U.S. patent application number 15/228150 was filed with the patent office on 2016-11-24 for process for the purification of heparan sulfate and use thereof in cosmetological and dermatological preparations.
The applicant listed for this patent is LABORATORI DERIVATI ORGANICI SPA. Invention is credited to Luigi DE AMBROSI.
Application Number | 20160338935 15/228150 |
Document ID | / |
Family ID | 41587870 |
Filed Date | 2016-11-24 |
United States Patent
Application |
20160338935 |
Kind Code |
A1 |
DE AMBROSI; Luigi |
November 24, 2016 |
PROCESS FOR THE PURIFICATION OF HEPARAN SULFATE AND USE THEREOF IN
COSMETOLOGICAL AND DERMATOLOGICAL PREPARATIONS
Abstract
The present invention relates to cosmetological and
dermatological compositions comprising heparan sulfate. The
invention further discloses a process for the purification of
heparan sulfate for dermatological and cosmetological applications,
which process comprises the following steps: solubilization of
heparan sulfate in water, adsorption on an anion exchange resin,
desorption from the resin by using conditions which result in
selective desorption of heparan sulfate. The cosmetological and
dermatological compositions according to the invention show, inter
alia, anti-age, lenitive, and whitening effect.
Inventors: |
DE AMBROSI; Luigi; (Santhia,
IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LABORATORI DERIVATI ORGANICI SPA |
MILANO |
|
IT |
|
|
Family ID: |
41587870 |
Appl. No.: |
15/228150 |
Filed: |
August 4, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13129030 |
Jul 28, 2011 |
9440099 |
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PCT/EP2009/062582 |
Sep 29, 2009 |
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15228150 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61Q 19/00 20130101;
A61K 8/73 20130101; A61K 2800/75 20130101; A61P 17/00 20180101;
A61P 17/16 20180101; A61K 31/727 20130101; A61K 2800/522 20130101;
A61K 31/726 20130101; A61P 17/04 20180101; A61Q 19/08 20130101;
A61P 29/00 20180101; A61Q 19/02 20130101; A61Q 19/004 20130101 |
International
Class: |
A61K 8/73 20060101
A61K008/73; A61K 31/727 20060101 A61K031/727 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 20, 2008 |
EP |
08169547.0 |
Claims
1.-13. (canceled)
14. A method of whitening skin, the method comprising applying a
formulation comprising heparan sulfate to a patient in need
thereof.
15. The method of claim 14, wherein the formulation comprises from
0.01 to 5% by weight heparan sulfate.
16. The method of claim 14, wherein the formulation comprises from
0.05 to 2% by weight heparan sulfate.
17. The method of claim 14, wherein the formulation comprises from
0.1 to 1% by weight heparan sulfate.
18. The method of claim 14, wherein the heparan sulfate has a
molecular weight between 6,000 and 12,000 Da.
19. The method of claim 14, wherein the formulation further
comprises one or more of the following ingredients: preservatives,
emulsifiers, stabilizers, humectants, anti-oxidants, and other
components which are included in cosmetological and dermatological
formulations.
20. The method of claim 14, wherein the formulation is an
emulsion.
21. A method of reducing itching, the method comprising applying a
formulation comprising heparan sulfate to a patient in need
thereof.
22. The method of claim 21, wherein the formulation comprises from
0.01 to 5% by weight heparan sulfate.
23. The method of claim 21, wherein the formulation comprises from
0.05 to 2% by weight heparan sulfate.
24. The method of claim 21, wherein the formulation comprises from
0.1 to 1% by weight heparan sulfate.
25. The method of claim 21, wherein the heparan sulfate has a
molecular weight between 6,000 and 12,000 Da.
26. The method of claim 21, wherein the formulation further
comprises one or more of the following ingredients: preservatives,
emulsifiers, stabilizers, humectants, anti-oxidants, and other
components which are included in cosmetological and dermatological
formulations.
27. The method of claim 21, wherein the formulation is an
emulsion.
28. A method for reducing free radicals as Reactive Oxygen Species
(ROS) and increasing cell proliferation and protein synthesis, the
method comprising applying a formulation comprising heparan sulfate
to a patient in need thereof.
