CA 02547745 1998-07-15
73512-12D
ANTIIRRITANT OR ANTIINFLAMMATORY COSMETIC COMPOSITIONS
This is a divisional application of Canadian
Patent Application No. 2,265,993 filed on July 15, 1998.
Field of the Invention
The invention relates to novel cosmetic
compositions. In particular, the invention relates to
cosmetic compositions having unique antimicrobial and
antiirritant properties.
Claimed in this divisional application are a
cosmetic and pharmaceutical formulation and method for
preventing or reducing irritation of skin. A cosmetic and
pharmaceutical formulation and method for inhibiting
inflammation in skin are also claimed. It should be
understood that the expression "the present invention"
encompasses the subject matter of both this divisional and
the parent applications.
Background of the Invention
Water miscible cosmetic and toiletry formulations
are frequently susceptible to microbial contamination, due
to their high water content and the nature of ingredients
that they often contain. This is particularly true with the
trend toward "natural" cosmetic ingredients, which are more
likely to be susceptible to contamination than synthetic
ingredients may be. The occurrence of microbial
contamination in a cosmetic formulation can result in an
unpleasant odor or the destabilization of an emulsion; this
in turn can lead to the necessity of reformulation or recall
of a commercial product.
1
CA 02547745 1998-07-15
73512-12D
To counteract this problem, it is often necessary
to add antimicrobial chemicals, such as preservatives or
biocides, to the formulation to prevent the growth of
microbes that may be introduced during the manufacturing,
filling or use of the product. However, such additives have
recently fallen into disfavor, in large part because many
preservatives are perceived as causing irritation and
consumers are now demanding preservative-free products.
Therefore, the market is shifting toward lower levels of
conventional preservatives, and also the replacement of
conventional preservatives with new molecules.
1a
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WO 99/03446 PCT/US98/14666
Thus, there is now a strong demand for cosmetic formulations
which are non-irritating and free of traditional preservatives,
but which will remain stable and free of contamination in the
hands of the consumer. The present invention provides such
formulations.
Summary of the Invention
The present invention relates to a cosmetic or
pharmaceutical formulation for topical application which
comprises an antimicrobial effective amount of 3-furan carboxylic
acid. In a preferred embodiment, the formulation also comprises
an anti-irritant effective amount of ferulic acid, or a
derivative thereof. In a particularly preferred embodiment, the
formulation also comprises an antimicrobial effective amount of
2-furan carboxylic acid. All such formulations are capable of
being made free of preservatives, and achieve the desired effects
by the use of naturally occurring materials.
The invention also relates to method for reducing the
irritant effect of a cosmetic or pharmaceutical formulation
comprising adding to the formulation an anti-irritant effective
amount of ferulic acid or a derivative or analog thereof, as well
as reducing or preventing irritation on the skin by topical
application to the skin of a formulation containing ferulic acid
or a derivative thereof. The invention further relates to a
method for reducing or preventing inflammation which comprises
applying to the skin an anti-inflammatory effective amount of 2-
furan carboxylic acid, 3-furan carboxylic acid, or a combination
thereof. Also encompassed is a method for inhibiting microbial
growth in a cosmetic or pharmaceutical formulation which
comprises adding to the formulation an antimicrobial effective
amount of 3-furan carboxylic acid.
Also provided by the invention are topical cosmetic and
pharmaceutical compositions useful in the treatment of
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WO 99/03446 PCTlUS98/14666
inflammation in the skin comprising effective amounts of 2-furan
carboxylic acid, 3-furan carboxylic acid, or a mixture thereof,
as well as methods of treating inflammatory reactions in the skin
comprising applying such compositions to the skin.
Brief Description of the Drawings
Figure 1 illustrates the inhibitory effect of (A)ferulic acid;
(B)2-furan carboxylic(2-furoic) acid; and (C)3-furan
carboxylic(3-furoic) acid on phospholipase AZ activity.
Figure 2 illustrates the inhibitory effect of 2-furan carboxylic
acid(2F) and 3-furan carboxylic acid (3F) on the inhibition of
lymphocyte adhesion to endothelial cells.
Detailed Description of the Invention
Furan carboxylic acids are naturally occurring degradation
products from lignin and cellulose. 2-furan carboxylic acid has
previously been disclosed as having antimicrobial activity, and
has been said to be particularly effective against
mycobacteria(US Pat. No. 5,387,605). However, antimicrobial
activity for 3-furan carboxylic acid has not previously been
reported; in the context of the present invention, it has now
been unexpectedly discovered that 3-furan carboxylic acid has
potent antimicrobial properties. Particularly surprising is the
observation that this acid has a greater level of antimicrobial
activity than 2-furan carboxylic acid. In contrast with the
latter, 3-furan carboxylic acid has sufficient antimicrobial
activity to be used as the sole antimicrobial in a cosmetic
formulation over a broad pH range. In particular, it is noted
that 3-furan carboxylic acid is capable of preventing growth of
Enterics, Pseudomonas, Staphylococcus and Mold, and substantially
reducing the growth of yeast; in contrast, although 2-furan
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CA 02547745 1998-07-15
WO 99/03446 PGT/US98/14666
carboxylic acid does exhibit inhibition of Enterics, Pseudomonas,
and Staphylococcus, it has a very poor level of activity against
Mold and yeast, making it inadequate on its own to preserve a
cosmetic formulation. To achieve the desired antimicrobial
effect, 3-furan carboxylic acid alone can be used in the
formulation in an amount of from about 0.05-5$, preferably 0.1-
3$, by weight of the total composition. Although the 2-furan
carboxylic acid alone is insufficient protection, it may be
desirable to use it in combination with 3-furan carboxylic acid
as a supplement to that acid's activity, in an amount of 0.5-5$.
