US010561702B2

(12 ) Campiche
United etStates
al.
Patent ( 10 ) Patent No.: US 10,561,702 B2
(45 ) Date of Patent: Feb. 18 , 2020
( 54 ) METHOD FOR THE TREATMENT OF SKIN (58 ) Field of Classification Search
DISORDERS USING DIPEPTIDE CPC ..... A61K 31/4164 ; A61K 31/4184 ; A61K
DIAMINOBUTYROYL BENZYLAMIDE 47/02 ; A61K 47/40 , A61K 9/0019 ; A61K
DIACETATE 9/19
See application file for complete search history.
(71 ) Applicant: DSM IP ASSETS B.V., Heerlen (NL ) (56 ) References Cited
(72 ) Inventors: Remo Campiche, Kaiseraugst ( CH ); U.S. PATENT DOCUMENTS
Dominik Imfeld , Kaiseraugst (CH );
Eileen Jackson, Kaiseraugst (CH ); 7,964,630 B2 6/2011 Imfeld et al .
Eliane Ursula Wandeler , Kaiseraugst 7,964,639 B2 * 6/2011 DeLuca A61K 8/671
(CH ) 514/529
2009/0111731 A1 4/2009 Imfeld et al.
(73) Assignee: DSM IP ASSETS B.V., Heerlen (NL ) 2010/0215726 A1 * 8/2010 Roth A61K 8/64
424/450
2012/0213845 Al 8/2012 Bernstein
( * ) Notice : Subject to any disclaimer, the term of this 2014/0219942 A1 * 8/2014 Mendrok -Edinger A61K 8/37
patent is extended or adjusted under 35 424 / 70.1
U.S.C. 154(b ) by 0 days . 2014/0309401 A1 10/2014 Hayashida et al.
2018/0360698 A1 * 12/2018 Boswell A61K 8/0208
(21) Appl. No.: 16 / 060,096 2018/0360722 A1 * 12/2018 Campiche A61K 8/64

(22) PCT Filed : Dec. 9 , 2016 FOREIGN PATENT DOCUMENTS

( 86 ) PCT No .: PCT/EP2016 /080469 CN 103 505 377 6/2015

DE 20 2012 002309 4/2012
$ 371 (c ) ( 1 ), WO 2010/003828 1/2010
(2 ) Date : Jun . 7 , 2018 WO 2016/154020 9/2016

( 87 ) PCT Pub . No .: WO2017 / 102591 OTHER PUBLICATIONS

PCT Pub. Date : Jun . 22 , 2017 International Search Report for PCT/ EP2016 /080469 dated Apr. 10 ,
2017, 5 pages .
(65) Prior Publication Data Written Opinion of the ISA for PCT/EP2016 /080469 dated Apr. 10 ,
US 2018/0353563 A1 Dec. 13 , 2018 2017, 10 pages.
Imfeld et al. “ 557 A peptide derivative with known anti-wrinkle
properties was identified as potent dipeptiylpeptidase-4 inhibitor” ,
(30 ) Foreign Application Priority Data Journal of Investigative Dermatology, vol. 136 , No. 5, May 1, 2016 ,
XP055345491, 2 pages .
Dec. 16 , 2015 (EP) 15200516 “ Instant Brightening Moisturiser” , MINTEL , Nov. 1, 2011 ,
Mar. 10 , 2016 (EP ) 16159702 XP002683631, 4 pages.
May 3 , 2016 (EP ) 16168111 “ Pentapharm Syn -Ake” ,Internet Citation , Feb. 26 , 2007,XP002472008,
4 pages.
(51) Int. Ci.
Thielitz et al. “ Inhibitors of dipeptidyl peptidase IV and aminopeptidase
N target major pathogenetic steps in acne initiation” , Journal of
A61K 38/05 (2006.01) Investigative Dermatology , Nature Publishing Group , vol. 127, No.
A61K 8/64 (2006.01) 5 , May 1 , 2007, XP002476229 , pp. 1042-1051.
A61Q 19/00 ( 2006.01 )
A61P 17/10 ( 2006.01) * cited by examiner
A61P 17/08 (2006.01)
A61K 8/06 (2006.01 ) Primary Examiner Marcela M Cordero Garcia
A61K 8/55 ( 2006.01) (74 ) Attorney, Agent, or Firm — Nixon & Vanderhye P.C.
A61K 9/107 ( 2006.01)
A61K 47/24 ( 2006.01) (57) ABSTRACT
(52 ) U.S. Cl. The present invention relates to a novel use of proline
CPC A61K 38/05 (2013.01); A61K 8/062 containing dipeptides for the prevention and /or treatment of
(2013.01); A61K 8/55 (2013.01 ); A61K 8/64 skin conditions associated with altered , damaged or mal
(2013.01); A61K 9/107 (2013.01 ); A61K 47/24 functioning sebaceous glands.
