benifit selected rom treating/preventing wrinkling, sagging, aged and/or hotodamaged skin; boosting collagen deposition in skin, oosting decorin production in. skin, enhancing tissue epair; soothing irritated, red and/or sensitive'skin; mproving skin texture, smoothness £nd/or firmness; ightening skin; and controlling oil/sebum secretion.

The inventive •compositions, methods and uses thus provide anti-aging benefits which result in the promotion of smooth and supple skin with improved elasticity and a reduced or delayed appearance of wrinkles and aged skin, with improved .skin colour. A general improvement in the appearance, texture and condition, in particular with respect to the radiance, clarity, ; and general' youthful appearance of' skin is achieved. The inventive, compositions, methods and uses are also: beneficial for. soothing _and calming sensitive, and/or irritated' skin, for lightening skin and for controlling oil/sebum secretion.- Thus the present invention advantageously provides a wide range of skin care benefits.
The term "treating" as used herein includes within its* scope reducing, delaying and/or preventing the above mentioned normal skin conditions such as wrinkled, aged, and/or photodamaged, and/or irritated skin and generally enhancing the quality of skin and improving its appearance and texture by preventing ■ or' reducing-, irritation', .wrinkling and • increasing flexibility firmness, smoothness, suppleness and elasticity .of the skin, all for cosmetic purposes.. The compositions, methods and uses according to. t"h:e; invention may be useful for treating skin which is already in a wrinkled, aged, photodamaged, irritated condition or for treating youthful skin to prevent or reduce those aforementioned undesirable changes due to the normal ageing/photoageing process-.

^Petroselinic acid
: Petroselinic acid (hereinafter referred to as PA) is a monounsaturated long chain (C18) fatty acid, having the . formula •CH3.(CH2)10 CH=CH(CH2)4COOH.
The. invention, also includes derivatives; of' ther free.a.acid which thus comprise petroselinic acid moieties. Preferable rderivatives include those derived from substitution of the [ carboxyl group of the acid, such as esters (eg triglyceride esters, monoglyceride esters, diglyceride esters, phosphoesters) amides (eg ceramide derivatives), salts (eg alkali metal and alkali earth metal salts, ammonium salts) and/or those derived from substitution of the C18 carbon chain, such as alpha hydroxy and/or beta hydroxy derivatives.
In the Case of 'triglyceride' ester derivatives, all positional is:orners of PA substituents :on the glycerol,' backbone are'incl'udedi.. The triglycerides !mus!t; contain ;at :, least one PA moiety. For example, of the three esterifiable positions on tfte glycerol backbone, the 1 and 2 positions may be esterified with PA and by another lipid at position 3 or as an alternative, the glycerol backbone could be esterified by PA at the 1 and 3 positions with another lipid at position 2.

Oils that are rich in petroselinic acid triglyceride are thus also suitable for use in the present invention. Such oils are commercially available and include parsley seed oil, carrot seed on, fennel fruit oil, parsnip seed oil, coriander seed oil,, chervil seed oil, caraway plant oil, and celery seed oil.
Wherever the:.term "petroselinic acid" or "PA" is used in this- specification, it is. to be understood that the derivatives thereof comprising.PA moieties are also included. "PA moieties" "refers to PA fatty acyl portion(s) of a PA derivative;
The PA to be .employed in accordance with the present invention is present in the topical composition in an effective amount. Normally the total amount of the active is present in an amount between 0.0001% and 50% by weight of the composition. More preferably the amount is from 0.01% to' 101 and-..most". preferably the amount 0.1% to 5:% • in-order-, to. maximize benefits at a minimum cost.
The term "retinoid' inter alia includes retinoic acid, retinoyl ester, retinoi; ret.inyl ester.
The term "retinoi" includes the following isomers of retinoi: all-trans-retinol, 13-cis-retinol, 11-cis-retinol, S-cis-retinol, 3, 4--didehydro-retinoi. Preferred isomers are all-trans-retinol, 13-cis-retinol, 3,4-didehydro-retinol, 9-

icis-retinol. Most preferred is all-trans-retinol, due to its 'wide commercial availability.
Retinyl ester is an ester of retinol. The term "retinol" has been defined above. Retinyl esters suitable for use in
the prese-nt.-. invention, are-C-I-GJO:-
preferably C2-C20 esters, and most preferably C2-C3, and C3.6'
esters because they are more' commonly available. The preferred esters for use i'n the present invention are selected.from, retinyl palmitate, retinyl acetate, retinyl propionate and retinyl linoleate, because.these are the most commercially available and therefore the cheapest. Retinyl ester is also preferred .due to its- efficacy.
Retinoyl ester is an ester of retinoic acid. Retinoyl esters suitable for use in the -present' invention include C1-C30 esters of retinoic acid, preferably C2-C2o esters and most
preferably- c2-o3and: c16 The--pre-ferned1- esters for user
in 'the present invention -are-selected from- retinoyl -linoleate, retinoyl palmitate, retinoyl oleate, retinoyl ascorbate,:- and retinoyl ; linolenaite-.
LRAT/ARAT Inhibitor
Retinol is an endogenous compound that occurs naturally in the human body and is essential for normal epithelial cell differentiation. Esters of retinol hydrolyze in-vivc to produce retinol. It is believed that retinyl esters and retinol are metabolically converted in the skin into retinoic acid according to the following mechanism

