Polyunsaturated fatty acid and diol ester as an anti-acne agent

13 Pages·2014·1.15 MB·English

US008623916B2
(12) United States Patent (10) Patent N0.: US 8,623,916 B2
Redoules et a]. (45) Date of Patent: Jan. 7, 2014
(54) POLYUNSATURATED FATTY ACID AND W0 WO 98/18751 5/1998
DIOL ESTER AS AN ANTI-ACNE AGENT W0 WO 01/38288 A1 5/2001
W0 WO 2006/019186 A1 2/2006
(75) Inventors: Daniel Redoules, Toulouse (FR); Sylvie OTHER PUBLICATIONS
Daunes-Marion, Toulouse (FR);
Abd El All et al., “Immunohistochemical Expression of Interleukin 8
Marie-Francoise Aries, Escalquens
in Skin Biopsies from Patients With In?ammatory Acne Vulgaris,”
(FR)
Diagnostic Pathology, vol. 2, No. 4, 2007, 6 pages.
Brown et al., “AcneVulgaris,” Seminar, The Lancet, vol. 351, Jun. 20,
(73) Assignee: Pierre Fabre Dermo-Cosmetique,
1998, pp. 1871-1876.
Boulogne-Billancourt (FR) Calder, “Polyunsaturated Fatty Acids, In?ammation, and Immunity,”
Lipids, vol. 36, No. 9, 2001, pp. 1007-1024.
Notice: Subject to any disclaimer, the term of this Deangelis et al., “Isolation and Expression of a MalasseZia Globosa
Lipase Gene, LIPl,” Journal of Investigative Dermatology, vol. 127,
patent is extended or adjusted under 35
2007, pp. 2138-2146.
USC 154(b) by 236 days. Downie et al., “Peroxisome Proliferator-Activated Receptor and
Farnesoid X Receptor Ligands Differentially Regulate Sebaceous
(21) Appl. N0.: 13/130,900 Differentiation in Human Sebaceous Gland Organ Cultures In Vitro,”
British Journal of Dermatology, vol. 151, 2004, pp. 766-775.
(22) PCT Filed: Dec. 21, 2009 Faergemann et al., “The Antimycotic Activity In Vitro of Five
Diols,”Sabouraudia, vol. 18, 1980, pp. 287-293.
(86) PCT N0.: PCT/EP2009/067701 Faergemann et al., “The In Vitro Activity of Pentane-1,5-diol against
Aerobic Bacteria. A New Antimicrobial Agent for Topical Usage?”
§ 371 (0X1)’ Acta Derm Venereol, vol. 85, 2005, pp. 203-205.
(2), (4) Date: May 24, 2011 Frankenfeld et al., “Preservation of Grain With Aliphatic 1,3-Diols
and Their Esters,” Journal of Agricultural and Food Chemistry, vol.
(87) PCT Pub. No.: WO2010/072738 23, No. 3, 1975, pp. 418-425.
Harb et al., “Inhibitory Effect of 1,3-Butylene Glycol on Microor
PCT Pub. Date: Jul. 1, 2010 ganisms,” Drug & Cosmetic Industry, May 1976, pp. 40-41 and
136-137.
(65) Prior Publication Data Ren et al., “Anti-In?ammatory Effect of ot-Linolenic Acid and Its
Mode of Action through the Inhibition of Nitric Oxide Production
US 2011/0230557 A1 Sep. 22, 2011
and Inducible Nitric Oxide Synthase Gene Expression via
NF-KB . . . ,” Journal of Agricultural and Food Chemistry, vol. 55,
(30) Foreign Application Priority Data
2007, pp. 5073-5080 (published on Web: Jun. 2, 2007).
Shalita, “The Integral Role of Topical and Oral Retinoids in the Early
Dec. 22, 2008 (FR) .................................... .. 08 58967 Treatment of Acne,” Journal of the European Academy of Dermatol
ogy and Venereology, vol. 15, Supple. 3, 2001, pp. 43-49.
(51) Int. Cl. Sugiura et al., “Evidence That the Cannabinoid CB1 Receptor Is a
A61K 31/22 (2006.01) 2-Arachidonoylglycerol Receptor,” The Journal of Biological Chem
A23D 9/00 (2006.01) istry, vol. 274, No. 5, Jan. 29, 1999, pp. 2794-2801.
C11C3/00 (2006.01) International Search Report dated Feb. 19, 2010 for PCT/EP2009/
(52) US. Cl. 067701 .
USPC .......................... .. 514/549; 554/172; 554/224
Primary Examiner * Deborah D Carr
(58) Field of Classi?cation Search
(74) Attorney, Agent, or Firm * Birch, SteWart, Kolasch &
USPC ................................. .. 514/549; 554/172, 224 Birch, LLP.
See application ?le for complete search history.