Tn preferred formulations of the invention, both acids are
combined, and in each case, throughout the specification and the
claims, when the acid is referred to, it is understood that this
refers to both the free acid and derivatives thereof having the
same activity. These compounds are available commercially, for
example, from Sigma Chemical.
It has also been observed that both 2- and 3-furan
carboxylic acids possess a surprising level of anti-inflammatory
activity. When tested in standard in vitro assays for anti-
inflammatory activity, both compounds have been shown to be
potent inhibitors of one or more steps in the inflammatory
cascade. Hoth 2- and 3-furan carboxylic exhibit the ability to
inhibit the adhesion of polymorphonuclear leukocytes(PMNs) to
activated endothelial cells, an indicator of chronic, rather than
acute, inflammation. In addition, both compounds are capable of
inhibiting the activity of Phospholipase A2 enzyme, which is a
stimulator of arachidonic acid production; this is an indicator
of inhibition of one of the early events in the inflammatory
cascade of events, indicating an ability to inhibit acute
inflammatory responses. In this particular property, however, 3-
furan carboxylic acid is exceptionally active, inhibiting
activity at a level of O.Olmg/ml. Therefore, in an additional
embodiment of the invention, a cosmetic or pharmaceutical
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WO 99/03446 PC'TNS98/14666
composition for treatment or prevention of inflammation is
provided, the composition comprising from anti-inflammatory
effective amounts of 2-furan carboxylic acid, 3-furan carboxylic
acid, or a mixture thereof. Generally speaking, the effective
amounts of these compounds will be in the same range as required
for their antimicrobial activity. Such compositions are useful
in the treatment and prevention of skin conditions or disorders
which comprise an inflammatory aspect, for example, psoriasis,
eczema, allergic contact dermatitis, or atopic dermatitis.
An additional useful component of the formulation is ferulic
acid or derivatives thereof. Ferulic acid is also a naturally
occurring material; it is found as a free acid in plants, and
also occurs in ester form in seeds, leave and bark of plants.
Esters are formed, for example, with long chain alcohols,
sterols; and hydroxyacids. Ferulic acid has previously been said
to exhibit a variety of biological activities, such as
antioxidant, deodorant, antiinflammatory, antimicrobial and
antipruritic. It has not, however, to the inventors' knowledge,
been identified as an antiirritant. In accordance with the
present invention, ferulic acid has been shown to have
substantial antiirritant properties, which render it particularly
useful in cosmetic formulations, and may be particularly
desirable in combination with an active acid component that has
the potential of causing irritation on the skin. The
antiirritant effect of ferulic acid can be achieved by
application shortly prior to, or simultaneously with, or shortly
after exposure of the skin to an irritating material. It may
also be used alone to generally soothe sensitive, easily
irritated skin. As used herein, the term "ferulic acid" will be
understood to encompass both the free acid and ester forms having
the same activity as the free acid, unless otherwise specified.
In the formulation, the ferulic acid is preferably used in an
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CA 02547745 1998-07-15
WO 99/03446 PCT/US98/14666
amount of from about .05-2.50, more preferably 0.1-1~ by weight
of the total composition.
It is particularly preferred that ferulic acid, or an ester
thereof, is combined with both 2-furan carboxylic acid and 3-
furan carboxylic acid, in the amounts specified above for each
individual component. The formulations containing this
combination exhibit sufficient antimicrobial control to avoid the
use of conventional cosmetic or pharmaceutical preservatives. By
"conventional" preservatives is meant those preservatives which
have been routinely used in the past for inhibition of microbial
growth; these include, but are not limited to, parabens,
propionates, formaldehyde releasers, benzoates, and cresols. An
added advantage of such formulations is that they are not only
inherently non-irritating, but also reduce irritation already
existing on the skin to which they are applied.
In an optional embodiment, the formulations may also
comprise vanillin, vanillic acid, or vanillic acid esters, in an
amount of from about .05-1~. Vanillin is also a naturally
occurring material, which is known to have activity as an
antimicrobial(Principles and Practice of Disinfection,
Preservation and Sterilization, Russell et al, eds., Second
Edition, Blackwell Scientific Publications; Cerrutti et al., J.
Food Sci. 62: 608-610, 1997.)
For topical application, the active components can be
formulated with a variety of cosmetically and/or pharmaceutically
acceptable carriers. The term "pharmaceutically or cosmetically
acceptable carrier" refers to a vehicle, for either
pharmaceutical or cosmetic use, which vehicle delivers the active
components to the intended target and which will not cause harm
to humans or other recipient organisms. As used herein,
"pharmaceutical" or "cosmetic" will be understood to encompass
both human and animal pharmaceuticals or cosmetics. Useful
carriers include, for example, water, acetone, ethanol, ethylene
6
CA 02547745 1998-07-15
glycol, propylene glycol, butane-1,3-diol, isopropyl myristate,
isopropyl palmitate, or mineral oil. Methodology and components
for formulation of cosmetic and_pharmaceutical compositions are
well known, and can be found, for example, in Remington's
Pharmaceutical Sciences, Eighteenth Edition, A.R. Gennaro, Ed.,
Mack Publishing Co. Easton Pennsylvania, 1990. The carrier may
be in any form appropriate to the mode of delivery, for example,
solutions, colloidal dispersions, emulsions(oil-in-water or
water-in-oil), suspensions, creams, lotions, gels, foams,
mousses, sprays and the like. The pH for the formulations of the
invention can be from about 3 to about 8, preferably about 4-7,
and most preferably about 9-6.