Retinyl Ester Retinol
Retinoic Acid
However, most of the- errdogenously applied-"retinol is' rapidly converted into inactive fatty esters for storage in epidermal • cells, (keratinocytes) .
Esterification of retinol into inactive retinyl esters is achieved in cells by transfer of a fatty acyl group from an acyl CoA, catalyzed by the enzyme acyl CoA retinol transferase (ARAT), or'by the transfer of an acyl group from phosphatidyl choline, catalyzed by the enzyme lecithin retinol.acyl transfrence (LRAT) These.esterification. reactions are very, efficient .in keratinocytes-the majority. (95%) of cellular retinoids are in the form of retinyl,fatty esters.
The term "LRAT/ARAT inhibitor" in the present application thus means an agent which inhibits these esterification reactions and thus potentiates the action of retinol by increasing the amount of retinol available for conversion to retinoic acid.
The LRAT/ARAT inhibitors within the scope of the present invention are identifiable as those compounds which at

100 μM concentration inhibit at least 20% of LRAT cr ARAT catalyzed retinol esterification as measured by the in vitro Microsomal Assay described below in Example 1. In a preferred embodiment of the invention, the LRAT/ARAT inhibitor is a compound that at 100 uM concentration inhibits at least 40% and' most perferablet at least 5:0r%! of LRAT or ARAT catalysed retinol esterification. The in vitro Microsomal'. Assay, employed,.for determining, whether' cr no.ta, compound' is. such. a. .LRAT./ARAT;.'. inhibitor is as', described in Example 1 below.
Thus if a compound passes this in vitro Microsomal assay, that is, it inhibits sufficiently an LRAT or ARAT catalysed retinol esterification as measured by the in vitro Microsomal Assay, it is included in the present invention Bven if it is not specifically mentioned herein.
Examples of such LRAT/ARAT inhibitors which satisfy the assay described.' in Example. 1 include.' fatty; acid amides hydroxy ,fatty., acid amides, ceramides, melinamide, Lmida'-z.olidin.O;ne.s., and. cyclic .aliphatic; unsaturated Cyclic Aliphatic Unsaturated Compounds
Suitable cyclic aliphatic unsaturated compounds are selected according to the in-vitro Microsomal Assay Test described above.

A preferred cyclic aliphatic unsaturated compound is selected from cyclic aliphatic unsaturated aldehydes, ketones, alcohols and esters such as alpha damascone, beta damascone, delta damascone, isodamascone, damascenone, alpha ionone, beta ionone, allyl alpha ionone, isobutyl ionone, alpha methyl ionone, gamma methyl ionone, brahmanol, sandanol, alpha terpineol, lyral, ethyl saffranate, and mixtures thereof. Preferably, in order to maximize performance at a minimum cost,,, a. cyclic alypathic, unsaturated compound is i selected from the group consisting of damascones and ionones.
Most preferably, the cyclic aliphatic unsaturated compound is a ot-Damascone and/or a-Ionone.
Suitable diterpenes are selected according to the in-vitro Microsomal Assay Test described above. A preferred diterpene compound is geran.yl geraniol which': is a potent inhibitor, of retinol este-rification.
Fatty Hydro-xethyl' Imidazoline Surfactant's
Fatty hydroxyethyl imidazoline surfactants included in the present invention pass the in-vitro Microsomal Assay test described above. Preferred fatty hydroxyethyl imidazolines have the following general structure:

wherein R is an aliphatic saturated or unsaturated, straight
or branched hydro-carbon chain containing from 8 to 20 carbon
; i : .
atoms ,
Preferably, R in the fatty hydroxyethyl imidazoline contains from 8 to 18 carbon atoms, more preferably from 11 to 18 carbon atoms. Most preferably, the fatty hydroxyethyl imidazoline is oleyl hydroxyethyl imidazoline, due to its commercial availability and efficacy.
Fatty Acid Amide
Preferably, the fatty acid amide contains at least 6 carbon atom's:. suratble fatty acidsd includes saturated" and unsaturated straight or branched fatty acids-. ' Suitable-fatty acids, preferably: contain from 8 to 24 carbon atoms, preferably from 12: to 20 rearbon atoms; andimost preferably.-from 12 to 18 carbon atoms, because longer chain fatty acid amides are more beneficial for conditioning of the skin. In the most preferred embodiment of the invention, amides of essential fatty acids are employed because essential fatty acids provide nutrition-for the skin. Examples of essential fatty acids include but are not limited to linoleic, iinolenic, arachidonic, gamma-linolenic, homo-gamma-

linolenic, and mixtures thereof. Linoleic acid is most preferred because it is also a precursor to ceramide.
The preferred amides included in the present invention are mono- and di-alkanol amides, particularly of essential fatty acids. Alkanol amides are more commonly available than alkyl amides.
; The..-most. fatty, acid amides ace selected, from .mono,--
| and diethanolamides and phosphatidylethanolamides of linoleic
acid, palmitic acid, and coconut oil; diethyl cocamide,
, linoleamidyl dimethylamine, dimethyl linoleamide, diethyl
linoleamide, dimethyl palmitide, myristoyl sarccsine.
Hydroxy Fatty Acid Amides
The structure of an amide of a hydroxy fatty acid is as follows:

therein Ri, R2 and R4 each is .independently selected from hydrogen and aliphatic saturated or unsaturated, straight or Dranched hydrocarbon chains which may be hydroxylated, ^onLaining.. fr.om. 1 to 20. .carbon atoms;
R3 is -(CH2)n where n is an integer from 0 to 18;