(57) ABSTRACT
(56) References Cited
The present invention relates to compounds of the following
U.S. PATENT DOCUMENTS formula (I): Where n is an integer betWeen 1 and 15, m is 0, 1,
2 or 3, and R is the hydrocarbon chain of a polyunsaturated
2008/0076699 A1 3/2008 Ley et al.
fatty acid selected from among omega 3 and omega 6, as Well
as to pharmaceutical or cosmetic compositions containing
FOREIGN PATENT DOCUMENTS
same, to a method for preparing and using same, in particular
EP 1902632 A1 3/2008 for treating acne and seborrhoeic dermatitis.
W0 WO 96/34846 11/1996
W0 WO 98/13330 A1 4/1998 19 Claims, 3 Drawing Sheets
US. Patent Jan. 7, 2014 Sheet 1 of3 US 8,623,916 B2
Figure 1
P. a C n e ipase, 33 kDa
Figure 2
US. Patent Jan. 7, 2014 Sheet 2 of3 US 8,623,916 B2
Figure 3
3000
Control PMA+A23187 “1-6 00-3
v-lenolenic acid ol-lenolenic acid
US. Patent Jan. 7, 2014 Sheet 3 of3 US 8,623,916 B2
Figure 4A
Figure 4B
Figure
6000
2000
US 8,623,916 B2
1 2
POLYUNSATURATED FATTY ACID AND R acnes, via receptors of the innate immune system, induce
DIOL ESTER AS AN ANTI-ACNE AGENT the production of NF-KB-dependent proin?ammatory cytok
ines such as IL-8, These mediators then in?uence in particular
The present invention relates to esters of an alkanediol and the migration of polynuclear neutrophils toWard the site of
a polyunsaturated fatty acid, more particularly an omega-3 or in?ammation, Where their mission is to kill bacteria. This
omega-6 fatty acid, as Well as to pharmaceutical and cosmetic in?ammatory response is normal and necessary for the elimi
compositions containing same, to a method for preparing
nation of the pathogen in the infected tissue. HoWever, exces
same and to the use of same, in particular to treat acne or sive and uncontrolled activation leads to in?ammatory acne
seborrheic dermatitis.
lesions. Thus, it has been shoWn that IL-8 level is correlated to
Alkanediols are compounds used in numerous ?elds such
the number of neutrophils mobilized in the in?ammatory
as cosmetics and agri-foods. Their use as a preservative, by
acne lesion (Abd El All H S et al. Diagn. Pathol. 2007; 2: 4).
virtue of their bacteriostatic properties, can be cited in par
Bacterial colonization in acne patients is most often asso
ticular. Thus, alkanediols constitute a means to control fungal
ciated With the appearance of in?ammatory acne lesions: for
and bacterial colonization and help protect many cosmetic or
example, a greater number of polymnuclear neutrophils and
agri-food products (Faergemann J, Fredriksson T. Sabourau
higher levels of IL-8 are found Within the highly-colonized
dia: 1980; 18, 287-293). These diols have a broad spectrum of
activity and are in particular effective With respect to species follicular canals compared to the loWly-colonized follicular
of fungi and Gram-positive bacteria (Harb N A, Toama M A. canals of subjects Without acne. The levels of these in?am
Drug Cosmet Ind: 1976; 1 18, 40). Moreover, the near absence 20 matory markers appear correlated With bacterial load. But
of acquired resistance in microorganisms enables alkanediols many questions remain unansWered such as the cause of
to be important tools in the development of anti-resistance colonization and the series of steps leading to lesion forrna
strategies, in particular With respect to Staphylococcus aureus tion. In any event, the role of bacterial colonization as a factor
(Faergemann J, Hedner T, Larsson P: 2005; 85, 203-205; WO in the progression of the disease is proven.
2004/112765). Finally, their very good tolerance enables 25 The current treatment for minor to moderate or in?amma
them to be used frequently and at doses exceeding several
tory acne corresponds to a topical application of antibacterial
percent.
active agents acting against colonization in particular by R
In particular, 1,2-alkanediols have bacteriostatic activities
acnes in combination With anti-in?ammatories (Shalita A., J.
and are Widely used as preservatives (JP-A-51091327) or in
Eur. Acad. Dermatol. Venereol. 2001; 15: 43).
the treatment of pathologies such as acne in Which the micro 30
bial component plays a key role in etiology (US. Pat. No. Treatments are generally initiated after the appearance of a
6,123,953). Other applications of 1,2-alkanediols are also certain number of in?ammatory acne lesions.
described, such as protective properties With respect to body One of the major problems of anti-acne therapy is to ?nd an
odors by virtue of their antiseptic (US. Pat. No. 5,516,510; adapted treatment, Which is to say one proportional to the
WO 2003/000220) or antifungal (WO 2003/069994) effect. 35 severity of the acne, that is begun as soon as possible, Which
Similarly, combinations of 1,2-alkanediols With other com is to say as of the initial colonization, against the development
pounds are described Wherein the result is a synergistic anti of in?ammatory acne lesions.
microbial effect. Thus, in this context, combinations to con
The Inventors thus discovered in a surprising Way that
trol microorganisms at the origin of body odors (US 2005/
esters of an alkanediol and a polyunsaturated fatty acid, and
228032) or microorganisms involved in the formation of acne 40
more particularly an omega-3 or omega-6 polyunsaturated
lesions (US 2007/265352, EP 1598064) have been claimed.
fatty acid, have an antibacterial and anti-in?ammatory action
Polyunsaturated fatty acids (PUFAs) are divided into tWo
categories: omega-3 (00-3) and omega-6 (00-6). Besides their as of the initial colonization by R acnes in the follicular canal,
metabolic effects, they are able to modify the expression of Wherein this action is in addition proportional to the coloni
genes coding for intracellular proteins. Such gene effects of 45 zation.