(2013.01 ); A61P 17/08 ( 2018.01); A61P 17/10
(2018.01 ); A61Q 19/008 (2013.01) 11 Claims, No Drawings
 US 10,561,702 B2
1 2
METHOD FOR THE TREATMENT OF SKIN wherein
DISORDERS USING DIPEPTIDE n represents 0 , 1 or 2 ,
DIAMINOBUTYROYL BENZYLAMIDE R and R4 - independently of each other — are selected
DIACETATE from the group consisting of H , C , -Coalkyl, amidino or
5 tetra -C7- Co- alkylamidinium ;
1

This application is the U.S. national phase of International R2 is H or C ,-Co- alkyl, or R1 and R2 together with the
Application No. PCT/EP2016 /080469 filed Dec. 9, 2016 , residue to which they are bound form a 5- to 7 -mem
which designated the U.S. and claims priority to EP Patent bered , saturated ring;
R is selected from the group consisting of C -C alkyl,
Application No 15200516.1 filed Dec. 16 , 2015 , EP Patent 10
arC , -Coalkyl and heteroary C.-Calkyl; and
Application No. 16159702.6 filed Mar. 10 , 2016 and EP R5 is H or, when n is 1, also NH2, or R $ and R 1 together
Patent Application No. 16168111.9 filed May 3, 2016 , the with the residue to which they are bound form a 5- to
entire contents of each of which are hereby incorporated by 7 -membered , saturated ring ;
reference . or a dermatologically acceptable salt thereof for use in the
The present invention relates to a novel use of proline prevention or treatment of disorders of the sebaceous glands
containing dipeptides for the prevention and /or treatment of 15 such as in particular sebaceous hyperplasia, hyperseborrhea ,
acne ( in particular acne vulgaris ), seborrheic dermatitis,
skin conditions associated with altered , damaged or mal atopic dermatitis and rosacea.
functioning sebaceous glands. In another embodiment, the present invention relates to a
Sebaceous glands are essential components of healthy cosmetic (nontherapeutic ) use of a DPP4 inhibitor of for
skin . They produce and secrete sebum , which is responsible 20 mula
for moisturizing and protecting skin and hair . Damaged or skin , blemishes(1) for the prevention or treatment of oily and/or shiny
malfunctioning sebaceous glands however play a central size . , blotchiness and/or reduction of skin pore
role in many unwanted dermatological conditions such oily As the compounds of formula (I) are highly active DPP4
and shiny skin , increased pore size , sebaceous hyperplasia as inhibitors , the present invention also relates to the use of
well as acne vulgaris .
Sebaceous glands can contribute to the development of 25 thereof connected
for preventing or treating illnesses or conditions
with an increased DPP4 activity or capable of
acne in several ways . One of the most common problems being prevented or alleviated by reducing the DPP - IV
faced by acne sufferers involves overactive sebaceous activity such as e.g. follicular hyperkeratosis , inflammation
glands and /or sebaceous hyperplasia ( enlarged sebaceous as well as hyproproliferative disorders (e.g. psoriasis ), with
glands). These conditions lead to an overproduction of 30 out being limited thereto .
sebum . The excess sebum however not only leads to oily /
shiny skin but also promotes the growth of bacteria that C -The term ‘ C -Coalkyl’as used herein refers to unbranched
Cgalkyl or branched Cz -Cgalkyl groups such as methyl,
contribute to acne symptoms like Propionibacterium acnes. ethyl, n -propyl , 1 -methylethyl, n -butyl, 1 -methylpropyl,
Furthermore, excess sebum can also contribute to kerati 2 -methylpropyl, 1,1 -dimethylethyl, n -pentyl, 1-methylbutyl,
nized plugs that block the follicle and spur the development 35 2-methylbutyl, 3 -methylbutyl, 2,2 -dimethylpropyl, 1 -ethyl
of inflammatory lesions. propyl, n -hexyl, 1,1-dimethylpropyl, 1,2 -dimethylpropyl,
DPP4 (Dipeptidyl Peptidase IV ) inhibitors are described 1 -methylpentyl, 2 -methylpentyl , 3 -methylpentyl, 4 -methyl
to suppress proliferation of sebocytes ( i.e. sebaceous gland pentyl, 1,1- dimethylbutyl, 1,2 -dimethylbutyl, 1,3 - dimethyl
cells ), to enhance their terminal differentiation and to butyl, 2,2 -dimethylbutyl, 2,3 -dimethylbutyl, 3,3 - dimethyl
decrease the total neutral lipid production . Thus, DPP4 40 butyl, 1-ethylbuty?, 2 -ethylbutyl, 1,1,2-trimethylpropyl, 1,2,
inhibitors are said to have the capacity to influence the major 2- trimethylpropyl, 1 -ethyl-1-methylpropyl, and 1- ethyl-2
pathogenic factors of acne such as sebaceous hyperplasia, methylpropyl groups .