Preferably, R1 R.2/ R4 each independently contains from 2 to 20 rarbon atoms, more preferably from 2 to 15 carbon atoms, most preferably from 3 to 13 carbon atoms.
Preferably the hydroxy acid amide is an amide of a- or β nydroxy acid, i.e.,.n is 0 or 1.
rhe most preferred- hydroxy fatty -acid amides to be included In: the inventive.1 compositions ar.e-:- lact.aiaid.e-nonoethanolamide, C13-β-hydroxy acid amide (2-hydroxy-Ci3-imide), N-hydroxyethyl-2-hydroxy-C16 amide, 12-hydroxy-N-(2-lydroxyethyl) octadecanamide, and monoethanolamide of castor ail.
olycyclic Triterpene Carboxylic acid (PTCA)
further example of a suitable LRAT/ARAT inhibitor is a PCTA which passes the in vitro Microsomal Assay.
Preferably.-the PTCA is a pentacyclic triterpene nono.carboxylie aoid.
tost preferably, PTCA is selected from the croup consisting ursolic acid, oleanolic acid, glycerrhetinic and jlycyrrhizic acid.
PTCA are commercially available from Aldrich and Sigma. 5Iant extracts containing PTCA are suitable for use in the resent invention e.g. Rosmarinus officinalis (rosemary), iospycos spp. (persimmon) , Forsythia suspense (forsythia) ,

Lavandula angustifolia (lavender), Prunella vulgaris (selfheal), Paeonia lactifolia, Glycyrrhiza glabra (licorice) .
It should be understood that depending on the pH of the composition, PTCA may be present in the composition as a salt, e.g. alkali or alkaline earth salt.
The ceramides may for example be naturally occurring ceramides, phyto ceramides short chain ceramides, pseudoceramides or neoceramides. The general structure of these molecules is described in EP A 711558 whose contents are hereby incorporated by reference.
The most preferred ceramide derivative is acetyl sphingosine due to its efficacy.
The retinoid ahd/dr LRAT/ARAT inhibitor can be"included.in the inventive, compositions in an amount ranging from 0.0001% to 501 by .weight; of: the composition,,. preferably .;i:t,'is used in . an amount of from 0.01% to 10%, most preferably from 0.1% to 5%.
Dermatologically Acceptable Vehicle
The composition used according to the invention also comprises a dermatologically/cosmeticallv acceptable vehicle to act as a dilutant, dispersant or carrier for the actives.

The vehicle may comprise materials commonly employed in skin care products such as water, liquid or solid emollients, silicone oils, emulsifiers, solvents, humectants, thickeners, powders, propellants and the like.
The vehicle will usually form from 5% to 99.9%, preferably from 25% to.,80% by weight of the composition, and, can, in the absence;'-of other cosmetic adjuncts, form the balance of the.- compos.ition..
Optional Skin Benefit Materials and Cosmetic Adjuncts
Besides the actives-, other specific skin-benefit actives such as sunscreens, other skin lightening agents, skin tanning agents may also be included. The vehicle may also further include adjuncts such as perfumes, opacif.iers, preservatives, colourants and buffers.
Product' Preparation,.-. Form-, CIs-e and Pack aging-.
To ^prepare ».the topical composition usedinthe method of the-present invention, -the lusual; manner; for preparing:- skin care products may be employed. The active components are generally incorporated in a dermatologically/cosmeticaily acceptable carrier in conventional manner. The active components can suitably first be dissolved or dispersed in a portion of the water or another solvent or liquid to be incorporated in the composition. The preferred compositions are oil-in-water or water-in-oil or water-in-cil-in-water emulsions.

The. composition.may be in the form of conventional skin-care products such as a cream, gel or lotion, capsules or the ;' like. The composition can also be in the form of a so-called "wash-off" product e.g. a bath or shower gel, possibly containing a delivery system for the actives to promote adherence to the skin during rinsing. Most preferably the product is a "leave-on" product, i.e. a product.to be applied to the skin without a deliberate rinsing; step so.on. after; its application to. the skin.
The composition may packaged in any suitable manner such as in a jar, a bottle, tube, roll-ball, or the like, in the conventional manner. It is also envisaged that the inventive compositions could be packaged as a kit of two separate compositions one containing the petroselinic acid and the second containing the retihoid/LRAT/ARAT inhibitor compound,
to be applied to the skin simultaneously or consecutively.
| The compo'.sitrion according to Che presentinvention; may' also be formulated in a form' suitable for oral ingestion' such' as a capsule, tablet or the like..
The method of the present invention may be carried out one or more times daily to the skin which requires treatment. The improvement in skin appearance will usually become ivisible after 3 to 6 months, depending on skin condition, the concentration of the active components used in the inventive method, the amount of composition used and the frequency with which it is applied. In general, a small quantity of the topical composition, for example from 0.1 to