PUFAs seem to be carried out via nuclear receptors called Indeed, it turned out that alkanediol and PUFA esters are
PPARs (peroxisome proliferator-activated receptors). PPARs recognized and cleaved speci?cally by the bacterial (R acnes)
belong to the family of nuclear steroid hormone receptors. lipase, thus enabling, by cleavage of the ester bond, the
They form heterodimers With the retinoid X receptor (RXR)
release of the tWo active agents With complementary activi
of retinoic acid and modulate gene expression. Thus, 00-3 50 ties, namely the antibacterial diol, capable of controlling
PUFAs Would be negative regulators of the in?ammatory
colonization by R acnes, and the anti-in?ammatory PUFA,
response by inhibiting the NF-KB activation pathWay via the
induction of the expression of IKBot, Which is the major Which blocks the recruitment of neutrophils and thus the
inhibiter of the NF-KB pathWay (Ren J and Chung S H. J in?ammatory cascade characteristic of acne. The release of
Agric Food Chem. 2007 55:5073-80). Moreover, 00-3 PUFAs 55 these tWo active agents thus induces an adapted response,
have an inhibiting action on the synthesis of arachidonic acid Which is to say one proportional to the colonization by R
to the bene?t of the synthesis of docosahexaenoic and eicosa acnes, as of the initial colonization and thus blocks the devel
pentaenoic acids (Calder P C. Lipids: 2001; 36, 1007-24). opment of this pathology responsible for the appearance of
During acne, excess sebum in the follicular infundibulum
in?ammatory acne lesions. In addition, since P acnes bacte
represents a favorable environment for colonization by Pro 60 ria are present in subjects Who do not have acne lesions, these
pionibaclerium acnes (P acnes). A correlation can thus be
esters also make it possible to prevent aggravation of the acne
established betWeen the degree of colonization by R acnes of
lesion by acting as of the formation of the comedo and by
the pilosebaceous canal and the appearance of a micro
inhibiting the in?ammatory cascade characteristic of acne.
comedo. Moreover, it has been shoWn that this colonization is
greater in subjects With acne compared to healthy subjects 65 Such esters have already been described in the literature
(BroWn S, Shalita A. Acne vulgaris. Lancet 1998; 351: 1871 (WO 98/18751; Sugiura et al., J. Biol. Chem. 1999, 274(5),
6). 2794-2801) but not for their biological properties.
US 8,623,916 B2
3 4
Thus, the present invention relates to a compound of the above, Wherein the ?rst double bond of the chain corresponds
following general formula (I): to the third carbon-carbon bond counting from the end oppo
site to the carboxylic acid function, as is illustrated in the case
of the ot-linolenic acid beloW:
(I)
HO
Said omega-3 fatty acids can be in particular ot-linolenic
acid, stearidonic acid, eicosatrienoic acid, eicosatetraenoic
Wherein: acid, eicosapentaenoic acid, docosapentaenoic acid, docosa
n is an integer betWeen 1 and 15, preferably betWeen 1 and hexaenoic acid, tetracosapentaenoic acid and tetracosa
10, hexaenoic acid, and preferably it is ot-linolenic acid or steari
mis 0,1,2 or 3, and donic acid, Which have anti-in?ammatory properties.
R is the hydrocarbon chain of a polyunsaturated fatty acid In the context of the present invention, “omega-6 fatty
selected from omega-3 and omega-6 polyunsaturated acid” refers to a polyunsaturated fatty acid, such as de?ned
fatty acids. above, Wherein the ?rst double bond of the chain corresponds
In the context of the present invention, “polyunsaturated 20 to the sixth carbon-carbon bond counting from the end oppo
fatty acid” refers to a linear carboxylic acid (R1CO2H) com site to the carboxylic acid function, as is illustrated in the case
prising from 10 to 28, preferably from 16 to 24, more prefer of the linoleic acid beloW:
ably from 18 to 22, carbon atoms (including the carbon atom
of the carboxylic acid function) and comprising at least 2,
preferably 2 to 6, C:C double bonds, Wherein said double
25
bonds preferably have a cis con?guration.