follicular hyperkeratosis and inflammation ( Thielitz et al. The term “ arC / -C alkyl’ as used herein refers to a C ,
JID (2007), 127; 1042-1051). Coalkyl- arylwherein the term “aryl’ is e.g. a phenyl, indanyl
Thus, there is an ongoing need for DPP4 inhibitors 45 or naphthyl group .
suitable for skin application which can be used for the The term 'heteroary1C -Csalkyl’ as used herein refers to
prevention or treatment of skin conditions associated with a —C , -Coalkyl- heteroaryl wherein the term " heteroaryl"
altered , damaged or malfunctioning sebaceous glands . refers to a 5- or 6 -membered aromatic ring containing one or
Surprisingly, it has been found that certain proline con more heteroatoms, viz ., N , O or S ; these heteroaromatic
taining dipeptides are highly effective DPP4 inhibitors
are able to reduce the sebum production as well as the skin
and 50 rings may be fused to other aromatic systems.
pore size. Examples for the 5- to 7-membered , saturated ring , that
Rand R2 or R1 and RS, respectively,may form together with
Thus, the first object of the present invention relates to a the residue to which they are bound, are pyrrolidinyl,
composition comprising at least one DPP4 inhibitor of piperidinyl , piperazinyl, morpholinyl, azepinyl, oxazolidi
formula (1) 55 nyl, thiazolidinyl and 1,2,3,4 - tetrahydroquinolinyl.
It is well understood , that the present invention encom
passes the compounds of formula (I) as optically pure
R5 NHR4
isomers such as e.g. as pure enantiomers or stereoisomers as
3-1
well as mixtures of different isomers such as e.g. as race
RÄHN . 60 mates, or mixtures of diastereoisomers.
HN NHR3, The term ‘or a dermatologically acceptable salt thereof'
R2 refers to compounds of formula (I) in the form of an acid
addition salt such as in the form of a chloride , an acetate or
a trifluoroacetate salt . Alternatively, the salt may be formed
65 by reaction with an alkali or earth alkaline base resulting in
the respective alkali or earth alkaline salt such as in par
ticular the respective lithium , sodium , potassium , magne
 US 10,561,702 B2
3 4
sium or calcium salts. Most preferred, in all embodiments of mode and route ofadministration ; the age, health and weight
the present invention , are the compounds of formula (I) in of the recipient; the nature and extent of the symptoms; the
the form of their acetates or trifluoroacetates . Such salts are kind of concurrent treatment; the frequency of treatment;
easily prepared by a person skilled in the art. and the effectdesired and can be adjusted by a person skilled
The term “ prevention ' as used herein , is not intended as an 5 in the art. Preferably , the amount of the composition to be
absolute term . Instead , prevention , e.g. , of acne , refers to applied to the skin is selected in the range of0.1 to 3 mg/cm²
delay of onset, reduced frequency of symptoms, or reduced skin , such as preferably in the range of 0.1 to 2 mg/cm² skin
severity of symptoms associated with the respective derma and most preferably in the range of 0.5 to 2 mg/cm² skin .
tological condition . Prevention therefore refers to a broad It is well understood that the uses herein , if not stated
range of prophylactic measures that will be understood by
those in the art. Similarly, the term “ treatment is not 10 otherwise , shall in particular refer to a cosmetic, non
intended to be an absolute term . In some circumstances, the therapeuticaluse intended to beautify the skin , preferably by
topical application of a compound according to the present
DPP4 inhibitors according to the invention seek to reduce invention
the sebum production that may e.g. lead to the symptoms of to the skin , preferably via a cosmetic composition .
acne. In some circumstances, treatment with the DPP4 15 The term ' cosmetic composition ' as used herein refers to
inhibitors of the invention leads to a reduction in the compositions , which are used to treat, care for or improve
frequency or severity of the symptoms. the appearance of the skin and /or the scalp . Particular
In all embodiments of the present invention R ', R + and R5 advantageous cosmetic compositions are skin care prepara
are preferably H. tions.