i ml is applied to the skin from a suitable container or .pplicator and spread over and/or rubbed into the skin using .he hands or fingers or a suitable device. A rinsing step lay optionally follow depending on whether the composition s formulated as a "leave-on" or a "rinse-off" product.
n order that, the present, invention may be more readily,-mder.stood, the following-examples are given, by way 'of .Llu.stra.tion .only..
ixample 1
his example demonstrates how LRAT/ARAT inhibitors within he scope of the present invention may be identified using he in vitro Microsomal Assay of the esterification of etinol.
ie'thod of in vitro microsomal 'esterification' of retinol:
Microsomes are obtained as described in J.;c.t Saari and D.L.! redberg, "CoA and Non-CoA Dependent Retinol Esterification n Retinal Pigment Epithelium" J. Biol. Chem. 23, 8C34-90 1988).
solution containing 0.1M sodium phosphate p.H 7 buffer, 5mM ithiothreitol, 2 rng/ml bovine serum albumin, 40 micromolar almitoyl CoA, 40 micromolar dilauroyl phosphatidyl choline,-0 micromolar retinol and a test compound or solven blank,

was-incubated for 1 hour at 37.c with a microsomal fraction isolated from bovine retinal pigment epithelial cells. After incubation, the reaction was quenched by addition of an equal volume of ethanol, and the retinyl esters formed (retinyl palmitate from the ARAT catalyzed reaction, and retinyl laurate from the LRAT catalyzed reaction) were extracted with hexane. The. hexane layer was removed, evaporated under nitrogen, and the residue analyzed by HPLC on a 3.9x300 mm' C18' revers.e.d.phas.e. column, using a 80% methanol, in. tetrahydrofuran mobile phase and fluorescence detection (325 nm excitation, 480 ran emission) to quantitate the retinyl esters. The quantity of ester formed in the presence of the solvent blank was taken as 100%, and this was used to calculate the percent inhibition of ester formation for the compounds tested. As a control, an aliquot of microsomes was inactivated.by boiling for 5 minutes, which resulted in at least 95% inhibition of ester formation.
The results that were obta'ine'.d'; axe,summarized in Table. I..




Acetyl Sphingosine 100 62
Acetyl Sphingosine 10 19
Linoleamide-DEA (LODEA) 100 43
10 Linoleamicle-DEA 10 ' 12
Linoleamide-MEA (LOMEA) 100 35
Linoleamide-MEA 10 0
oleyl hydroxyethyl imidazoline 100 90
oleyl hydroxyethyl imidazoline 10 14
\i 15 caprylic hydroxyethyl imidazoline 100
diazolidinyl urea 100 0
thiamine 100 0
caffeine 100 0
adenine 100 0
20 phenyl benzimidazole sulfonic acid 100 0
uracil 100 0
tryptophan 100 0


It can be seen that acetyl sphingosine, LODEA, LOMEA and hydroxyethyl imidazoline surfactant are a potent retinol esterification inhibitors, while other surfactants and other heterocyclic compounds were essentially inactive. Caprylic hydroxyethyl imidazoline (R = CH3(CH2)6) did not sufficiently inhibit LRAT.
The: in vitro Microsomal Assay Test was run on the compounds listed in Tables 2Aand 2B
The compounds in Table 2A were tested at a lOOuM concentration. The compounds in Table 2B were tested at a lOuM concentration.


Alpha damascone 83 98
Beta damascone 84 92
J Delta damascone 87.. 9.5. .
Isodamascone 80 92
; D.amascenon.e 70 79
Alpha ionone' 45 49.
Beta ionone 22 24
Allyl alpha ionone 22 36
Isobutyl ionone 8 45
Alpha methyl ionone 67 77
Gamma methyl ionone 21 38
Brahmanol 70 75
Sandanol 15 43
Alpha terpineol 26 25
T.imb'eraT... 34 33
!L'yral 76 71
Tonalid 50. 33
Ethyl saffranate 1 51 496
Traseolide 41 21
Sandalone 23 12


alpha damascone 67 87
beta damascone 45 52
delta damascene. 58-- . 64
damascenone 23 29
allyl alpha ionone 16 . 17
It can be seen from the results in Tables 2A and 2B that rertain cyclic aliphatic unsaturated compounds in particular :he ionones and damascones are potent inhibitors of LRAT' and \RAT catalyzed retinol esterification. These contain the :rimethyl cyclohexene ring system present in retinol.
?he in-vitro Microsomal Assay test was conducted with idditional cyclic aliphatic unsaturated compounds. The results;, that: were obtained are- summarized' ;i'n- Table. 3...
/he compounds, in: Table 3 .were tested at a :100 pM :oncentra;tion.


dihydro alpha ionone 13 18
alpha ionol 0 0
beta ionol 0 0
cinnamaldehyde • 0 0
: vanillin- 0 0
i' ■
eucalyptol 0 0
menthol 0 0
thymol 0 0
carvone 0 0
camphor 0 0
mentone 0 0
fenchyl alcohol 12 4
isocyclogeraniol 18 16
dimethyl ionone 0 9
delta methyl, ionone 0, ■■-■ 10

It .can.; be, .seem from the results, in..Table 3 that not all' cyclic aliphatic unsaturated compounds inhibit or sufficiently.inhibit LRAT and ARAT catalyzed retinoi esterification.
The in-vitro Microsomal Assay test was conducted with a diterpene compound, geranyl geranioi or farnesol.