In the context of the present invention, “hydrocarbon chain HO
of a polyunsaturated fatty acid” refers to the hydrocarbon
chain (R1) linked to the acid function of the polyunsaturated Said omega-6 fatty acids can be in particular linoleic acid,
fatty acid (R1 COZH). R1 thus represents a linear hydrocarbon 30 y-linolenic acid, eicosadienoic acid, dihomo-y-linolenic acid,
chain comprising from 9 to 27, preferably from 15 to 23, more arachidonic acid, docosatetraenoic acid, docosapentaenoic
preferably from 17 to 21, carbon atoms and comprising at acid, adrenic acid and calendic acid, and preferably it is
least 2, preferably 2 to 6, C:C double bonds, Wherein said linoleic acid, Which has sebum control properties.
double bonds preferably have a cis con?guration. Thus, in the In particular, n can be 1, 2, 3, 4 or 5, preferably 5. Advan
case of the linoleic acid of the folloWing formula: 35 tageously, n23 and preferably n25.
Advantageously, m is 0 or 1.
Advantageously, n+mz3 and preferably n+mz5.
Advantageously, the hydrocarbon chain comes from a
polyunsaturated fatty acid selected from ot-linolenic acid,
40 stearidonic acid, eicosatrienoic acid, eicosatetraenoic acid,
eicosapentaenoic acid, docosapentaenoic acid, docosa
hexaenoic acid, tetracosapentaenoic acid, tetracosahexaenoic
acid, linoleic acid, y-linolenic acid, eicosadienoic acid,
dihomo-y-linolenic acid, arachidonic acid, docosatetraenoic
45 acid, docosapentaenoic acid, adrenic acid and calendic acid.
Preferably, the polyunsaturated fatty acid Will be selected
from ot-linolenic acid, stearidonic acid and linoleic acid, more
preferably from ot-linolenic acid and linoleic acid.
In the context of the present invention, “omega-3 fatty In particular, the compounds of the invention can be
acid” refers to a polyunsaturated fatty acid, such as de?ned selected from the folloWing molecules:
bow
W
OH
US 8,623,916 B2
-continued
W0
OH
W
15
The present invention further relates to a compound of the The present invention further relates to a method for the
formula (I) as de?ned above for the use of same as a drug, in cosmetic treatment of acne or seborrheic dermatitis compris
particular intended to treat acne or seborrheic dermatitis. ing the application on the skin of a cosmetic composition such
The present invention further relates to the use of a com as de?ned above.
pound of the formula (I) as de?ned above for the manufacture The present invention further relates to a method for pre
20
of a drug, in particular intended to treat acne or seborrheic paring a compound of the formula (I) as de?ned above by
dermatitis. coupling a polyunsaturated fatty acid selected from omega-3
The present invention further relates to a method for treat and omega-6 fatty acids, Whose carboxylic acid function is in
ing acne or seborrheic dermatitis comprising the administra free or activated form, and a diol of the folloWing formula (II):
tion of an effective quantity of a compound of the formula (I) 25
as de?ned above to a person in need thereof.
The present invention further relates to a pharmaceutical or (H)
cosmetic composition comprising at least one compound of Wort.
the formula (I) as de?ned above in combination With at least
one pharmaceutically or cosmetically acceptable excipient, 30
adapted in particular to percutaneous administration.
In the present invention, “pharmaceutically or cosmeti
Wherein n is an integer betWeen 1 and 15, preferably betWeen
cally acceptable” refers to that Which is useful in the prepa
1 and 10, andmis 0,1,2 or 3.
ration of a pharmaceutical or cosmetic composition and is
In particular, n can be 1, 2, 3, 4 or 5, preferably 5. Advan
35
generally safe, non-toxic and neither biologically nor other
tageously, nz1.3 and preferably n25.
Wise undesirable and is acceptable for therapeutic or cosmetic
Advantageously, m is 0 or 1.
use, in particular by topical application.
Advantageously, n+mz3 and preferably n+mz5.
The pharmaceutical or cosmetic compositions according to
In the context of the present invention, “free form” means
the invention can be provided in the forms commonly used for 40 that the carboxylic acid function of the PUFA is not protected
topical application, Which is to say in particular lotions,
and is thus a COZH group. The PUFA is thus of form R1 COZH
foams, gels, dispersions, emulsions, shampoos, sprays, such as previously de?ned.
serums, masks, body milks or creams, With excipients that In the context of the present invention, “activated form”
enable in particular cutaneous penetration in order to improve refers to a carboxylic acid function that is modi?ed so as to
the properties and the accessibility of the active principle. 45 make it more active With respect to nucleophiles. Said acti
Advantageously, said compositions are in the form of a vated forms are Well knoWn to those persons skilled in the art
and can be in particular an acid chloride (COCl). The acti
cream.