29. The method of claim 28, wherein the formulation comprises from
0.01 to 5% by weight heparan sulfate.
30. The method of claim 28, wherein the formulation comprises from
0.05 to 2% by weight heparan sulfate.
31. The method of claim 28, wherein the formulation comprises from
0.1 to 1% by weight heparan sulfate.
32. The method of claim 28, wherein the heparan sulfate has a
molecular weight between 6,000 and 12,000 Da.
33. The method of claim 28, wherein the formulation further
comprises one or more of the following ingredients: preservatives,
emulsifiers, stabilizers, humectants, anti-oxidants, and other
components which are included in cosmetological and dermatological
formulations.
Description
[0001] The invention relates to a new process for the purification
of heparan sulfate. The present invention also relates to
cosmetological and dermatological preparations making use of
heparan sulfate.
[0002] Heparans are extracted from heparinoids which constitutes a
by-product in the purification of heparin from pig intestinal
mucosa. They are structural isomers formed during synthesis of
glycosaminoglycanes (GAGs); highly sulfated and epimerised
structures are typical of heparin, while highly epimerised and less
N- and O-sulfated structures are typical of heparin-like structures
presenting low anticoagulant activity.
[0003] It is known that endogenous proteoglycans (PG) play an
important role in wound healing (V. Prathiba and S. Gupta:
Cutaneous wound healing: Significance of proteoglycans in scar
formation". Current Science, vol. 78, no 6, 2000). However, the
prior art does not describe the effect of administration of
proteoglycans and, more specifically, of heparan sulfate in
cosmetological and dermatological applications.
[0004] The present invention relates to cosmetological and
dermatological preparations comprising heparan sulfate, and their
use as whitening, anti-wrinkle, lenitive and anti-oxidant agent. It
has been surprisingly found that heparan sulfate is effective in
the treatment of the above mentioned conditions.
[0005] In a further embodiment, the invention is directed to
medical devices making use of heparan sulfate in the cosmetological
and dermatological field.
[0006] In another preferred embodiment, the present invention is
directed to a process for the purification of heparan sulfate,
which process allows the use of pure heparan sulfate in
dermatological and cosmetological applications.
[0007] In a preferred embodiment, the cosmetological and
dermatological preparations according to the invention make use of
purified heparan sulfate. In fact, purification of heparan sulfate
is an important step in order to remove residual compounds which
are extracted with heparan sulfate but present different
characteristics.
[0008] The purification process of the invention is characterized
by the following steps: solubilization of heparan sulfate in water,
adsorption on an anion exchange resin, desorption from the resin
under conditions which result in selective desorption of heparan
sulfate.
[0009] The anion exchange resin is preferably a strong base
macroporous anion exchange resin such as, for example, Lewatit.TM.
S 6328 A. The adsorption is performed by mixing a sufficient amount
of resin beads. Preferably the amount of beads is higher than 15 g
of resin per gram of heparan sulfate, more preferably is comprised
between 18 and 25 g resin per gram of heparan sulfate.
[0010] After adsorption on the anion exchange resin, heparan
sulfate is selectively desorbed by using an alkaline or alkaline
earth metal salt, preferably an halogenide or an acetate. Examples
of suitable salts are magnesium dichloride, magnesium diacetate,
sodium chloride, sodium acetate, potassium chloride and potassium
acetate. The pH of the salt solution is preferably comprised
between 7.0 and 10.0. The concentration of the salt is important in
order to achieve complete and selective desorption of heparan
sulfate. A preferred range of concentration is comprised between
0.3 M and 1.0 M, more preferably between 0.5 M and 0.8 M. In fact,
if the concentration of the salt is too low, the desorption of
heparan sulfate is incomplete. If the concentration is too high,
the desorption is not selective and, together with heparan sulfate,
also highly sulfated compounds are desorbed.
[0011] After desorption from the anion exchange resin, heparan
sulfate is optionally further passed on a cationic resin and on a
anionic resin. An example of suitable anionic resin is Amberlite
Forte IR 120.TM.. An example of a suitable cationic resin is
Amberlite Forte IRA 410.TM..
[0012] Purified heparan sulfate according to the invention was
tested in various dermatological and cosmetological applications.