The results that were obtained are summarized in Table 4.

Geranyl Geraniol1 100 81 77
Geranyl Geraniol 10 38 16
Farnesol2 100 43 43
Farnesol 10 20 10
Obtained from TCI America (Portland, Oregon) . Also-available from Sigma and CTC Organics (Atlanta, Georgia).
2 Available' from Givaudan Co., Bedoukian Co., or Dragoco "Co.
It can be seen from the results in Table 4 that both geranyl geraniol and farnesol- inhibit' retinol esterification. Geranyl geraniol is a substantially more potent esterification inhibitor, than farnesol.
Example 2
Identification of Procollagen-'I and Decorin Upregulation In Skin In Vivo Following Topical Retinoic Acid Treatment for Comparative. Ruxpos.es.:
Collagen, the predominant matrix skin protein is known to impart tensile strength to skin. Decorin is a proteoglycan

which is known to be important for controlled and correct deposition of collagen in the extracellular matrix of skin. It is also known in the art that' the levels of collagen and decorin in skin are significantly reduced with aged and/or jphotodamaged skin. Many studies have shown that the levels
of collagen type I in skin is decreased with age and/or with increased- p.hotodarnage, (for -example: Lav/ker, R.
J. Inv. Derm. , (.1979) ,73,.79-66; Griffiths et al. N. Eng.. J.
med. (1993)329, . 530-535) In the case -of decorin-,:. it-has Ibeen shown; that' mRNA express! oirr and" expression, of Che. proteoglycan is greatly reduced in photodamaged skin in itro (3ernstein et al. Lab. Invest. (1995)72,662-669). The reduction of the levels of these skin proteins is •accordingly associated with a decrease in the tensile, 'strength of the skin causing wrinkles and laxity.
lit is well known in the art that retinoic acid is a potent
|anti-aging active and induces dermal repair of photodamaged
s.skin. It has been shown that wrinkle effacement and dermal

frep.air. foil owing topical, treatment- of s.kin.with: retinoic
lacid arises through new collagen deposition and synthesis in
!the skin (fox example, .Griffiths .et al.. N.. Eng. J. med. (1993) 329, 530-535). It is widely accepted that
strengthening of the dermal matrix by boosting the level or
collagen in skin using retinoic acid provides anti-ageing/dermal repair benefits. Procollagen I is a precursor of collagen. Increased production of procollagen I in response to a test compound application is a marker of an increased collagen level.

Two groups of women were recruited with identical or nearly identical degrees of mild to moderate photodamage on each outer forearm. They were supplied with 0.05% retinoic acid in a moisturising base (Retinova®) and also with a colour matched moisturising cream with similar sensory characteristics (Dermacar'e® lotion), but no active ingredients:, . as; a pi ace ho: contra]!'., Each' participant of. the two groups applied the Retinova® to one outer forearm and placebo (Dermacare®) to the other:outer forearm. Group 1 applied the products daily to their outer forearms for 14 weeks and the Group 2 applied the products to., their outer forearms for 28 weeks. At the end of the studies two full . thickness 4mm punch biopsies were taken from the treated areas of each forearm. Immunohistochemical analysis of the biopsy tissue taken from the participants was performed to identify the effect of retinoic acid treatment on the expression of the skin extracellular matrix components, decorin and procollagen-I, as compared with the placebo treated- forearms. The" following pxocedu're was followed:
Antibody dilution buffer for wax sections was composed of Tris Buffered Saline (TBS), 3% bovine serum albumin (BSA), 0.05% Triton X-100 and 0.05% sodium azide. Primary antibodies for procollagen-I (amino terminal) were obtained from Chemicon"International Inc. (cat# MAB 1912, rat IgGl) and used on wax sections at a dilution of 1:300, overnight at 4°C after the section had been pre-treated with trypsin (0.5 mg/ml, 25 minutes, 37°C). Primary antibodies for

decorin were obtained from Biogenesis (rabbit polyclonal) and used on wax sections at a dilution of 1:800, overnight
iat 4°C. Anti-rat biotinylated secondary antibodies, (Obtained from DAKO (cat# E0468, rabbit polyclonal), were 'applied to wax sections at a dilution of 1:400. Anti-rabbit •biotinylated secondary antibodies, obtained from'Amersham (cat>#. RPN.1004., donkey polyclonal ),.„, were., applied to wax. sections at a- dilution of 1:400. Streptavidin conjugated aLkaline phosphatase,, obtained from Zymed (cat# 43-4322), 'was used ast a conection of 1:2500 % Fast Red chromogen was obtained from DAKO (cat# K597). Gills #3 Haemotoxylin nuclear counterstain obtained from Sigma. (cat# GHS-3), was filtered and used without dilution. Trypsin was obtained from Sigma (cat*.T-7186) and slides were mounted with Glycergel from DAKO (cat# C563)V
Wax sections of the biopsy tissue were mounted on silane coated slides:'and baked for- 18: hours at 55°C . The slides were de-waxed- through xylene and alcohol and brought to -water and-.their transferred to TBS.. DAKO®, pen was used to , ring the section's. The sections were processed" for antigen retrieval using trypsin where necessary, as indicated for each antibody. Where antigen retrieval was necessary, the slides were incubated for 25 minutes at 35°C with trypsin at 0.5 mg/ml (Sigma Cat # T-7186). The protease was subsequently rinsed off (2x2 minutes) with TBS. Following antigen retrieval, if necessary, or otherwise directly after ringing the sections, non specific antibody binding was blocked with 5% solutions of secondary antibody host serum