Said compositions generally contain, in addition to the one vated PUFA in the form of an acid chloride thus has the
or more compounds according to the present invention, a formula RlCOCl.
physiologically acceptable medium, in general Water-based 50 According to a ?rst particular embodiment of the inven
tion, the polyunsaturated fatty acid is used in its free acid
or solvent-based, for example based on alcohols, ethers or
form. In this case, the coupling reaction Will be carried out in
glycols. Said compositions can also contain surfactants,
sequestrants, preservatives, stabiliZers, emulsi?ers, thicken the presence of a coupling agent such as diisopropylcarbodi
imide (DIC), dicyclohexylcarbodiimide (DCC), 1-(3-dim
ers, gellants, humectants, emollients, trace elements, essen ethylaminopropyl)-3-ethylcarbodiimide hydrochloride
55
tial oils, fragrances, colorants, matti?ers, chemical or mineral
(EDC), carbonyldiimidaZole (CDI), 2-H-benZotriaZole-1
?lters, moisturizers or thermal Waters, etc.
yl) -1 , 1 ,3 ,3 -tetramethyluronium hexa?uoropho sphate
Said compositions can further contain other active prin (HBTU), 2-(1H-benZotriaZole-1-yl)-1,1,3,3-tetramethyluro
ciples leading to a complementary or possibly synergistic
nium tetra?uoroborate (TBTU) or O-(7-aZobenZotriaZol-1
effect. 60 yl) -1 , 1 ,3 ,3 -tetramethyluronium hexa?uoropho sphate
Advantageously, the compositions according to the present (HATU), optionally combined With a coupling auxiliary such
invention Will comprise from 0.01% to 10% by Weight, pref as N-hydroxysuccinimide (N HS), N-hydroxybenZotriaZole
erably from 0.1% to 1% by Weight, of one or more com (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benZotriaZole
pounds of the formula (I) compared to the total Weight of the (HOOBt), 1-hydroxy-7-aZabenZotriaZole (HOAt), dimethy
composition. 65 laminopyridine (DMAP) or N-hydroxysulfosuccinimide
Said compositions are more particularly intended to treat (Sulfo-NHS). Advantageously, the coupling Will be carried
acne or seborrheic dermatitis. out in the presence of a carbodiimide (in particular DIC, DCC
US 8,623,916 B2
7 8
or EDC) and dimethylaminopyridine. Preferably, the cou from light. The organic phase is then extracted With CH2Cl2
pling Will be carried out in the presence of 1-(3-dimethylami and then Washed With a saturated NaCl solution, dried on
nopropyl)-3-ethylcarbodiimide hydrochloride or diisopropy MgSO4, ?ltered and concentrated.
lcarbodiimide and dimethylaminopyridine. The crude product is a yelloW oil Which is puri?ed by open
According to a second particular embodiment of the inven column chromatography using silica gel (Q: 22x35 cm) and
tion, the polyunsaturated fatty acid is used in its activated CHCl3 as solvent.
form, and more particularly in the form of an acid chloride. In The folloWing three products Were prepared according to
this case, the coupling reaction Will advantageously be car method A With diisopropylcarbodiimide as coupling agent.
ried out in the presence of pyridine and dimethylaminopyri
dine. 3-Hydroxybutyl
The present invention Will be better understood in the light (9Z,12Z,15Z)-octadeca-9,12,15-trienoate
of the nonrestrictive examples Which folloW.
(C22H38O3) (3-hydroxybutyl ot-linolenate)
DESCRIPTION OF THE FIGURES
FIG. 1: Detection of R acnes lipase (33 kDa) in in?amma (a)
tory acne lesions (M corresponds to siZe markers; A corre H c
3 \ (6)
sponds to in?ammatory acne lesions; B corresponds to come
Ho H20 0 HZC_CH (d)
dos With no in?ammation). / (s) \\
FIG. 2: Study of the hydrolysis of the esters of the invention 20 cH—cH2 (r) 0 /CH
by recombinant R acnes lipase after 2 h of incubation. The / (1‘) U) H c (6)
3c H20 (q) 2 \
light gray column represents the results obtained With 3-hy
(V) \ (p) CH O)
droxybutyl ot-linolenate, the dark gray column represents the /C H2 (I) (k) (h) //
results obtained With 2-hydroxypentyl ot-linolenate, the black HZC @ H2C—CH2 H2C—CH
column represents the results obtained With 2-hydroxyoctyl 25 HC CH Q)
ot-linolenate, the White column represents the results obtained H2C—CH2 W — (I)
(n) (m)
With 3-hydroxynonyl ot-linolenate and the hatched column
represents the results obtained With glyceryl trilinolenate.
FIG. 3: Activity of PUFAs on the release of interleukin 8 by Puri?cation conditions: elution gradient: CHCl3/AcOEt:
human HaCaT keratinocytes stimulated by PMA/A23187 30 95/5 then CHCl3/AcOEt: 9/1
(proin?ammatory agents). Translucent oil (yield: 69%)
FIGS. 4A, 4B and 4C: Effects of PUFAs on the production Rf (CHCl3/AcOEt: 9/1):0.5
of eicosanoids (CYCLO: cyclooxygenase metabolites, NMR (1H, CDCl3) 6 (ppm): 0.98 (t, 3H, CH3 (61)); 1.22 (d,
LIPOX: lipoxygenase metabolites, AA: arachidonic acid) 3H, CH3 M); 1.31 (m, 8H, CH2 (0,”, m, I)); 1.6 (m, 2H, CH2 (m);
during an in?ammatory response induced by PMA/A23187. 35 1.8 (m, 2H, CH2 (0); 2.1 (m, 4H, CH2 (2k, 17)); 2.3 (t, 2H,
The White columns represent the control (a reaction medium CH2 ((1)); 2.8 (m, 4H, CH2 (h, 8)); 3.85 (m, 1H, CH (14)); 4.1-4.3
Without PUFAs); the light gray columns correspond to the (m, 2H, CH2 (5)); 5.4 (m, 6H, CH (c, da? g, I.’ . .