The fonaulation, in addition to the carries and the
antimi crobial/antiirritant components, also can compris a other
optional materials which may be chosen depending on the carrier
and/or the intended use of the formulation. Additional
components include, but are not limited to, water solubl a
colorants (such as FD&C Blue #I); oil soluble colorants (such as
D&C Green #6); water soluble sunscreens (such as Eusolex* 232);
oil soluble sunscreens (such as Octyl Methoxycinnamate);
particulate sunscreens (such as Zinc Oxide): antioxidants (such
as HHT); chelating agents (such as Disodium EDTA); emulsion
stabilizers (such as carbomer); preservatives (such as Methyl
Paraben); fragrances (such as pinene); flavoring agents (such as
sorbitol); humectants (such as glycerine); waterproofing agents
(such as PVP/Eicosene Copolymer): water soluble film-formers
(such as Hydroxypropyl methylcellulose); oil-soluble film formers
(such as Hydrogenated C-9 Resin): cationic polymers (such as
Polyquaternium 10); anionic polymers (such as xanthan gum);
vitamins (such as Tocopherol); and the like.
The antimicrobial/antiirritant/antiinflammatory components
are well-suited for combination with other active components
intended for topical application. In particular, the ferulic
*Trade-mark
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WO 99/03446 PCT/US98/14666
acid may assist in reducing the irritating effects of other
active components in a formulation. Examples of known irritants
that are frequently used for therapeutic purposes topically are
retinoids, such as retinol and retinoic acid, and hydroxyacids,
such as glycolic, lactic, or salicylic acids. Examples of other
types of actives which may form part of the composition include,
but are not limited to, those that improve or eradicate age
spots, keratoses and wrinkles, analgesics, anesthetics, anti-acne
agents, antibacterials, antiyeast agents, antifungal agents,
antiviral agents, antidandruff agents, antidermatitis agents,
antipruritic agents, antiemetics, antimotion sickness agents,
anti-inflammatory agents, antihyperkeratolytic agents, anti-dry
skin agents, antiperspirants, antipsoriatic agents,
antiseborrheic agents, hair conditioners and hair treatment
agents, antiaging agents, antiwrinkle agents, antiasthmatic
agents and bronchodilators, sunscreen agents, antihistamine
agents, skin lightening agents, depigmenting agents, wound-
healing agents, vitamins, corticosteroids, tanning agents,
sunscreens or hormones. More specific examples of useful active
agents include retinoids, topical cardiovascular agents,
clotrimazole, ketoconazole, miconozole, griseofulvin,
hydroxyzine, diphenhydramine, pramoxine, lidocaine, procaine,
mepivacaine, monobenzone, erythromycin, tetracycline,
clindamycin, meclocyline, hydroquinone, minocycline, naproxen,
ibuprofen, theophylline, cromolyn, albuterol, retinol, retinoic
acid, 13-cis retinoic acid, hydrocortisone, hydrocortisone 21
acetate, hydrocortisone 17-valerate, hydrocortisone 17-butyrate,
betamethasone valerate, betamethasone diproprionate,
triamcinolone acetonide, fluocinonide, clobetasol, proprionate,
benzoyl peroxide, crotamiton, propranolol, promethazine, vitamin
A palmitate, vitamin E acetate, DHEA and derivatives thereof,
alpha- or beta-hydroxy acids, and mixtures thereof. The amount of
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WO 99/03446 J PCTNS98/14666
active agent to be used in any given formulation is readily
determined in accordance with its usual dosage.
Particularly preferred embodiments of the present
formulations are moisturizing creams or lotions. To that end,
the antimicrobials/antiirritant components are combined with
agents that are moisturizers, emollients or humectants. Examples
of useful combinations are oils, fats, waxes, esters, fatty acid
alcohols, fatty acid ethoxylates, glycols, sugars, hyaluronic
acid and hyaluronates, dimethicone, cyclomethicone, and the like.
Further examples can be found in the International Cosmetic
Ingredient Dictionary, CTFA, Sixth Edition, 1995.
The invention is further illustrated by the following non-
limiting examples.
Application of the formulations of the invention is achieved
in accordance with the nature and intended use of the final
product. For example, a moisturizing, cleansing, or skin-
soothing formulation may be used on a daily basis, or more or
less frequently depending upon need. If the formulation contains
a pharmaceutical or cosmetic active, the application will be in
accordance with the recommended regimen for the active.
Determination of other appropriate application regimens is a
matter of routine optimization.
EXAMPLES
I. This example illustrates the efficacy of 3-furan carboxylic
acid in retarding microbial growth in a cosmetic formulation.
A nonionic oil-in-water emulsion is prepared containing
0.973$ 3-furan carboxylic acid. The formulation is divided into
five portions, each one getting an inoculation at day 0, and a
reinoculation after three weeks, of one of the following
microbial cultures: (1)Enterics; (2)Pseudomonas;
(3)Staphylocorcus; (9)Yeast; and (5)Mold. The concentration of
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WO 99/03446 PCT/US98/14666
microbes in each individual formulation is calculated on the day
of the inoculation, and then again calculated on day 2, and at
the end of one, two, three(pre-reinoculation) and four weeks, at
a variety of pH values. These are also compared with formulations
containing 2-furan carboxylic acid and an identical series of
microbes, and an acid pH control emulsion without a furan
carboxylic acid added. The results are shown in Tables 1, 2 and
3. All numbers, other than those listed as "<10", are log base
10; thus, for example, "6.6" is equivalent to the presence of 8
million bacteria. Entries of <10 indicate less than 10 colony
forming units, the lowest level of detection.