More specifically, heparan sulfate was tested for whitening,
lenitive and anti-age effects. In all these applications heparan
sulfate showed a significant activity.
[0013] A preferred embodiment of the present invention is directed
to a cosmetological composition comprising heparan sulfate.
[0014] Preferably, the cosmetological compositions according to the
invention comprise from 0.01% by weight to 5% by weight of heparan
sulfate, more preferably from 0.05% by weight to 2% by weight, even
more preferably from 0.1% by weight to 1.0% by weight.
[0015] The molecular weight by weight (Mw) of the heparan sulfate
suitable in the cosmetological and dermatological applications of
the invention is preferably comprised between 6,000 and 12,000
Da.
[0016] The cosmetological compositions according to the invention
optionally comprise other substances such as preservatives,
emulsifiers, stabilizers, humectants, anti-oxidants, which are
usually included in cosmetological and dermatological
compositions.
[0017] In a preferred embodiment, the compositions according to the
present invention are used as anti-age product. Many anti-age
products often claim their ability to induce an increase in cell
proliferation or in the protein synthesis on fibroblasts, with a
linked improvement in cell firmness and thickness.
[0018] Fibroblasts are the main cell component in the connective
tissue of the dermis and they are able to synthesize high amount of
collagen and elastin, the main components of the dermis that play
an essential role in the skin thickness and appearance. The
increase in protein amount for cell stimulates the ability of
fibroblasts to synthesize collagen and elastin.
[0019] Another claim for anti-aging and protective cosmetics is
their activity in counteracting oxidative stress and free-radicals
release in the skin. Oxidative stress is one of the main cause of
aging process and is involved in the development of many serious
human diseases. Exposure to UVA rays, pollutants, tobacco's smoke,
as well as diet, drugs intake or pathologies can increase the level
of oxidant activity in the skin cells or decrease the effectiveness
of endogenous antioxidant systems. Following exposure to oxidative
stress Reactive Oxygen Species (ROS) formation, damages to the
cells membrane and to DNA may occur in the cell, leading to cell
transformation or even death. The induction of fibroblasts
proliferation and protein synthesis in dermis cells are of great
concern for actives targeted to anti-age topical products.
[0020] It has been found that compositions comprising heparan
sulfate are effective both as anti-wrinkle compositions and as
anti-oxidant compositions.
EXPERIMENTAL
[0021] Organic sulfur, Uronic acid, Glucosamine, Optical rotation,
total nitrogen and APTT were measured in accordance with the
European Pharmacopeia, 4.sup.th edition. Electrophoresis was
performed in accordance with R. Cappelletti et al. "A new
Electrophoretic method for the separation of all Gags" Anal.
Biochem. 99:311-315 (1979).
[0022] Molecular mass (M.sub.w) was determined by size exclusion
chromatography (European Pharmacopoeia 4.sup.th ed.: 2.2.30 and
2.2.46 for chromatography techniques and 01/2002:0828 p. 1297 for
method).
Purification of Heparan Sulfate
[0023] 5 g of heparan sulfate from pig intestinal mucosa were
dissolved in 200 ml of demineralised water. 90 g of Lewatit S 6238
A.TM. were added to the solution. The mixture was slowly stirred at
room temperature for 72 h and then filtered. The filtrate was
analysed and no heparan sulfate was detected.
[0024] The resin was washed, transferred in a column (diameter 3
cm, length 40 cm) and eluted with a 5% by weight solution of
MgCl.sub.2 at pH 8.5.
[0025] The first 300 ml are collected, after which the solution did
not show presence of heparan sulfate (negative reaction to
quaternary ammonium).
[0026] The solution is brought to pH=6 with a 10% solution of
hydrochloric acid and heparan sulfate is precipitated by adding 600
ml of acetone. The suspension is left at 15.degree. C. for 12 h.
Then the solid is collected, dissolved in 50 ml of water and
precipitated by adding 100 ml of acetone.
[0027] Heparan sulfate is then dissolved in 100 ml of water and the
solution is passed over a resin Amberlite Forte IR 120.TM. and then
over a resin Amberlite Forte IRA 410.TM.. The eluate is neutralized
by adding a 5% by weight solution of NaOH until pH 6. Water is
evaporated under vacuum, the solid is filtered on a 0.45 .mu.m
filter and lyophilised. Yield: 4.12 g.