activity of 2.3 ug/ml PUFA; the dark gray columns corre NMR (13C, CDCl3) 6 (ppm): 14.24 (CH3 (61)); 20.46
spond to the activity of 11.5 ug/ml PUFA; and the black (CH2 (17)); 23.42 (CH3 M); 25.01 (CH2 (m); 25.5 (CH2 (8));
columns correspond to the activity of 23 ug/ml PUFA. FIG. 40 25.59 (CH2 (,0); 27.16 (CH2 GO); 29.06 (CH2 (0, m); 29.07
4A corresponds to ot-linolenic acid, FIG. 4B corresponds to (CH2 ("0); 29.53 (CH2 (1)); 34.3 (CH2 ((1)); 38.1 (CH2 (0); 61.51
stearidonic acid and FIG. 4C corresponds to linoleic acid. (CH2 (5)); 64.89 (CH (14)); 127.08 (CH (6)); 127.6 (CH 0.));
128.22 (CH w); 128.26 (CH Cg)); 130.22 (CH (1.)); 131.93
(CH (do); 174.2 (C:O (0).
45 MS: ESI+ [M+H]+:351.2 (100%); [M+Na]+:373.3 (48%)
ABBREVIATIONS USED: APCI+ [M+H]+:351.2 (calculated M:350.2)
APCI Atmospheric-pressure chemical ionization 2-Hydroxypentyl
MIC Minimum inhibitory concentration
DPM Disintegrations per minute (9Z,12Z,15Z)-octadeca-9,12,15-trienoate
ESI Electrospray ionization
50
HPLC High-performance liquid chromatography (C23H4OO3) (2-hydroxypentyl ot-linolenate)
NMR Nuclear magnetic resonance
Rf Ratio to front
MS Mass spectrum
CFU Colony-forming unit
55
EXAMPLE 1
Synthesis of Compounds of the Invention
60
1.1. General Method A: from an Unsaturated Fatty Acid
The unsaturated fatty acid is put into solution in 20 ml
anhydrous CH2Cl2 under circulating N2. A carbodiimide
(coupling agent) (1.1 eq) and dimethylaminopyridine (0.5 eq)
are then added directly. After stirring the medium at room 65
temperature for 5-10 minutes, the diol (5 eq) is added. The
medium is stirred vigorously for 18 hours, under N2, aWay
US 8,623,916 B2
9 1 0
Puri?cation conditions: CHCl3/AcOEt: 98/2 2-Hydroxyoctyl
(9Z,12Z,15Z)-octadeca-9,12,15-trienoate
Translucent oil (yield: 45%)
Rf (CHCl3/AcOEt: 97/3):0.58 (C26H46O3) (2-hydroxyoctyl ot-linolenate)
NMR (1H, CDCl3) 6 (ppm): 1 (m, 6H, CH3 (W, 0)); 1.25 (m,
10H, CH2 (0, n, m, I, 0); 1.5 (m, 4H, CH2 (P, Z4)); 1.65 (m, 4H,
CH2 (17%)); 2.1 (m, 2H, CH2 (8)); 2.4 (t, 2H, CH2 ((1)); 2.8 (m,
2H, CH2 (,0); 3.9 (m, 1H, CH (0); 41-43 (m, 2H, CH2 (5)); 5.4
(m, 6H, CH (d, d,j”, g, i,j))'
NMR (13C, CDCl3) 6 (ppm): 14.2 (CH3 (a, W)); 18.6
(CH2 (v, m); 25.5 (CH2 (m); 25.5 (CH2 (m); 27.16 (CH2 (8));
28.9 (CH2 (,0); 29.1 (CH2 GO); 29.4 (CH2 (0am); 29.5 (CH2 (m));
34.17 (CH2 ((1)); 35.41 (CH2 (14)); 68.53 (CH (0); 69.76
(CH2 (S)); 128.22 (CH (6)); 129.58 (CH (1.)); 130.22 (CH m
CH Cg)); 130.81 (CH (1.)); 131.93 (CH (a0); 174.05 (C:O (0).
MS: APCl+ [M+H]+:365.1 (calculated M:364.3)
20 Puri?cation conditions: CHCl3/AcOEt: 98/2
3-Hydroxynonyl Translucent oil (yield 37%)
Rf (CHCl3/AcOEt: 98/2):0.72
(9Z,12Z,15Z)-octadeca-9,12,15-trienoate
NMR (1H, CDCl3) 6 (ppm): 0.88 (m, 3H, CH3 (2)); 0.97 (t,
3H: CH3 (ms CH2 (0, n, m, I, v, w, x, (ms
(C27H48O3) (3-hydroxynonyl ot-linolenate) 25 2H, CH2 (14)); 1.65 (m, 2H, CH2 (‘0); 2.02 (m, 4H, CH2 (1,, k));
2.4 (t, 2H, CH2 ((1)); 2.8 (m, 4H, CH2 (8J0); 3.9 (m, 1H, CH (0);
41-43 (m, 2H, CH2 (5)); 5.4 (m, 6H, CH (c, (L?g, 1w).