In all embodiments of the present invention R2 is prefer- 20 The term “pharmaceutical composition ' as used herein
ably H and methyl, most preferably H. refers to compositions, which are used to treat skin disorders
In all embodiments of the present invention Rº is prefer of the sebaceous glands . Particular advantageous pharma
ably arc , -C alkyl, most preferably benzyl. ceutical compositions are skin treatment preparations.
In all embodiments of the present invention n is prefer The compositions according to the invention are prefer
ably 0 or 1 , most preferably 1. 25 ably intended for topical application , which is to be under
Most preferred in all embodiments of the present inven stood as the external application to keratinous substances,
tion is a compound of formula (I), wherein R1, R², R4 and such as in particular the skin .
R $ are H , R3 is benzyl and n is 1 . As the compositions according to the invention are
The compounds of formula (1) can be prepared as e.g. intended for topical application , they comprise a physiologi
disclosed in US 2009/0111731. 30
cally acceptable medium , that is to say a medium compatible
Particular advantageous in all embodiments of the present with
invention is the dipeptide having the sequence H-(beta - Ala ) braneskeratinous substances, such as the skin , mucous mem
Pro -Dab -NH -benzyl, in particular as diacetate . This com cally acceptable medium fibers
, and keratinous
is a
. In particular the physiologi
cosmetically , respectably phar
pound is also known as Dipeptide Diaminobutyroyl Benzy maceutically acceptable carrier.
lamide Diacetate (INCI) [CAS 823202-99-9 ], and is 35 The term “ cosmetically acceptable carrier ' respectively
commercially available as SYN® - AKE from DSM Nutri *pharmaceutically acceptable carrier ' as used herein refers to
tional products Ltd. a physiologically acceptable medium which is compatible
In another object, the invention relates to a method for the with keratinous substances . Suitable carriers are well known
prevention or treatment of disorders of the sebaceous gland
such as in particular sebaceous hyperplasia, hyperseborrhea , 40 in the art and are selected based on the end -use application .
acne, seborrheic dermatitis, atopic dermatitis , and rosacea, Preferably , the carriers of the present invention are suitable
said method comprises topically administering an effective for application to skin (e.g. , sunscreens, creams, milks,
amount of a pharmaceutical composition comprising at least lotions, masks, serums, hydrodispersions, foundations,
one DPP4 inhibitor of formula (I) to the appropriate skin creams, creamgels , or gels etc.). Such carriers are well
area of a person in need of such treatment and optionally 45 known to one of ordinary skill in the art, and can include one
appreciating the effect. or more compatible liquid or solid filler diluent, excipient ,
In a further object, the invention relates to a method for additive or vehicle which are suitable for application to skin .
the prevention or treatment of oily and /or shiny skin , blem The exact amount of carrier will depend upon the levelof the
ishes and /or blotchiness and/ or to the reduction of skin pore compound of formula (I) and any other optional ingredients
size, said method comprises topically administering an 50 that one of ordinary skill in the art would classify as distinct
effective amount of a cosmetic composition comprising at from the carrier ( e.g., other active components ). The com
least one DPP4 inhibitor of formula (I) to the appropriate positions of the present invention preferably comprise from
skin area of a person in need of such treatment and option about 75 % to about 99.999 % , more preferably from about
ally appreciating the effect. 85 % to about 99.99 % , still more preferably from 90 % to
The amount of the at least one compound of formula (I) 55 about 99 % , and most preferably, from about 93 % to about
in the respective compositions according to the present 98 % , by weight of the composition, of a carrier.
invention is preferably selected in the range of 0.5 ppm to The compositions of the present invention can be formu
5'000 ppm , preferably in the 2.5 ppm to 250 ppm , most lated into a wide variety of product types , including creams,
preferably in the range of 25 ppm to 100 ppm , based on the waxes, pastes, lotions, milks, mousses, gels , oils, tonics, and
total weight of the composition . 60 sprays. Preferably the compounds of formula (I) are formu
The term “ an effective amount refers to an amount lated into lotions, creams, gels , and tonics. These product
necessary to obtain the physiological effect. The physiologi forms may be used for a number of applications, including ,
cal effect may be achieved by one application dose or by but not limited to , hand and body lotions, facialmoisturizers ,
repeated applications. The dosage administered may, of anti -ageing preparations, makeups including foundations,
course , vary depending upon known factors, such as the 65 and the like. Any additional components required to formu
physiological characteristics of the particular composition late such products vary with product type and can be
comprising the at least one compound of formula (I) and its routinely chosen by one skilled in the art .