CA 02547745 1998-07-15
WO 99/03446 PCT/US98/14666
Table 1: 0.473 3-furan carboxylic acid
A. pH 3.0
:: ':':::;:::;-'.~2;;;~ . .:.:.::::;, ...:.>.; :: ;; _ ; . ,; . ,:..::
:;.:;.. ~ ....,.:;...;;.>;":::'3~~e~t ~ :.. ~'e~lC
'1 ~ ..... .'fit '~ .. . ... '~te~ ~i .
..............,...............~ ::.............: . ~ ..
.......
. .. .::............. ....... ...... ...
.:,.:.......... .. .. . .. ...:.v..:.
. ..............:. :.. .....:......,....:.:.:v. ,~a,.yp:.;;
'::a.::,.r,.... .:v->:::>:::>.o::.~::::.::.:.>-,..:,~-
::
....;r:.'a~:: :!!\.:>,>7::,va ~b
'::::: "v \ ,
:::'..::'.::.n:...s:......m:t; v!~> .. .
v::: w:.'>n~.::~!wl:.,;!_;a!,.::~-.:~:!.ri:::..:.!~'ss~4.v:..,.-.
..:w...~ \ ::
~::n: ..T
.:... ':.'.'::f::::<:.':'..'.'.....'.'.'a...:-. .:.: v:
av::::::::.>_; ::>:.. ..,
. .. .:::..:y:::n.,~.~X :.: ~X.,s.. _.:,
......su...:.....-....--..............: v::!H::::m:vn:::::-.: ..,
,n.
:>: : . ......... ..':i.:~.'~'.......,.. -
,:,...a...s..,.\,.
.. ..,. .:..: ...........sn...'.'.';.','.'.:-
...\.......\\\\...........,..''.'.-.:,...'.....-..n...,.5. J
:..., .........,.......-..............-........ ... ..,...:~..."
n .< .... ... .::.......-.................... ..:.
...................\\............
. . . .,.... :.. ...........:.. \ :.......
.: ...: . .. ... ,
... . . . ... .
.... . ...-...
1 6.6 <10 <10 v<10 <10 <10
2 6.3 <10 <10 <10 <10 <10
3 6.3 <10 <10 <10 <10 <10
4 5.7 <10 <10 <10 <10 <10
5.2 <10 <10 <10 <10 <10
B. pH 4.0
,, ,.,:."..~.:. .-,::.:.::.::.. : . ... .. .. . . .... .. .
:: v. ::. . .:,. :: .~>.. :.::. . . .. ..
: : :::::..: . . .:.;~.;~.... . ,\x . . :: :: :>:..!.
~.:v. :,::::,:.~..:-:- .;:.:.. :.....:.::;,>: ,: : :.:?:!::.:.
:. . .... ":..:.: .....-...... .:>::. .;...:::.::: ..>?:
... . :.:::::::.::::.;.:.:.:.::.:..: ~::::~o:::::>:.~) .!.>::..>;R.:,.x:
. . ....,.:.. . .:..:.:.~:. .::::::.:::...::..:. .... ..D,,.", .. ....
-:.:.:.......:~::~:::...:::....... .~ :........... ...:~.........
:: :. :.:... ............. .:..~::..a..:_:~-.~:.>.:...
.~...~........... .... -. . .... . ............:.~.... .~ ~~
... ....:::::::..:.:..:. .... ..........- ..............
.... ..........:... . ........-.~::>:::::.~: ~ ..~..........
.. .r. ... ..: .. ....
:>:Y.a..~:.,~ . ..:: ;.:;a:a::::';::.:. .....~_ ............
-::. .. ..............:: f ..
. .. o-:::)x.~::i
: n . ...........:
.. > ::! b.:: :vi<V-:JY>M>>:ihv.-
> !2- ... :b.:;!.f ..:of
. "2f:?:i'f:'vf V:!: :6:0i'2:i:i
..;....-....--.-,~'.: S!.~ :Y!-~\
,...:~.~:.r: ".a:.~.:!.:':<
7 v:-:f.!-(C~-.r?:~>~::. :'af::
. .
a... .!...;-:..: ...............
,...s... a. . . ::>i>
~: .... . t,.. .... .:>:
-.,-.!-..,..i...:(.. w: -':
....,:"..: ~::.,.., . .. .:
....,-. .x!!?.",..::.. \2>.'...
!v,W v::.....:..,': :::$Y.Va:?:....... -1' ._,.::::::::::..
.... ..... .:.. as ..,.2 .,...
:: . . '. . ..., '>'
... ... ..-. .,........::;_
..:..-..:,.:..,. . .,......... . .., ~
:...........................,... ..:. ...
....R?3...>.,.
........ 6.6 :.. ,...:.....,x2 ... . . -.: .-
....v..........,.,......
'"~'-~:'~~ eas<2e::Y.'-.>f:..:...:' , .... ....
1 .::......::.:>.::::::::..
.......................\...............
' . :.,::.:....!. . <10
:.... :::.:..............:... .. ."......rno-.w....,-........,.,.
.........,~.~.a...n:<10 .................................
, <10
~ <10
..............................
~.