in TBS/0.5% BSA/0.1% sodium azide as the blocking solution for at least. 20 minutes, at room temperature in a humid chamber. The excess blocking solution was drained off, but the sections were not allowed to dry. The sections were then incubated with the primary antibody (appropriately diluted as indicated above) in a humid chamber overnight at 4°C. Antibody was. subsequently, drained from. the. section without allowing them to dry. The slides were then washed with TBS to" remove unbound primary antibody - a one minute rinse.followed by three five. minute .washes- and then incubated with the appropriate secondary antibody (approoriately diluted as indicated above) in a humid • chamber for 1 hour at room temperature.
The antibody solution was subsequently drained from the slides without allowing the section to dry. The slides were washed in TBS, a one minute rinse followed by 4 x 5 min washes, in order to remove the unbound secondary antibody. For the biotinylated secondary antibody the sections were subsequently incubated with streptavidin conjugate- for-45 • minutes at 37°C and then washed in TBS to remove unbound streptavidin: conjugate. The chromogen was added and the colour developed withi observation to.'avoid ; over staining The sections were then counterstained and mounted.
Differences in the expression of procollagen-I and decorin between retinoic acid (Retinova®) and placebo (Dermacare®) treated sites-were—determined by visual assessment of the immunohistochemically stained sections using light microscopy:

This analysis identified marked upregulation of both procollagen-I and decorin in the photodamaged skin following topical application of retinoic acid (Retinova®), as set out in. Table 5 below.
Table 5
Effect of Retinoic Acid Treatment on expression of procollagen I and decorin in skin In Vivo

Total No. of Participants No. of Participants showing marked increase in expression of procollagen-I No. of Participants showing marked increase in expression of decorin
Group 1 after 14 weeks 16 9 10
Group 2 after 28 weeks- 15 10 15
The extra cellular matrix components procollagen 1" and decorin are thus clearly identifiable markers of retinoic acid induced dermal repair.
Example 3
Procedure For Measuring Procollagen-I and Decorin Synthesis In Human Dermal Fibroblasts

Preparation of Dermal Fibroblast Conditioned Medium

Primary human foreskin fibroblasts at passage 2 (P2) were seeded into 12-well plates at 10000 cells/cm2 and maintained for 24 hours- in an atmosphere of 5% carbon dioxide and 4% oxygen in Dulbeccos Modified Eagles Medium (DMEM) supplemented, with 1.0..% foetal calf serum. After: this, time-,-the cells were washed with serum free DMEM and then incubated in. fresh serum free DMEM for a further 60 hours'. The fibroblast monolayers were then washed again with s'erum free DMEM. Test reagents and vehicle controls were added to the cells in triplicate in a final volume of 0.4ml/well fresh serum free DMEM and incubated for a further 24 hours. This fibroblast conditioned medium was either analysed immediately or snap frozen in liquid nitrogen and stored at -70°C for future analysis. The cells were then counted and data from the dot-blot analysis subsequently standardised to cell number.
Example 4
Dot Blot Assay for P.rocollaqen-I and. Decorin Protein in Dermal Fibroblast Conditioned Medium
Samples of conditioned medium from dermal fibroblasts treated with vehicle (as a control) or test reagents were supplemented with 20mM dithiothreitol (1:10 dilution of 200mM stock solution) and 0.1% sodium dodecylsuiphate (1:100 dilution of 10% stock solution), mixed well and then incubated at 75°C for 2 minutes. A standard for the assay was generated by serial dilution of neat fibroblast

conditioned medium from fibroblasts seeded at 10000 cells/cm2
in a 175cm flask and maintained in serum free DMEM as
described above.
Assay samples were subsequently applied in triplicate to a prewetted sheet of Immobilon-P transfer membrane using the ', 96-well Bio-Dot Apparatus' from Bio-Rad as described' in the manufacturers guidelines. Approximately 200ul of medium was applied per well The medium was allowed to filter through the membrane under gravity (JO minutes) after which the membrane was washed twice with PBS (200(il) . These P3S washes were allowed to filter through the membrane under gravity (2x15 minutes). The Bio-Dot apparatus was then attached to a vacuum manifold and a third and final PBS wash carried out under suction. The apparatus was disassembled, the membrane removed and quickly cut as required before being placed in blocking buffer overnight at 4°C. Membranes prepared for decorin analysis were blocked with 3% (w/v) BSA/ 0..1%' (v/;v) Tween 20: irr PBSv whilst thfosev for procollagen-! analysis., were .blocked with. 5-.% (w/.v). non.fat. dried milk powder/ 0. 05% Tween 20 in PBS. •
The following day, the membranes were probed with 1:10000 dilution of primary antibodies to either human procollagen-I (MAB1S12; rat monoclonal; Chemicon Int. Inc., Temecula, CA) or human decorin (rabbit polyclonal; Biogenesis) for 2 hours at room temperature. The membranes were subsequently washed with T3S/ 0.05% Tween 20 (3 x 5 minutes) and then incubated with 1:1000 dilution of 125I-con j ugated anti-rat or anti-rabbit F(ab')2 fragments (Amersham) as required for 1 hour