 US 10,561,702 B2
5 6
If compositions of the present invention are formulated as pickering emulsion , then the amount of the oily phase
an aerosol and applied to the skin as a spray -on product, a present in such cosmetic emulsions is preferably at least 10
propellant is added to the composition . wt.- % , such as in the range of 10 to 60 wt.- % , preferably in
The compositions according to the present invention can the range of 15 to 50 wt.- % ,most preferably in the range of
be prepared by conventional methods in the art such as e.g. 5 15 to 40 wt.- % ,based on the totalweightof the composition .
by admixing a compound of formula (1) with all the defi In one embodiment, the compositions according to the
nitions and preferences given herein with the cosmetically present invention are advantageously in the form of an
acceptable carrier. oil- in -water (O /W ) emulsion comprising an oily phase dis
The compositions of the invention ( including the carrier) persed in an aqueous phase in the presence of an O / W
may comprise further conventional adjuvants and additives, 10 emulsifier. The preparation of such O / W emulsions is well
such as preservatives/antioxidants, fatty substances/oils, known to a person skilled in the art.
water , organic solvents, silicones, thickeners, softeners, If the composition according to the invention is an O / W
emulsifiers , antifoaming agents , aesthetic components such emulsion , then it contains advantageously at least one O /W
as fragrances, surfactants , fillers, anionic, cationic , nonionic or Si/W - emulsifier selected from the list of, glyceryl stearate
or amphoteric polymers or mixtures thereof, propellants , 15 citrate , glyceryl stearate SE ( self- emulsifying ), stearic acid ,
acidifying or basifying agents , dyes, colorings/colorants, salts of stearic acid , polyglyceryl-3 -methylglycosedistear
abrasives, absorbents , chelating agents and /or sequestering ate . Further suitable emulsifiers are phosphate esters and the
agents, essential oils, skin sensates, astringents, pigments or salts thereof such as cetyl phosphate (e.g. as Amphisol® A
any other ingredients usually formulated into such compo from DSM Nutritional Products Ltd.), diethanolamine cetyl
sitions . 20 phosphate (e.g. as Amphisol® DEA from DSM Nutritional
In accordance with the present invention , the composi Products Ltd.), potassium cetyl phosphate (e.g. as Amphi
tions according to the invention may also comprise further sol® K from DSM Nutritional Products Ltd.), sodium
cosmetically active ingredients conventionally used in cos cetearylsulfate , sodium glyceryl Oleate phosphate , hydroge
metic and/or pharmaceutical compositions. Exemplary nated vegetable glycerides phosphate and mixtures thereof.
active ingredients encompass skin lightening agents ; UV- 25 Further suitable emulsifiers are sorbitan oleate, sorbitan
filters, agents for the treatment of hyperpigmentation ; agents sesquioleate, sorbitan isostearate, sorbitan trioleate, cetearyl
for the prevention or reduction of inflammation ; firming , glucoside, lauryl glucoside , decyl glucoside, sodium
moisturizing , soothing , and /or energizing agents as well as stearoyl glutamate, sucrose polystearate and hydrated poly
agents to improve elasticity and skin barrier . isobutene. Furthermore, one or more synthetic polymers
Examples of cosmetic excipients , diluents , adjuvants, 30 may be used as an emulsifier. For example , PVP eicosene
additives as well as active ingredients commonly used in the copolymer, acrylates/C10-30 alkyl acrylate crosspolymer,
skin care industry which are suitable for use in the cosmetic and mixtures thereof.
compositions of the present invention are for example The at least one O /W , respectively Si/ W emulsifier is
described in the International Cosmetic Ingredient Diction preferably used in an amount of 0.5 to 10 wt. % , in particular
ary & Handbook by Personal Care Product Council (http :// 35 in the range of 0.5 to 6 wt.- % , such as more in particular in
www.personalcarecouncil.org/), accessible by the online the range of 0.5 to 5 wt.- % , such as most in particular in the
INFO BASE (http://online.personalcarecouncil.org/jsp/ range of 1 to 4 wt.- % , based on the total weight of the
Home.jsp ), without being limited thereto . composition .