<10
2 6.3 <10 <10 <10 <10 <10
3 6.3 <10 <10 <10 <10 <10
4 5.7 2.5 <10 <10 <10 <10
5 5.2 <10 <10 <10 <10 <10
C. pH 5.0
_;:...:;:,,.,,;:..,-~:: .;~9!!!~!!!!n!!!~,sw, ..
.......!.~!!!!~!~!~!!!............ ..
~ :-.. ."-,, t-._ ......
~;~~~:. .:...:ww!P._.... .... ;: ...::..,.-:.;;
., ::xr':?,.:', , : .:~: . ........! .. .:.::-::~:.;
! ,. . :., .. :: ~n2:' 2;:::
' ..'.'... : .>.,.;>;>....:... ' ::a::::.;!.~.;::
r.:7.2!,...: . .. . :.::.. : :, .,-~: ~.>>
::: . ::.. : .::.~::::.u.'. : .,y..: : .,;
' . . ..... ':;t Q, :.~ ...::
..... ." .> : : .. : ..,2!:>:o-y ' ' '::>_.
............ .M..\.S.:.......:. ::;::22>?;:.;:;,
~>. . :.:. :. .;;..!..-: ~ ::f:Y.,. .. ,
:!! :, : :ro-, ....~:.:...,.:....;::: :-:~:G:.~.:::.:-.
::~:a ~ ;:......::::::. o-::.. .. ::.'f::. . ., ,q.
::!n>:>:~>:.... .. - ..;r. .~::>~.....:.:.,.::.
.: . ::~~IB~r::"~.:
i.,,.r.. . > . ...,TN : :.
...:.~ ..... ::.;;;y :.
... ..r:: .",~::::::
.~..,b> n ' ..;..,c ...- :! :2:fiaiia;;:fffv''ri%%:::
i.l...:.:.,.~'aTw.::.'...:
;:; :: f:?aA
:., N:fFi.'WV.... ::-.oT>:i
.3'~::V%- .. ?rr,~r-:.:!
.::.G~:&..: .,y..:.:.o-% _
!i>:v iw !J'.2:ii::,"..
-:."S.. ....
5.:.:.......... <ff:'v'?A:~<
., '~.o-a~;:wo: ::: :iC!>
tto-: '>. !~!:rii8?Jr':-:-:%%;:if>.,..2J::. ..: r:!
%~>f~:~. ~ ~.f:.::~:v;.'......5 .o-.
:: : , ....:.:.:.:
............,:...'~:.:-::o: ..!.x.:2~:o->. .::..,
.:::i?::.:.......
,. >.....:' :. ........".....-..,.':.. ".,
. .- .. . .. . . ....
.:....:..-.... ....:....;::..r....:;.: . :... .
.............
...........................n...........:.::.,:.::>!:..,.::.'4:u..............~,
a.-................. !.. :....a.n ,. .
...:.' . .. . ..
... .......................... ~ ..........s...t..... .,
.. . .......-:.~..: .na. ....................,.":......-.
... :::.::: .... ... ......
::.._:.:..o-r ..:......
:
Y
1 6.6 <10 <10 <10 <10 <10
2 6.3 <10 <10 <10 <10 <10
3 6.3 <10 <10 <10 <10 <10
4 5.7 2.5 2.7 1.7 2.0 1.8
5 5.2 <10 <10 <10 <10 <10
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WO 99/03446 PCT/US98/14666
Table 2: 0.375 2-furan carboxylic acid
A. pH 3.0
_. ~w.w.....,.w....., .. ...... .:. ..
' .. . .....;::::.. .. :::: ~.:: . . .: ...:
..; ' ::.::<.a. :::y~::": ~ .
: ~ ~ ;:.. ~ ~ ~ ~e~lr~t ~8~
~0~3~ '~'3T . ~ ::
.
....
:'
~
>'t~
. ... ... ....................:.:.... . ..
:.................:..:,. .
.....:...........:...............:
..................................:.:..:.........: ....... ::.:..:....;
:::........................,..............::...::.. .
..........::............. ,................... .. ,.....:::..,......._,_.,
.................. ,.. ... .........:.v.v...,...,.. ,
.....:...:.., ,..,..:...,...
.............v~..:.............:.. ....................... . ...
...,........ :.............. ...........
... ...........:.. .................:....:~ ....
....:.....:.>.:.::......:.........". ... ........ ....
.................... ._. :.....:,...,.v.:.:.:.. ... .
..,.......... ...
:.,........,.................... ........................,. . . :
.. . ..
............~.."...,. : .....:.:......:
:...."...:.:.,.,.........:::>.. . .:,:
..4~........t..... .............:._::.:.<:.... ......... ~. .. .
............,.........:...................................... .v.
~ .....
~ . : :.......,:.:.:...,..,......"....
...:.:........................,. ...............:..::,.:::.,..
. :~
1 6. <10 <10 <10 <10 <10
6
2 6.3 <10 <10 <10 <10 <10
3 6.3 <10 <10 <10 <10 <10
4 5.7 <10 <10 <10 <10 <10
5.2 <10 <10 <10 <10 <10
B. pH 4.0
"it ::::
;, .: :a ....:: :, :: .:
: : .. .. ;> . .:. : ...:.:~....::.. :.:.::; ..
...: :.. .. . ..::::::YY:.:,.<.. >< >>:
.pl ~. ...Y ..:.. . 3c...............:< . :..,...:..:..
t..,.
.::.. . .. .......: ..... :. :::
................. . .................... ....: .. ...