NMR (13C, CDCl3) 6 (ppm): 14.2 (CH3 (a, W)); 18.6
(CH2 (v, m); 22.7 (CH2 w); 25.5 (CH2 (m); 25.5 (CH2 (m);
(a) 27.16 (CH2 (8)); 28.9 (CH2 (,0) 29.1 (CH2 GO); 29.4
H3C 30 (CH2 (0, n,)); 29-5 (CH2 (m); 29-9 (CH2 (“0); 31-7 (CH2 ()0);
\ (b) (6)
34.17 (CH2 ((1)); 35.41 (CH2 (14)); 68.53 (CH (0); 69.76
HO H2C—O HZC—CH (a)
\ l (5) \\CH (CH2 (5)); 128.22 (CH (6)); 129.58 (CH 0.)); 130.22 (CH (f)
CH_CH2 (r) O / CH Cg)); 130.81 (CH (1.)); 131.93 (CH (d)); 174.05 (C:O w).
/ (u) H C (6) MS: APCl+ [M+H]+:407.3 (calculated M:406.34)
H20 H20 (q) 2 \
(V)\ \ (p) CH 0) 35 1 .2. General Method B: from a Polyunsaturated Fatty Acid
CH2 CH2 (1) (k) (h) / Chloride
(X) / (W) / /
Linoleoyl chloride (6.7><10_3 mol) and dimethylaminopy
HZC HZC (o) H2C—CH2 H2C—CH
\ \ / \ / (3) ridine (DMAP) (0.1 eq) are added, under circulating nitrogen,
CH2 (y) H2C—CH2 HC—CH to a solution of diol (5 eq) in pyridine (20 ml). After Vigorous
/ (n) (m) (1) (1)
(z) H2C\ 40 stirring for 18 hours under N2, at room temperature and aWay
from light, the reaction medium is reduced dry.
CH3 The organic phase is then extracted With AcOEt and then
(m)
Washed With H20 and NH4+Cl_, dried on MgSO4, ?ltered
and concentrated.
Puri?cation conditions: CHCl3/AcOEt: 96/4 45 The crude product is a broWn oil Which is puri?ed by open
column chromatography using silica gel (Q: 22x35 cm) and
Translucent oil (yield 73%)
CHCl3 as solvent.
Rf (CHCl3/AcOEt: 97/3):0.64 The folloWing product Was prepared according to method
B With pentylene glycol as the diol.
NMR (1H, CDCl3) 6 (ppm): 0.95 (m, 3H, CH3 (M1)) 1 (m,
50
3H, CH3 (d)); 1.4 (m, 16H, CH2 (0,”, m, I, W’XJ’ZQ; 1.5 (m, 2H, 2-Hydroxypentyl (9Z,12Z)-octadeca-9,12-dienoate
CH2 M); 16-18 (m, 4H, CH2 (P, 0); 2 (m, 4H, CH2 (k, m); 2.3
(t, 2H, CH2 ((1)); 2.8 (m, 4H, CH2 (h, 8)); 3.7 (m, 1H, CH (14)); (C23H42O3) (2-hydroxypentyl linoleate)
4.1-4.3 (m, 2H, CH2 (5)); 5.4 (m, 6H, CH (d, da? g, I”).
NMR (13C, CDCl3) 6 (ppm): 14 (CH3 (a, mo); 20.4 55
(CH2 (17)); 22.5 (CH2 (2)); 25.09 (CH2 (m); 25.26 (CH2 (M);
25.58 (CH2 (8, ,0); 27.17 (CH2 GO); 29.06-29.3
(CH2 (x, 0, n, m, I)); 31.7 (CH2 w); 34.5 (CH2 ((1)); 37.45
(CH2 (0); 37.58 (CH2 (v); 61.59 (CH2 (5)); 68.72 (CH (14));
60
127.08 (CH (6)); 127.71 (CH (1.)); 128.21 (CH w); 128.26
(CH Cg)); 130.22 (CH (1.)); 131.94 (CH (do); 174.9 (C:O w).
MS: ES1+ [M+H]+:421.1 (100%); [M+Na]+:443.1 (92%)
The following product Was prepared according to method
65
A With 1-(3 -dimethylaminopropyl) -3 -ethylcarbodiimide
hydrochloride as coupling agent.
US 8,623,916 B2
1 1 12
Puri?cation conditions: CHCl3/AcOEt: 98/2 Second, the action of recombinant R acnes lipase on the
Translucent oil compounds of the invention Was studied as described beloW.