[0028] Analysis: Rotation=+50.620. Organic S=9.07%. Uronic
acid=31.97%. Glucosamine=37%. Total N=2.26%. APTT=6.31 U/mg.
Mw=7538 Da (Before purification 8729 Da).
Test for Whitening Efficacy Evaluation
In Vitro Evaluation of the Bleaching Activity of a Raw Material on
Cell Cultures--Test Procedure
[0029] The in vitro whitening effect of HS was detected by
measurement of melanin content on melanoma cells B16
(fibroblast-like cells able to produce melanin) after 6 days of
contact with HS at 2 different concentrations: 0.1 mg/ml (0.01%)
and 0.05 mg/ml (0.005%).
[0030] The negative control (NC) was represented by untreated cells
(cell without any substance). The positive control (PC) was
represented by cells treated with Hydroquinone 5 .mu.g/ml
(0.0005%). On the basis of results obtained, heparan sulphate
sodium salt (HS) shows in vitro whitening effect.
TABLE-US-00001 TABLE I Melanin Content (.mu.g melanin/mg % Melanin
Synthesis protein) Inhibition vs NC HS 0.01% 41.9 10.3 HS (0.005%)
44.4 5.2 PC: Hydroquinone (0.0005%) 10.8 76.8 NC: untreated cells
46.8 . . .
In Vivo--Whitening Efficacy Evaluation of a Cosmetic Formulation on
20 Volunteers
Test Procedure:
[0031] The test was performed in the single blind mode. 20
volunteers (age between 40-50 years) with aged spots where present
(hand, forehead, arm, face, neck). The region with aged spots was
divided in two zone: one for the treatment with tested substance,
one with placebo. The spots were treated for 30 consecutive days: 1
application (1 mg of substance)/die on tested area (150-200
cm.sup.2) [as per Colipa guideline].
[0032] The tested substance was an emulsion of the following
composition: aqua, ethylhexyl palmitate, cetearyl alcohol, 0.9%
heparan sulfate sodium salt, carbomer, acrylates/c-10-30 alkyl
acrylate crosspolymer, sodium hydroxide, phenoxyethanol, propylene
glycol, methylparaben, ethylparaben, propylparaben, disodium
EDTA.
[0033] Placebo was an emulsion of the following composition: aqua,
ethylhexyl palmitate, cetearyl alcohol, carbomer, acrylates/c-10-30
alkyl acrylate crosspolymer, sodium hydroxide, phenoxyethanol,
propylene glycol, methylparaben, ethylparaben, propylparaben,
disodium EDTA.
[0034] The evaluation (MI--Melanin Index by Mexameter MX 18) was
made in the following way: [0035] T.sub.0--basal value, value of
Melanin Index before the application of substance. [0036]
T.sub.15--value of Melanin Index after 15 days of substance
application. [0037] T.sub.30--final value, value of Melanin Index
at the end of test (after 30 days of substance application).
TABLE-US-00002 [0037] TABLE II T.sub.0 MI .+-. std dev T.sub.15 MI
.+-. std dev T.sub.30 MI .+-. std dev HS 0.9% 229.09 .+-. 44.06
222.00 .+-. 58.37 220.81 .+-. 54.25 Placebo 199.89 .+-. 55.89
205.84 .+-. 55.95 215.07 .+-. 75.45
[0038] The decrease of MI (Melanin Index) in the area treated with
tested substance, versus MI treated with placebo, after 30 days of
application, resulted statistically relevant (p<0.01). Hence the
Whitening Effect of tested substance (emulsion with 0.9% Heparan
Sulphate sodium salt) was confirmed.
Test for Lenitive Efficacy Evaluation
[0039] In Vitro--Degranulation Test on Basophil Cells with Cosmetic
Raw Materials--Test Procedure
[0040] Basophil cells, when come in contact with irritant
substances, give their biochemical response that is the secretion
(by degranulation) of substances (such as histamine, leukotriens,
proteolytic enzymes as hexosaminidase) that contribute to
inflammatory events.