:.::t.::..:
. .........:.Q...:.............~''...:.......... .. . ~c...~.........:~:
#t
.. ... .......... ............ ' .' . ... .C....
.................. .......... . ......................:......:::
..........
.. . ...... . ........."~ ...........................
.. . .... ......................~...3..... . ....
.........,... ~' ... .....'.................... :
. .'... . ...................... ,
..:...... ...........
:
.................... ......... .. ....................... ....... .
...................
....... .... .;A;::.:.Y:. ............ ... .. . .
... .....
........::.:5:...S.:.YY:;:S,.::...:Y<.:..;:::.:><s...:>.:>:......... . .....
...::4: ...
::::.:_'..i.;.;::;.:ii:.::>::::.:>;SS.... .. .
Y:S.~..,.:,:.:.,Y:.Y,...::.::::a4: . . .:.:..:.:.:.,'.:.;a:.:'.-
:.YYA::Y,;
.. . .Y S.Yi:.Y:i:::::::....................... :
.......:,..Y::..::..:._.,:.:::..,.
....................>.:::..::.~....:... ... .:.:.,...................,.......
.:.:_:. . . ::.::::....:.
................................~ .
....................:..,...............:...... ..::i:.x:.,>.".::..,::.i.,~. .
..
.. . ~..........~ ........... ... r.............. ....
.. ...,.......... .. ..:..".. . .,. ..
..x. .~ . . .. . ......
.. .... ...... .. .:.s... ........
..........:..~ ........,..........t...........
..... ..:.. .. ."
......".................... . :.:..:.:..
...:............c.....::..:..:.;:.. .
.........,............:..:..........:::. .
:::>:.:.,Y~:..:...................
:.........
.T
....:..................,..........
.:.
.......................
l 6.6 <10 <10 <10 <10 <10
2 6.3 <10 <10 <10 <10 <10
3 6.3 <10 <10 <10 <10 <10
4 5.7 3.7 3.7 <10 <10 >5.7
5 5.2 1.6 <10 <10 <10 <10
C. pH 5.0
:::. .: ... :.;:::::..:.:..... ..... .. ~!w!!!!~.~w.wwww www
-::: . .:: .. .............. r- :-:::: ......::x:r
: -:: :. ::.: . ~! .. ..... .: .., :,.;;.,;;.
-:. ~ ,.::.:. ................ :.iii:Y,.._._. :::-..:.c:
~. 7 .. :....;~,:~::~:;.~.___.:.Y;~::r....... ' '~ .....::";:::;
:.... .. . k ,v:. : ~~:.::.::YY\4,: .,::_~.
:: :. .,.,. :~ :.. .----;,-~~;-:..Y;.atv::J6' .a:.;ac>;;.i
...Y.,i:~:::::::.;::::.......:.~ ~ .: . . :4SlY.SJ:'.
.Yl~. i ~:n !-. ... . .. . ~::v. ' :1v'
., . )j .. . . . ..............;........ . .:\
':':. HI;I :.:J . ~ .. .. . ::. v::n~:
~.: ~ t,K .......-.. ............. ... .... S~.
:. :.. , ~~ .~. ::: :........~....
:>..:::........... .. . . :...,.: ...~....................
:..:::: ..., . .............. .n. ._::.::..:.. . .
~: : .,.::::::::..v.~::. ............ . .. :... . . .....
.,::.,.. w:n:~ . .. ...... . ..........
..... ..... ...............: . :....
... . ..... v... . ................
..........
-:::::::::: ..... .... -_: :t4i~..:
:v:: n . :;: ... t.'o'"::
..nv: ..., :ttt?i ........-'i'YR~2'.':::J:;w:~i~':.
w:::R:::v:.......:n ...
~: .. .i:Y1"?'..i::<ty,:\>Y~!Y~...:::::::::i.i
>.. ' ~" -
.::3Ja;5iii::igS::~.. Y iY: s..n~24R
.,:J- . t, .~.:_:-:... .tfi=:.,... 1..~..:::a...
. 4._.'tax... : 't' ... t,:
. ~a X. .Y.~LYi~:: . .. .
!:~:. ~'...............TiiiZw...:2:w . ..
s:;:'v,'!::: .415.:.,:Y::.~.:vvi~ ......,'/.
~~...n..................::':$ . ' i.k~.:" .., .: -: :3
,:'~'.~ii:~..,~.:..~......aw....n...............:L:,...........f.'N.?o.::.
.;:,_.,y,,..~:::,:::Y:::'~'...'
. .......n..~.:: w . .'~ <10
....:::::~........::::...................:..... ...:.....:: . <10
:................ .:..." >6 .. ~
:.a,.. :~ .
1 ..................:4iJ>6 ...4...
.......... ..n.
., ...
:.::... ..
. ..?m:-.........::::.Y
6.6 <10
2 6.3 >6 <10 <10 <10 <10
3 6.3 2.9 <10 <10 <10 <10
4 5.7 >5.7 >5.7 >5.7 >5.7 >5.7
5 5.2 5.2 5.2 -5.2 3.0 >5
12
CA 02547745 1998-07-15
WO 99/03446 PCT/US98/14666
Table 3: Control emulsion
A. pH 3.0
J
_: ::~:;>::<::. .,;'7.
::. 5.,.:. .: .,n.,,?? > ~ : ~~ ,\
,. ~ ,. '. ~ k1i'25:' ~ \.y~
...::f.. -. .v.: xi-i' .. . v:. -.v. .. ~::.