The enzymatic reaction Was carried out by incubating the
Rf (CHCl3/AcOEt: 98/2):0.54
substrate at the desired concentration (500 uM) in TRIS
NMR (1H, CDCl3) 6 (ppm): 0.9 (m, 6H, CH3 (a, W)) 1.3 (m,
buffer (left at room temperature for 1 h before the tests began)
16H, CH2 (0,”, mahbacada 0); 1.45 (m, 2H, CH2 (14)); 1.65 (m, 2H,
and then initiated the reaction by adding the enZyme and
CH2 (m); 2 (m, 4H, CH2 (k, 8)); 2.3 (t, 2H, CH2 ((1)); 2.8 (m, 2H,
incubating at 370 C. The tests are carried out in 2 ml amber
CH2 01)); 3.8 (m, 1H, CH (0); 4.1-4.3 (m, 2H, CH2 (5)); 5.4 (m,
glass vials. A control is carried out by not adding the enZyme
4H, CH (? g, I”).
in order to evaluate the possible spontaneous hydrolysis of the
NMR (13C, CDCl3) 6 (ppm): 14.2 (CH3 (a, W)); 18.6 substrate in the buffer. A 10 ul sample is taken at each desired
(CH2 M); 22.6 (CH2 ([0); 25 (CH2 (m); 25.6 (CH2 01)); 27.2 time during the reaction and then froZen to quench the reac
(CH2 (8, ,0); 29 (CH2 (0)); 29.1 (CH2 (,0); 29.3 (CH2 (do); 29.4 tion. The linolenic acid released is then derivatiZed With
(CH2 ("0); 29.6 (CH2 (1)); 31.4 (CH2 (6)); 34.1 (CH2 ((1)); 36.2 anthryldiaZomethane (ADAM) Which forms a ?uorescent
(CH2 (14)); 69 (CH2 (5)); 70 (CH (0); 127.78 (CH Cg)); 127.91 complex With the fatty acid. The product formed is separated
(CH (1.)); 129.94 (CH 0.)); 130.22 (CH (1.)); 174.05 (C:O m).
by HPLC and then quanti?ed using a standard range of com
MS: ESI+ [M+H]+:367.3 (100%); [M+Na]+:389.3 (80%) mercial linolenic acid derivatiZed under the same conditions.
20 As shoWn in FIG. 2, the compounds of the invention give
EXAMPLE 2 rise to a hydrolysis reaction in the presence of R acnes, since
the formation of ot-linolenic acid is observed.
Composition According to the Invention Thus, hydrolysis of the compounds of the invention by R
acnes releases both the diol and the PUFA. Expression of the
25
A formulation according to the invention, in cream form, lipase is thus a necessary condition to observe the cleavage of
With the following composition (the quantities are given in compounds of the invention and thus to obtain a therapeutic
Weight percentages compared to the total Weight of the com effect. In addition, since the R acnes lipase is essentially
position): present in subjects With acne, as shoWn in FIG. 1, the com
30
pounds of the invention should enable an adapted response
for each person.
Quantity Compound Function 3.2. Study of the Antibacterial Activity of the Diols
Obtained after Hydrolysis of the Compounds of the Invention
3 Glycerin Humectant 35
0.1 Disodium EDTA* Sequestrant Said diols are of the folloWing general formula (II):
0.35 Phenoxyethanol Preservative
1 Polyacrylate- 1 3; Gellant and
Polyisobutene; Polysorbate stabilizer
20; Water (mixture sold
under the name Sepiplus ®
40
400 by SEPPIC)
4 Glyceryl stearate; PEG- 100 Emulsi?er (11)
stearate (mixture sold Wort.
under the name Simulsol ®
165 by SEPPIC)
1 Cetyl alcohol Consistency factor
5 Cyclopentasiloxane Emollient 45
3 Glyceryl tri-2- Emollient
ethylhexanoate
2 Dicaprylyl carbonate Emollient
1 Diol and PUFA ester Anti-acne agent
Wherein n is an integer betWeen 0 and 15 and m is 0, 1, 2 or 3.
according to the invention
0.27 Chloiphenesin Preservative 50 Tests Were carried out With eight microbial strains using the
2 Polymethyl methacrylate Mattifying poWder
0.1 Fragrance Fragrance dilution method described beloW.
Minimum inhibitory concentrations (MICs) are deter
*EDTA: ethylenediaminetetraacetic acid
mined using a micromethod in liquid medium. Successive 1/ 2
55 dilutions of the test products in culture medium (Trypcase
EXAMPLE 3 Soy Broth) are carried out in 96-Well microplates in a ?nal
volume of 0.1 ml. The Wells are inoculated With 0.01 ml of the
Results of Biological Tests bacterial suspensions titrated at roughly 1><107 CFU/ml. The
microplates are incubated in optimal groWth conditions and
60
the MIC is read visually.
3.1. Study of the Action of R acnes Lipase on the Com
pounds of the Invention Table 1 beloW presents the results obtained With various
First, it Was shoWn by Western blot that the R acnes lipase diols. The columns labeled “BS” represent the bacteriostatic
is strongly induced in the in?ammatory lesions of subjects activity and the columns labeled “BC” represent the bacteri
65
With acne compared to samples taken from healthy subjects cidal activity of the diols (1% corresponds to a concentration
(see FIG. 1). of 10 mg/ml).