The necessary amounts of the active ingredients as well as Particular suitable O / W emulsifiers to be used in the
the excipients, diluents , adjuvants , additives etc. can , based 40 compositions according to the invention encompass phos
on the desired product form and application , easily be phate ester emulsifiers such as advantageously 8-10 alkyl
determined by the skilled person . The additional ingredients ethyl phosphate , C9-15 alkyl phosphate , ceteareth -2 phos
can either be added to the oily phase, the aqueous phase or phate, ceteareth -5 phosphate, ceteth -8 phosphate, ceteth - 10
separately as deemed appropriate . phosphate , cetyl phosphate , C6-10 pareth -4 phosphate, C12
The cosmetically active ingredients useful herein can in 45 15 pareth - 2 phosphate , C12-15 pareth -3 phosphate , DEA
some instances providemore than one benefit or operate via ceteareth - 2 phosphate , DEA -cetyl phosphate , DEA -oleth - 3
more than one mode of action . phosphate , potassium cetyl phosphate, deceth -4 phosphate,
Ofcourse , one skilled in this art will take care to select the deceth -6 phosphate and trilaureth -4 phosphate .
above mentioned optional additional ingredients, adjuvants, A particular suitable O /W emulsifier to be used in the
diluents and additives and /or their amounts such that the 50 compositions according to the invention is potassium cetyl
advantageous properties intrinsically associated with the phosphate e.g. commercially available as Amphisol® K at
combination in accordance with the invention are not, or not DSM Nutritional Products Ltd Kaiseraugst.
substantially, detrimentally affected by the envisaged addi Another particular suitable class of O /W emulsifiers are
tion or additions. non -ionic self-emulsifying systems derived from olive oil
The compositions according to the present invention may 55 e.g. known as (INCI Name) cetearyl olivate and sorbitan
be in the form of a suspension or dispersion in solvents or olivate ( chemical composition : sorbitan ester and cetearyl
fatty substances, or alternatively in the form of an emulsion ester of olive oil fatty acids) sold under the tradename
or micro emulsion ( in particular of oil -in -water ( O /W ) or OLIVEM 1000 .
water - in -oil (W /O ) type, silicone -in -water (Si/ W ) or water In one particular embodiment, the invention relates to
in - silicone ( W /Si) type , PIT-emulsion , multiple emulsion 60 compositions with all the definitions and preferences given
(e.g. oil- in -water - in oil (O / W / O ) or water-in -oil - in -water herein in the form of O /W emulsions comprising an oily
( W /O /W ) type ), pickering emulsion , hydrogel , alcoholic gel, phase dispersed in an aqueous phase in the presence of an
lipogel, one- or multiphase solution or vesicular dispersion O / W emulsifier wherein the O /W emulsifier is potassium
or other usual forms, which can also be applied by pens, as cetyl phosphate. The amount of oily phase in such O / W
masks or as sprays . 65 emulsions is preferably at least 10 wt.- % ,more preferably in
If the composition is an emulsion , such as in particular an the range of 10 to 60 wt.- % , most preferably in the range of
O / W , W / O , Si/ W , W /Si, O /W /O , W /O /W multiple or a 15 to 50 wt.- % , such as in the range of 15 to 40 wt.- % .
 US 10,561,702 B2
7 8
The compositions according to the invention in general TABLE 2 - continued
have a pH in the range of 3 to 10 , preferably a pH in the
range of 4 to 8 and most preferably a pH in the range of 4 Results of the protease assay with N - Pal-Gly - His- Lys/ N
Pal-Gly -Gln -Pro - Arg (2 : 1 ) mixture
to 7.5. The pH can easily be adjusted as desired with suitable 5
acids, such as e.g. citric acid , or bases, such as sodium C ( N - Pal -Gly -His -Lys /N -Pal-Gly -Gln -Pro - Arg ; 2 : 1 ) * DPP4 activity
hydroxide (e.g. as aqueous solution ), triethanolamine ( TEA [ppm ] [UM ] [% ]
Care ), Tromethamine ( Trizma Base ) and Aminomethyl Pro
panol (AMP -Ultra PC 2000), according to standard methods 25.00
50.00
34.37
68.74
123
116
in the art. 10 100.00 137.48 112
The following examples are provided to further illustrate *Reference (commercially available as MATRIXYL TM 3000 )
the compositions and effects of the present invention . These
examples are illustrative only and are not intended to limit As can be retrieved from table 1 and 2 , only the dipeptide
the scope of the invention in any way . according to the present invention was capable to inhibit
15 DPP4, whereas the reference even stimulated the DPP4
EXPERIMENTAL PART activity .