.:. .n::: .y.5:.:,:..::;. , . ..: . . .:'v . ..:.:.,
n .. .... , ::: ......; .::..
:..,x..vb............ .. .,..'....:....Jx:..x: ...
. ....5.,. h-x-::. .-
.i'$:-~:~ulr!u.. . T..... . ,x .,\ ,:
..:: ~~~ .
/ 'aG,.tv 5x.....,.o:,v.~t'v::a:,:'::\bv.
1:5. ... '- :S x. 5:.
J:i't~. -.v.v
~ '- 7. .\c t'": i:%"v:
::" ~ ::J '~:', ~.''-:
:. i~.~\'-~;'; ,.. 7 . ..'.A
_=:=\ a . :.
:::- ... "2:"..tx, . . S. . , 5:.:
-.u 't'::::xv. :: ":n: S ~'::
.. w ~
. \-: .. :' . v-::::._:
,-:..., '~ '~sx'=i~:::_': C \..... v:. .~
' ~x. =.'-:.':"'to:-,:
r..~\\..:-... \- . ' "' v:..::w :> ...\
....~i. -.. . ~.
... v'=\ fr ?~~it::2~:..~4...._\
...ax\\\,:::....,~15.....L\
:=s. :-._:-.\ ~~: r:~- .\, .
~a::::. ~ :: ...\ ~n .... .:... ,.\\4......x.J\w~.....-.x'\\.-.,
. \.. -.. ~~....-..x.........x:v. ._....
.. .::..:'h'~'~''~~w;>:....~ ..... . ....._... .. .....
.. ...'1 \ , .,.. . .
- ..757::. ...... .'S\Y1" .
:._" ,. .. .
., .. ,.....:x.
.'~~-.
1 6.6 3.7 <10 <10 <10 <IO
2 6.3 >6 <10 <10 <10 <10
3 6.3 2.0 <10 <10 <10 <10
4 5.7 5.7 5.7 5.7 4.5 >6
5.2 5.2 5.2 5.2 5.2 >5
B. pH 9.0
.:.:.: ::...:t:.::_::::::... .:::._:.. ............ __......... . .
-:: .. . -.::._::.. . _.:. . ........ ...._.. ..
... ~-..".-:.: . >...~::.::57 .. ... ...........,:::\r:
:::,J-:3 x.. . i.: ~,p;p: ...
d. . '':J' ...5 ...
.., d:w . t.~.,.:::
, . ,.. -.! '. ...
,x '..::.. .: . ......... . , bv:;'
.......... .... .... ....x 1..;.,'S;.Sj
~:7x ::?v.'. . . ::.';:-.'!!::i...a '. :)-y . '.
.- t:..-'.;... ; ' ....:. 7 xv :.:557JJ a
.i .. . . . . ... .:~.. .< x . ': .v ..:7,
:::.,.;...::,' :.........,.. :.....v. r. . . .,..
....:....~7vo. ,: .Y.?....~ ...:. ' , . .
-::.v7 t. :. .... .. ,......._:: _,
.:,. : ,..x,.......: , : c5:t ... ~2, ::! ~:
~\ :.v..?4y
'5 ' y '..... ~'~ . . .. : t
'b :;a v'. . ..
% C ' ~~ ,; x!wS.w:t.::iY::x
~ ! ~ :.:: .~ ;
~ ,.
' :.. ~
~:
v
-5:5:.:J:?
7: : ..
, ;;?7
. Y.".. ~.. "': 5jt;~~vjt:;ii:!'' :::. .a<a:::v.
.... ....x , - O:W
. .ijj s -~'> < t
::v .) S: a% ~:=is%iv.
:f ~7 ~ .:4>tiv
x . i7 5r t. :g: . :: ..~. \-:::b.
..\,....,~a .~ ....:Ge::. -.O. ?
'? -. t i :5>x. r.
.. 3 wW:~''"~:;."S:J
I ... :C::xY'... , ..k~.
C::.x.:. ::~Y:: :a:.f,:.. .'."5
v a..:. . . '\::f~:y;,.'~,.
v. 2.x
:..C,",.x
:1\:b..y:- ~.'!::. .: :.:...~w.....'~-:':: ,...:..,:,t..x~,
H axi~~:~:'t: .yy::!x
?. x. .::v\t' ' .':.:,.::a.::.~,,tt:x,.u>
:J ...... '~iy~~..:t..::5~.,,\:.:-.::
,.n..:57::-:.. ;cr:....x
:::.
1 6.6 > >6 6 <10 >6
3.2
2 6.3 >6 >6 <10 <10 <10
3 6.3 >6 2.0 2.0 <10 <10
d 5.7 >6 >6 >6 >6 >6
5 5.2 5.2 5.2 5.2 3.3 >5
5
C. pH 5.0
...5:.7:::7_'~ - v~:::--:: .. ".y....,w
w -:J: \ .. 1 . , ':.,
, \\--' ...: \ --:::
:..'.4. p n:!p.~ :
: .4.
.. _ '..a
\.. :i::'~
-"~.
-n' . .v.v .x
'
,5 v::.v.v ._
. .. ,. :.:..:.:. ;~_ . .- :..\_.,:
:: _
7 S ._.. A. .:~: a
... 9-. ~'~ _~-.
h .. ' :I7~ ::."-\ ,;
:z>7~:' H 4.5 .i -. .:.:~. _
7 ~,::.-x [~ --.~ .-
. ' '' 1 . . . .s
~~R7- itt?-= ~_ ' ...y .:
3e: - -.g. f5.t 'S r
:.. . -5i: ' :.<noy7~, J
'as:... ~_:._. ~
f.'~'~4.-::.~:::....~':'<''.:. .: .: ,. ~.....