at room.temperature. Following this the Immobilon strips were again washed with TBS/Tween 20 (3x5 minutes) before being allowed to dry in air at room temperature. The dried membranes were wrapped in cellophane and exposed to a Molecular Dynamics storage phosphor screen for 16-18 hours. At the end of this time the exposed screen was scanned by a pho.sphorimager (Molecular. Dyaamics Pho.sphorimag.er.. S.F.). using-ImageQuant™ software. Dot intensity was assessed by computer-assisted image analysis using the quantification tools in. ImageQuanttm standardised to cell number, and. the effects of various test reagents on decorin and procollagen-I synthesis-were determined relative to a vehicle treated control value of 100 arbitrary units.
Example 5
The table below indicates the synergistic effect of petroselinic acid in. combination .with the LRAT/ARAT . inhibitors Ceramide 6 or LOMEA on procollagen-I and !decorin syntnesis- in human dermal, fibroblasts and the amounts, in which the actives were applied. In order to normalise the results the effects of the test substances were determined relative to a vehicle treated control value of 100 arbitrary units. The concentrations of reagents used in the trials had no influence on cell viability.

Table 6

The Synergistic Effect on Procollagen-I and/or Decorin 1 Synthesis by Petroselinic Acid in combination with a LRAT/ARAT Inhibitor

Treatment . Eracollagen-I. Decorin:
Control (Vehicle) 100 100
■0.01 nM- PA 85.1 %
1 0.01 ΜM Ceramide 6 94.3- %
0.01 μM PA + 0.01 uM Ceramide 6 125.6% -
0.01 μM PA 101.8%
r' 0.1 μ /ml LOMEA 133.9%
0.1 μM PA + 0.1 ug/ml LOMEA 239.1%
The results in table. 6 indicate that the combination- of . petroselinic acid with a LRAT/ARAT inhibitor significantly upregul:ate:s,' the synthesis of procollagen iand/or decorin in human dermal fibroblasts as compared to the control.
The level of decorin in skin is associated with improved condition and appearance of skin. . Increasing the level of decorin in skin is important for controlled and correct deposition of collagen in skin which is associated with many-skin benefits such as wrinkle effacement and dermal repair of photodamaged skin.

Synergy of Petroselinic Acid with Retinoids
The table below indicates the synergistic effect of petroselinic acid in combination with the retinoids on procollagen-I and/or decorin synthesis in human dermal fibroblasts, and the amounts in,whoich the result actives were, applied. In order to normalise the results the effects the test substances were determined relative to a vehicl treated control value of 100 arbitarary units The! concentrations of reagents, used in the trials had no influence on cell viability.
Table 7
Untreated control = 100%. All results normalised to this value.

Actives Tested Procollagen 1 Decorin
0.01 μM PA
0.01 μM trans Retinoic acid
0.0.1 uH- PA. + .0.01 μM.-trans., Retinoic acid 100.5%
' 133.,4%
0.01 μM PA 0.01 μ.M Retino.i 0.01.μM PA + 0.01 uM Retinol 86.0%
0.01 μM PA
0.1 uM Retinyl Linoleate
0.01 μM PA + 0.1 μM Retinyl
Linoleate 107.5% 109.1% 122.3%
TM'ei result sin table 7 indicate that thei combination of ' petroselinic acid with a retinoid significantly upregulates the synthesis of procollagen-I and/or decorin in human dermal fibroblasts as compared to the control.
The level of decorin in skin is associated with improved condition and appearance of skin. Increasing the level of decorin in skin is important for controlled and correct deposition of collagen in skin which is associated with m-ny

skin benefits such as wrinkle effacement and dermal repair of photodamaged skin.
Example 6
This example, illustrates.. oil-inrw.ater. creams accoxding., to. the invention.

Petroselenic acid (triglyceride) ex NU Check Prep 1.15 1.15 3 2 1..
Retinyl Linoleate 0.15
i Retinoic acid
l 0.001
i Retinol . 0.15 . 0.15
Mineral oil - • 4' • t- 4 4 ' ' 4
a-ionone 1 -
Isodamascone 0.3 - •
3rij 56* 4 4 4 4 4
Alfol 16RD* 4 4 4 4 4
Triethanolamine ' 0.75 0.75 0.75 0.75 0.75
Butane-1,3-diol 3 3 3 3
Xanthar. gum 0.3 0.3 0.3 0.3 0.3
Per.fume qs. qs- qs Qs :• . qs •■
Butylated hydroxy toluene
■ 0.01 0.01 0.01 o.oi 0.01
Water: to. 100: ■to. 10.0: „ to ioo; To, ioo ;■ . ..to^IOOi
* Brij 56 is cetyl alco
Blfol UDn i c- l~ai-\,'\ air hol POE ( 10)

Example 7
This example illustrates alcoholic lotions according to the invention.