[0041] Inhibition in basophil cells degranulation is a measure of
lenitive (anti-itching) effect of a substance. The in vitro
lenitive effect of HS was detected by measurement of the release of
.beta.-hexosaminidase after 2 h of contact with HS at 4 different
concentrations: 10 mg/ml (1%), 5 mg/ml (0.5%), 1 mg/ml (0.1%), 0.2
mg/ml (0.02%).
[0042] The negative control (NC) was represented by untreated cells
(cells without any substance). The positive control (PC) was
represented by basophil degranulation stimuli (by crosslinking the
receptor Fc.epsilon.RI with a specific polyclonal antibody against
to the .alpha.-chain stimulate the degranulation). The maximal
stimulation (degranulation) is: cell treated with 1% Triton X-100.
The results are reported as Optical Density (OD) at 405 nm.
TABLE-US-00003 TABLE III release of .beta.-hexosaminidase OD at 450
nm NC: untreated cells 0.054 PC: basophil degranulation 0.117 Max
Stimulation 0.377 HS 10 mg/ml 0.066 HS 5 mg/ml 0.059 HS 1 mg/ml
0.062 HS 0.2 mg/ml 0.061 HS 10 mg/ml + PC 0.061 HS 5 mg/ml + PC
0.055 HS 1 mg/ml + PC 0.110 HS 0.2 mg/ml + PC 0.112
[0043] On the basis of results obtained, heparan sulphate sodium
salt (HS) shows in vitro lenitive effect.
[0044] The .beta.-hexosaminidase "natural" level, level without any
contact with irritant substance is 0.054 (OD at 450 nm). HS alone
(at different concentration) has the same effect of negative
control, that means leaves the cell with their "natural" level of
.beta.-hexosaminidase. This confirms that HS has no effect on
basophil cells degranulation.
[0045] When the cells come in contact with an irritant substance
(positive control) the value increase at 0.117, or higher to 0.377
when in contact with Triton X-100 (1%).
[0046] When the cells come in contact both with an irritant
substance (positive control) and HS the level of
.beta.-hexosaminidase decrease in a dose dependent way, with best
efficacy with HS at 0.5% and 1%, hence HS has a lenitive
(anti-itching) effect.
In Vivo--Lenitive Efficacy Evaluation on a Cosmetic Formulation on
20 Volunteers--Short Term Test--Test Procedure
[0047] The test was performed in single blind mode. 20 volunteers
(age between 36-52 years) were treated on a circumscribed treated
zone of 1 cm.sup.2 on the back of each volunteer: one for test
substance application, one for placebo application, and one for
control (untreated zone). The test was performed by a single
application (2 mg of substance) on tested area (1 cm.sup.2) [as per
Colipa guideline]. Total time: 60 minutes after application
[0048] Tested substance was an emulsion of following composition:
aqua, ethylhexyl palmitate, cetearyl alcohol, 0.9% heparan sulfate
sodium salt, carbomer, acrylates/c-10-30 alkyl acrylate
crosspolymer, sodium hydroxide, phenoxyethanol, propylene glycol,
methylparaben, ethylparaben, propylparaben, disodium edta.
[0049] Placebo was an emulsion of following composition: aqua,
ethylhexyl palmitate, cetearyl alcohol, carbomer, acrylates/c-10-30
alkyl acrylate crosspolymer, sodium hydroxide, phenoxyethanol,
propylene glycol, methylparaben, ethylparaben, propylparaben,
disodium edta.
[0050] The transient erythemagenic reaction is induced by exposure
to a known amount of UVB radiation coming from a Solar Stimulator.