1:..~5: ~H~-. :;a ":o' :a
. ~. . .:
-vi "- ': ~'.'l >T d5.
:::..~".'..7.N...~:... %: ~ . . . . .. k:.. '~
r .. ~...a~.~...,..
, :......pp..a . :,~C'.'-:.... ,.'..\tn;!. : -.,~, ~ .7
~y; :yp'% ~ t~. ...h 2-0 '.6.
' . .
: t.J '%:. >6
h :J'y '.
- :,6
..~:~:$
...:..~,,....c
"?t'.h!
:..:.w......
:...>6
.
2 6.3 >6 >6 <10 <10 <10
3 6.3 >6 >6 >6 4.2 <10
4 5.7 >6 >6 >6 >6 >6
5 5.2 >5.2 5.2 5.2 3.0 >6
Comparison of the antimicrobial effects of the two furan
carboxylic acids shows that the 3-furan carboxylic acid is
superior to 2-furan carboxylic acid in its ability to retard the
growth of mold and yeast. The control shows that the
antimicrobial effect of these two acids is due to the
13
CA 02547745 1998-07-15
characteristics of the specific acid, rather than the acid pH or
the vehicle used.
Example 2: This example illustrates the antiirritant effect of
the components of the invention:
Seven volunteers with a history of skin sensitivity to
Balsam of Peru are chosen for the study: The test compounds
studied are as follows: Ferulic acid, 0.1$ in hydroalcohol 1:1:
2-furan carboxylic acid, 0.1~ in hydroalcohol 1:1: and 3-furan
carboxylic acid, 0.1$ in hydroalcohol, 1:1.
The test compounds are applied to the ventral forearms of
panelists. The material is allowed to absorb for twenty minutes
and then Balsam of Peru, an irritant is applied on the test
sites. Skin irritation is measured in terms of increase in skin
redness. The degree of redness is measured with a Minolta
Chromameter and compared with the controls, the positive control
being skin treated with Balsam of Peru alone, and the negative
control being a skin site treated with 10~ cola solution(a known
antiirritant), and challenged like the test products.
Ferulic acid is 72~ effective in preventing the onset of
irritation. This compares favorably with the cola solution,
which shows 69~ reduction. Interestingly, both 2- and 3-furan
carboxylic acid show some reduction in the onset of irritation,
at 49~ and 48~, respectively.
Example 3: This example illustrates the anti-inflammatory effect
of 2- and 3-furan carboxylic acids, in two different aspects of
the inflammatory cascade.
A. PLAN (phospholipase AZ) inhibi_ti_on
The enzyme reaction is accomplished in a cuvette containing 2m of
phosphate buffered saline. Fluorometric substrate NHD-C6-HPC is
*Trade-mark
19
CA 02547745 1998-07-15
73512-12D
solubilized 1 mM in EtOH, and is diluted in the reaction mixture
to yield a lO~tM final concentration. The assay is initiated with
the addition of .001 units per ~tl of PLA~ enzyme obtained from
snake venom. Enzyme activity is monitored either with or without
the presence of potential inhibitors. Materials tested are
solubilized as follows: ferulic acid and 2-furan carboxylic acid
are solubilized lOmg/ml in EtOH, and 3-furan carboxylic acid is
solubilized 100mg/ml in EtOH; each sample is diluted
appropriately to yield assay concentrations of .01, .1 and 1
mg/ml. Prior to enzyme addition, samples are measured
fluorometrically at excitation 470 and emission 570 to determine
background level of fluorescensce. Following enzyme addition,
samples are monitored every 15 minutes over a 60 minute period.
EC-50 values are calculated, but are limited to those
samples which best yield a linear fit. Of those calculable in
this manner, 2-furan carboxylic acid yielded a .5mg/ml
valueiFigure 1B). Although it was not possible to calculate an
EC-50 for 3-furan carboxylic acid, as shown in Figure 1C, it
provides the greatest inhibition by blocking the enzyme activity
at .lmg/ml dose. Ferulic acid also shows some inhibitory effect,
but is the weakest of the three tested(Figure 1A).
B. Inhibition of lymphocyte adhesion
Endothelial cells are grown to confluence and washed thoroughly
with basal media prior to the experiment to remove all fetal calf
serum. The test compounds are then added to the endothelial
culture well and incubated for 2 hours. IL-1~ is added to
stimulated expression of adhesion molecules on the endothelial
cells before lymphocytes are added.
Lymphocytes are isolated from blood collected from adult
human subjects and added to the endothelial culture wells. PHA
is added to stimulate adhesion molecule expression in the
lymphocytes and the cultures incubated overnight. After a brief
CA 02547745 1998-07-15
WO 99/03446 PGTNS98/14666
washing procedure, cells are incubated with Rose Bengal, and the
number of adherent cells assessed by measuring the absorbance at
570nm.
Figure 2 illustrates that both 2- and 3-furan carboxylic acid are
very potent inhibitors of lymphocyte adhesion at concentrations
of 0.5o and 1~. The first bar shows the absorbence of
endothelial cells plus any adherent lymphocytes under non-
stimulated conditions. The second shows the increase in
absorbance after overnight incubation with PHA. The next four
bars show the decrease in absorbance back to non-stimulated
conditions in cultures preincubated with either 0.5$ or to of the
two acids.
16