PA (triglyceride)' ex NU Check Prep




0. 1

Geranyl Geraniol












Butylated hydroxy toluene






to 100

to 100

To 100.'

to .100.

Example 8
This example illustrates a suncare cream incorporating the composition of the invention:

Coriander, seed. oil. ex. Lo.de.rs.. Croklaah (PA triglyceride- about "60 - 75'. % o:f. total fatty acids) •: 4% ■
Retinyi- Linoleate 0.01 ■
Cocoylhyroxyethylimidazolie 0.1
Silicone oil 200 cts 7.5
Glycerylmonostearate 3
Cetosteryl alcohol 1.6
Polyoxyethylene-(20)-cetyl alcohol 1.4
Xanthan" gum' 0.5- .
Parsol 1789 1.5
■Octyl, met.hoxycinnat.ej (PARS.OL .MCX) ,
i i •'". 7
Perfume Qs
Color Qs
Water to 100

Example 9
This example illustrates a high internal phase water-in-oil emulsion incorporating the inventive composition.

Petroselinic acid (triglyceride) ex WU Check Prep 1 2 0.5 3
Retinol 0.5 - - -
LODEA 2 - -
LOMEA ex. Rhone Poulenc - 1 - -
Fully hydrogenated coconut oil 3.9 3.9 3.9 3.9
Brij 92*, 5 5 5 5
Bentone 38 0.5 0.5 0.5 0.5
MgS0.7H20 0.3 0.3 0.3 0.3
Butylated hydroxy toluene 0.01 0.01 0.01 0.01
Perfume Qs Qs Qs 05
Water To 100 ,. To 100 To 100 To 100

* Brij 92 is polyoxyethylene (2) oleyl ether
Examples 6 to 9 illustrate topical, compositions according to the present invention. The compositions can be processed in conventional manner-. They are suitable -for cosmetic use. In particular the compositions are suitable for application to wrinkled, rough, dry, flaky, aged and/or photo-damaged skin to improve the appearance and the feel thereof as well as

1. A leave on topical composition comprising:
(a) petroselinic acid and/or derivatives thereof; (b). a r.etinoi.d.. and/or. a. LRAX/ABAT inhibitor;, and. (c) a dermatologically acceptable vehicle.
2. A topical composition according to claim 1 wherein the retinoid is selected f rom -.retinoic acid, retinol and/o: a retinyl ester.
3. A topical composition according to claim 1 or claim 2 wherein the retinoid is retinol.
4. A topical composition according to claim 1 or claim 2 wherein the retinyl ester is retinyl linoleate.
5.. A topical. composition according to any of the precedinc claims/' wherein the LRAT/ARAT inhibitor is a fatty amide, a hydroxy fatty, acid';- amide,.;.a- ceramdde,- \& melinamide, an imidazolidihones, a cyclic aliphatic unsaturated hydrocarbon, a terpens, or a fatty hydroxyethyl imadazoline surfactant, or mixtures thereof.
A topical' composition according to claim 5, wherein the cyclic aliphatic unsaturated compound is selectee from cyclic aliphatic unsaturated aldehydes, ketones,

alcohols and esters such as alpha damascone, beta damascone, delta damascone, isodamascone, damascenone, alpha ionone, beta ionone, allyl alpha ionone, isobutyl ionone, alpha methyl ionone, gamma methyl ionone, brahmanol, sandanol, alpha terpineol, lyral, ethyl saffranate, and mixtures thereof.
A topical composition according to claim 6, wherein the-cyclic aliphatic unsaturated compound is-- an a damascone or a.n A topical composition according to claim 5, wherein the fatty acid in the fatty acid amide is selected from linoleic acid, linolenic acid, arachidonic acid, gamma-iinolenic acid, homo-gamma-linolenic acid, and mixtures thereof.
A'topical composition according to claim 8, wherein the fatty acid in the fatty acid amide is linoleic acid.
A topical.composition according .to.claim.8, wherein.the fatty -acid amide is linoleamide monothanolamide (MEA)-.
A topical composition according to claim 5, wherein the hydroxy fatty acid amide is lactamide-monoethanolamide, C13-p-hydroxy acid amide (2-hydroxy-Ci3-amide) , N-hydroxyethyl~2-hydroxy-C16 amide, 12-hydroxy-N-(2-hydroxyethyl) octadecanamide, . and monoethanolamide of castor oil, and mixtures thereof.

12. A topical composition as claimed in claim 5, wherein the terpene is a pentacyclic triterpene monocarboxylic acid.
13. A topical composition as claimed in claim 5, wherein the ceramide is a ceramide derivative which is acetyl sphingosine.

Dated this 29th day of January, 2002



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Patent Number 208862
Indian Patent Application Number IN/PCT/2002/00117/MUM
PG Journal Number 35/2007
Publication Date 31-Aug-2007
Grant Date 14-Aug-2007
Date of Filing 29-Jan-2002
# Inventor's Name Inventor's Address
PCT International Classification Number A61K 7/48
PCT International Application Number PCT/EP00/06594
PCT International Filing date 2000-07-11
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 9918022.6 1999-07-30 U.K.