2. Ex Vivo Assay with Sebaceous Glands (SG )
1. Dipeptiyl Peptidase 4 (DPP4) Protease Assay Method Organ culture of human sebaceous glands has been per
formed in order to verify the modulatory activity of H-(beta
The potential ofH-(beta - Ala )-Pro -Dab -NH -benzyl [CAS 20 Ala )-Pro -Dab -NH -benzyl on sebum secretion . A viability
823202-99-9), respectively N - Pal-Gly -His- Lys [ 147732-56
7 ]/N -Pal-Gly -Gln -Pro - Arg [221227-05-0 ] (2 : 1 ) to inhibit test has been performed in parallel. Human sebaceous
glands from skin sample od labia minora have been used
DPP4 was tested using the DPP4 Drug Discovery Kit from (donor: female 37y ). The epidermis of the full thickness
Enzo Life Sciences. In brief: DPP4 is pre- incubated for 10 25 sample has been carefully removed and the sebaceous
minutes with inhibitors and then added to a quenched glands have been micro - dissected .
fluorogenic substrate (H -Gly - Pro -AMC ). Cleavage of the The sebaceous glandshave been pooled in groups of eight
AMC -moiety from the c -terminus of the peptide substrate by and cultured up to day 6. SGs are cultured in 24 well plate
DPP4 increases its fluorescence intensity at 460 nm . The immersed in 500 ul of SGs medium (modified Williams’E
AMC signal is then recorded using a fluorescence 30 medium ). After 24 hours the culture medium has been
microplate reader at Ex /Em = 380/460 every minute for a changed and substituted with the medium containing the
total time of 20 min . A “ best fit ” line is generated with the substance to be tested . The medium has been renewed at day
data points of the first 10-20 min and the slope calculated . 3 and 5 of culture . At day 6 the glands have been collected
The remaining activity in the presence of inhibitor was and used for the quantification of lipids and proteins. Briefly
calculated as follows: % activity remaining (with inhibitor) 35 the SGs have been homogenized in isopropyl alcohol
= ( slope of inhibitor-sample / slope of control-sample ) * 100. (IPOH ) in order to extract lipids and let the protein undis
solved . An aliquot of IPOH has been withdrawn to measure
TABLE 1 the lipids whereas the rest has been evaporated in a vacuum
centrifuge . The remaining dry pellet has been minced again
Results of the protease assay with H-(beta - Ala )-Pro -Dab -NH -benzyl 40 in presence of protein lysis buffer. The lipids dissolved in
c (H-(beta -Ala - Pro -Dab -NH -benzyl) DPP4 activity IPOH and the protein dissolved in the lysis buffer have been
quantified by infrared spectroscopy (Millipore Direct
[ppm ] [UM ] [% ] Detect). The total lipid amount has been obtained by nor
100
malizing the quantified lipids upon the quantified proteins
0
0.77
0
1.56 74 45 ( i.e.mg of lipids/mg of proteins).
1.55 3.13 58
3.10 6.25 40 TABLE 3
6.19 12.50 24
12.39 25.00 14 Total fat quantification (mg lipid /mg protein )
24.78 50.00 7
49.55 100.00 4
50 Capsaicin H-( beta- Ala)-Pro - Dab- NH
99.10 200.00 2 SG test sample untreated 5 UM benzyl 10 uM
1 18.4 15.5 14.7
2 18.6 15.4 14.8
3 17.7 15.3 16.3
TABLE 2 55 4 19 15.1 15.0
5 16.6 15.8 13.2
Results of the protease assay with N - Pal-Gly - His- Lys/ N 6 16.8 15.7 13.3
Pal-Gly -Gln -Pro -Arg ( 2 : 1 ) mixture 7 16 15.7 14.6
8 17.1 15.5 13.5
C ( N - Pal-Gly -His - Lys/N - Pal-Gly -Gln - Pro -Arg; 2: 1)* DPP4 activity 9 17.3 15.7 12.9
60
10 17.5 15.6 13.0
[ppm ] [um ] [% ] 11 16.6 15.6 14.4
12 17.8 15.4 13.2
0 0 100 Average ( 1-12 ) 17.5 15.5 14.2
0.78 1.07 119 S.D. 1.0 0.2 1.1
1.56 2.15 118 P -value vs untreated t - test < 0.01 < 0.01
3.13 4.30 106
6.25m 8.59 92 65 Conclusion : H-(beta - Ala )-Pro-Dab-NH -benzyl (10 uM ) reduced the sebum secretion ex
12.50 17.19 116 vivo in sebaceous glands by 19 % vs untreated .
 US 10,561,702 B2
9 10
3. Clinical Study TABLE 5 - continued
A double blind parallel group study was performed with Results from the computational analysis of gloss
Caucasian female volunteers aged 40-55 . 30 volunteers Specular Gloss
(mean age 46 +/– 1 year ) received a placebo formulation and 5
29 volunteers (mean age 48 +/– 1 year) received a verum Placebo -2.37 %
formulation containing SYN® - AKE. The placebo respec Verum -2.53 %
tively verum formulation was applied twice daily for 28 days
to face. At the end of the study pictures were taken for
computational analysis of pores: 11 photograph of the entire 10 oily-As/shininess
can be retrieved from table 5 the gloss (reflecting the
of the skin) was effectively reduced by the
face (90 °) was taken with Newtone® Color face for the pore verum formulation .