The evaluation (EI--Erythema Index by Mexameter MX 18) were made in
the following way: [0051] T.sub.0--basal value, value of Erythema
Index after UVB radiation and before the application of substance
[0052] T.sub.15--value of Erythema Index after 15 minutes from
substance application [0053] T.sub.30--value of Erythema Index
after 30 minutes from substance application [0054] T.sub.60--value
of Erythema Index after 60 minutes from substance application
TABLE-US-00004 [0054] TABLE IV T.sub.15 EI .+-. std T.sub.30 EI
.+-. std T.sub.60 EI .+-. std T.sub.0 EI .+-. std dev dev dev dev
Tested 455.75 .+-. 64.63 439.55 .+-. 82.67 421.59 .+-. 72.31 406.39
.+-. Sub- 77.06 stance Pla- 453.21 .+-. 71.50 430.26 .+-. 76.22
415.69 .+-. 67.42 415.79 .+-. cebo 75.18
TABLE-US-00005 TABLE V T.sub.0 vs T.sub.15 T.sub.0 vs T.sub.30
T.sub.0 vs T.sub.60 Tested Substance -4.19 -8.29 -11.24 Placebo
-5.36 -8.29 -8.58
[0055] The decrease of EI (Erythema Index) in the area treated with
tested substance, versus EI treated with placebo, after 60 minutes
from application, resulted statistically relevant (p<0.1), and
is statistically relevant (p<0.01) the decrease after 60 minutes
versus basal value. Hence the Lenitive (redness reduction) Effect
of tested substance (emulsion with 0.9% Heparan Sulphate sodium
salt) has been confirmed.
Test for Anti-Wrinkle Efficacy Evaluation
[0056] In vitro Evaluation of the anti-ageing activity of cosmetic
products through the investigation of their anti-oxidant and
protective activity against UVA on human keratinocytes cell
cultures.
Test Procedure
[0057] The antioxidant effect of HS is detected by measurement of
reduction of free radicals as Reactive Oxygen Species (ROS) after
exposure to 3 different UVA irradiation for 1 minute and 30 seconds
(1' 30''), for 3 minutes (3') and for 4 minutes and 30 seconds (4'
30''), with HS at 7 different concentrations: 0.031 mg/ml
(0.0031%), 0.063 mg/ml (0.0063%), 0.125 mg/ml (0.0125%), 0.250
mg/ml (0.0250%), 0.500 mg/ml (0.0500%), 1.000 mg/ml (0.1000%) and
9.000 mg/ml (0.9000%). The negative control (NC) was: untreated
cells (cells without any substance). The positive control (PC) was:
Vitamin C (0.15 mg/ml) (0.015%).
[0058] Table VI shows the results obtained. It is evident that
heparan sulphate sodium salt (HS) shows in vitro antioxidant
effect. The efficacy of HS at 0.1% concentration is comparable or
even higher than the efficacy of Vitamin C. The efficacy of HS is
higher that Vitamin C even at prolonged exposure to UVA radiation
(4' 30'').
TABLE-US-00006 TABLE VI Heparan Sulphate Sodium Salt Vit C mg/ml
mg/ml Exposure 9.000 1.000 0.500 0.250 0.125 0.063 0.031 0.15 ROS
1'30'' UVA 30.7 38.7 30.6 28.0 23.5 25.4 25.3 33.5 Inhibition %
3'00'' UVA 31.6 35.7 29.1 22.9 14.2 21.8 20.1 31.4 4'30'' UVA 26.3
30.4 24.7 18.1 10.3 13.7 6.2 12.7
In Vitro--Mitogenic Activity Evaluation of a Raw Material
Test Procedure:
[0059] The mitogenic effect of HS is detected by measurement of
fibroblast cells proliferation, measurement of protein synthesis
after exposure for 48 hours and 72 hours, using HS at 2 different
concentrations: 0.1 mg/ml (0.01%) and 0.05 mg/ml (0.005%). The
negative control (NC) is represented by untreated cells, the
positive control (PC) is: EGF (Epidermal Growth Factor) (10
.mu.g/ml) (0.001%).
TABLE-US-00007 TABLE VII % cell proliferation vs NC 48 h 72 h PC:
EGF (10 .mu.g/ml) (0.001%) 112.5 121.6 HS 0.1 mg/ml (0.01%) 110.5
118.1 HS 0.05 mg/ml (0.005%) 106.7 118.2 % protein synthesis vs NC
48 h 72 h PC: EGF (10 .mu.g/ml) (0.001%) 116.6 119.0 HS 0.1 mg/ml
(0.01%) 103.1 113.2 HS 0.05 mg/ml (0.005%) 110.2 120.4
[0060] On the basis of results obtained, heparan sulphate sodium
salt (HS) shows in vitro cell proliferation and protein synthesis
efficacy. The highest effect (18.2% for cell proliferation and
20.4% for protein synthesis) is observed after 72 hour from
exposure at 0.05 mg/ml.
* * * * *