size analysis. Furthermore a questionnaire was filled out by
the volunteers . TABLE 6
TABLE 3 15 Results from the questionnaire
A B Agree
Phase Ingredients INCI Name Placebo Verum
Pore Size Reduction
A AMPHISOL ® K Potassium cetyl phosphate 1.00 1.00
Placebo 60 %
Ecorol 16 /98P Cetyl alcohol 3.00 3.00 20
Cutina CP Cetyl palmitate 1.50 1.50 Verum 69 %
Eutanol G Octyldodecanol 3.00 3.00 Non Shiny Skin
B Pemulen TR - 1 Acrylates/C10-30 alkyl 0.10 0.10
acrylate cross -polymer Placebo 60 %
? 1,3 -Butylenglycol Butylene glycol 3.00 3.00 Verum 83 %
Water dem . Aqua Ad 100 Ad 100
25
D Dow Corning 345 Cyclopentasiloxane , 2.50 2.50
Fluid cyclohexasiloxane As can be retrieved from table 6 , a perceivable pore size
E Natriumhydroxid Aqua, sodium hydroxide 0.09 0.09 reduction as well as an effective reduction of oily /shiny skin
F
30 % soln .
SYN ? - AKE Dipeptide Diaminobutyroyl 0 4.00
was observed by the volunteers .
The invention claimed is :
Benzylamide Diacetate , 30 1. A method for the treatment of sebaceous gland disor
Glycerin , Aqua
G Euxyl PE 9010 Phenoxyethanol, 1.00 1.00 ders selected from the group consisting of sebaceous hyper
ethylhexylglycerine plasia, hyperseborrhea, acne, seborrheic dermatitis , atopic
G Frag 49424902 Perfume 0.05 0.05 dermatitis and rosacea, wherein the method comprises topi
Chloe cally administering to skin area of a person in need of such
35 treatment an effective amount of a pharmaceutical compo
sition comprising a dipeptidyl peptidase IV (DPP4 ) inhibi
TABLE 4 tory effective amount of dipeptide diaminobutyroyl benzy
lamide diacetate .
Results of the computational analysis of the pores 2. The method according to claim 1 , wherein the amount
Number of Pores DO to D28 40 of the dipeptide diaminobutyroyl benzylamide diacetate in
the composition is in a range of 0.5 ppm to 5,000 ppm , based
Placebo + 3.2 % on the total weight of the composition .
Verum
Conspicuous surface of each pore DO to D28
-0.8 % 3. The method according to claim 1, which comprises
topically applying the composition to the skin area in an
Placebo -3.2 % 45 amount of 0.1 to 3 mg/cm² skin .
Verum -4.4 % 4. The method according to claim 1, wherein the compo
Conspicuous surface of all pores DO to D28 sition is an O / W emulsion comprising an oily phase dis
Placebo + 1.5 % persed in an aqueous phase in the presence of an O / W
Verum -2.7 % emulsifier.
Conspicuous volume DO to D28 50 5. The method according to claim 4 , wherein the O / W
Placebo + 3.8 %
emulsifier is potassium cetyl phosphate .
Verum -1.3 % 6. A method for the treatment of oily, shiny, blemished
and /or blotchy skin and /or to reduce skin pore size, wherein
the method comprises topically administering to skin area of
As can be retrieved from table 4, the pore size was 55 cosmetic
a person incomposition
need of suchcomprising
treatmentaandipeptidyl
effective peptidase
amount ofIVa
effectively reduced by the treatment with the verum formu
(DPP4) inhibitory effective amount of dipeptide diaminobu
lation containing a DPP4 inhibitor according to the present tyroyl
invention . benzylamide diacetate .
7. The method according to claim 6 , wherein the cosmetic
TABLE 5 60 composition is a skin care preparation .
8. The method according to claim 6 , wherein the amount
Results from the computational analysis of gloss of the dipeptide diaminobutyroyl benzylamide diacetate in
Contrast Gloss the composition is in a range of 0.5 ppm to 5,000 ppm , based
on the total weight of the composition .
Placebo -10.24 % 65 9. The method according to claim 6 , which comprises
Verum -11.55 % topically applying the composition to the skin area in an
amount of 0.1 to 3 mg/cm² skin .
 US 10,561,702 B2
11 12
10. The method according to claim 6, wherein the com
position is an O /W emulsion comprising an oily phase
dispersed in an aqueous phase in the presence of an O /W
emulsifier .
11. The method according to claim 10 , wherein the O /W 5
emulsifier is potassium cetyl phosphate .