Note: Descriptions are shown in the official language in which they were submitted.
CA 03001437 2018-04-09
WO 2017/080945 PCT/EP2016/076807
1
ANTI-PERSPIRANT COMPOSITION
Field of the invention
The invention relates to an anti-perspirant composition that contains low or
no
aluminium salt.
Background of the invention
Current anti-perspirant ingredients are based on aluminium, but inorganic
salts have
the effect of leaving white patches on clothes. Additionally there is a
perceived health
risk associated with aluminium. The current approach is to reduce the amount
of
aluminium in antiperspirants or to use additional metal salts such as those of
zirconium.
However, this approach tends to lower the efficacy of the formulation and
hence prove
more expensive. Zirconium-based antiperspirants tend to leave yellow patches
on
clothes.
US 2009/0016978 Al (Courtois et al.) describes an antiperspirant composition
comprising a carrier substance and a water-soluble or water-dispersible
thiolated
polymer. The prior art inventors believe that the thiol groups of the thiomer
enable or
enhance the polymer's ability to act as a mucoadhesive and that this ability
enables or
enhances the antiperspirant activity of the thiomer. "Mucoadhesives" are
materials that
can attach to mucin in a biological surface. The prior art inventors further
believe that
the antiperspirant activity results, at least in part, from the ability of the
thiomers to act
as pore blockers. The thiomers, when swollen by water, are thought to serve to
as
plugs that may, at least in part, block the exit of sweat from eccrine sweat
glands. It is
essential for the invention that the thiomer is water-soluble or water-
dispersible in order
for it to dissolve or disperse in eccrine sweat.
WO 03/042251 (The Procter & Gamble Company) discloses compositions comprising
chitosan in the form of a network of nano-sized fibres. Traditional chitosan
is usually
semi-crystalline and only soluble in acidic medium, typically in a pH range of
from 1 to
5 limiting homogeneous formulation. A process for producing the network of
nano-
sized fibres is described involving the steps of forming an aqueous solution,
CA 03001437 2018-04-09
WO 2017/080945 PCT/EP2016/076807
2
neutralising the chitosan just to the point of precipitation, and homogenising
the
resulting suspension. It was observed that the minimum concentration of
chitosan to
inhibit Malassezia furfur (yeast implicated in dandruff) was lower than
expected. This
document also discloses an anti-dandruff composition comprising from about
0.01 % to
about 5 %, preferably from about 0.5 % to about 2 % of chitosan by weight of
the
composition as the active anti-dandruff agent. The chitosan can be used in
different
applications, such as hair care, skin care, personal cleansing, odour control,
wound
care, blood management, oral care, film formation, controlled release of
hydrophobic or
hydrophilic materials, hard surface, fabric treatment, plant care, seed,
grain, fruit and
food protection, water purification and drug delivery. The chitosan
compositions
provide hair care benefits when formulated into products such as shampoos,
conditioners, hairsprays, styling mousses and gels, hair tonics and hair
colorants,
especially anti-dandruff benefits and reduction of hair damage caused by the
process
of hair bleaching, permanent waving or coloration. Additionally, the
compositions
provide scalp benefits and conditioning properties such as softening,
manageability
and stylising of the hair. Specific examples are a shampoo, a conditioner, a
dentifrice, a
mouthwash, a non-abrasive gel, a chewing gum and a plant care composition.
WO 2006/040092 (Beiersdorf AG) discloses an aerosol formulation comprising one
or
more anti-perspirants and/or deodorising substances and chitosan having a
degree of
deacetylation of 75 to 98 %, a viscosity of 5 to 10 mPas, a weight average
molecular
weight distribution of less than 300 000 Da and a number average molecular
weight
distribution of less than 100 000 Da. It appears that the disclosed chitosan
preserves
the skin flora rather than acting purely as a bacteriocide. In particular, the
chitosan
appears to bind to the bacteria preventing microbial decomposition of sweat
leading to
odour. Anti-perspirants reduce sweat formation with the aid of astringent
compounds
in them, which are predominantly aluminium salts, such as aluminium
hydrochloride,
activated aluminium chlorohydrate or aluminium zirconium. It is customary to
combine
astringents with antimicrobials in the same composition. Aerosol products
generally
contain active anti-perspirant substances in the form of solids, which are
suspended in
an oil phase. Conventional active deodorant substances include ethyl hexyl
glycerol,
methyl phenyl butanol and polyglycery1-2-caprate. One aim of the invention
described
in WO 2006/040092 is to reduce whiteness on skin or clothes. The formulation
comprises 0.001-2, preferably 0.01-1, especially 0.015-0.3% w/w chitosan. The
CA 03001437 2018-04-09
WO 2017/080945 PCT/EP2016/076807
3
formulation comprises 1-35, preferably 1-25, especially 1-20 % w/w anti-
perspirant
component. The formulation comprises preferably 0.01-10, especially 0.05-5 %
w/w
deodorant component. Examples disclosed are anhydrous compositions. WO
2006/040092 further discloses that the pressure container used for the aerosol
can be
made of a metal, protected glass, non-shatter glass or some other glass, or
else of a
plastic. The propellant gas is preferably chosen from a long list of suitable
gases.
US 2003/0133891 (Cognis Corporation) discloses a deodorising preparation
containing
nanoscale chitosans and/or chitosan derivatives with a particle diameter in
the range
from 10 to 300 nm. Chitosans have a bacteriostatic effect and a synergistic
deodorising
effect with esterase inhibitors and aluminium chlorohydrates. It is disclosed
that
absorption of nanoscale chitosans and/or chitosan derivatives by the Stratum
Corneum
is increased leading to long-lasting deodorising effect. The chitosan is
normally used
at levels of 0.01-5, preferably 0.1-1, more particularly 0.2-0.6% w/w. The
document
provides long lists of anti-perspirants based on salts of aluminium, zirconium
or zinc,
and deodorants. The preparations may contain 1-50, preferably 5-30,
particularly 10-
% w/w anti-perspirants. Specific examples of anhydrous anti-perspirant or
deodorant suspension sticks and soft solids, deodorant cream emulsions, and
oil-in-
water roll-on and sprayable anti-perspi rants / deodorants are provided. In
particular a
20 composition (composition 2 in table 2) is disclosed comprising the
nanoscale chitosan,
distearyl ether and dioctyl carbonate.
WO 03/072610 (Cognis Deutschland GmbH & Co. KG) discloses transparent cosmetic
preparations containing chitosan and having a pH of below 6, comprising a)
chitosan
25 and/or chitosan derivatives, b) at least one anionic surfactant, c) at
least one alkyl
oligoglycoside, and d) water. Chitosans are valuable raw materials for use in
cosmetics, because they have film-forming and moisturizing properties. They
are also
known to inhibit the activity of esterase-producing bacteria, so they are
often
incorporated into deodorants as well. Previously, it had been difficult to use
them
simultaneously with anionic surfactants, owing to the positive charge on them,
leading
to precipitation, which made the resulting preparation turbid. The document
provides
lists of anti-perspirants and esterase inhibitors. The preparations may
contain 1-50,
preferably 5-30, particularly 10-25 % w/w anti-perspirants. Transparent anti-
CA 03001437 2018-04-09
WO 2017/080945 PCT/EP2016/076807
4
perspirants are claimed in claim 9. Examples of water-based clear cosmetic
preparations containing chitosan and anionic surfactants are provided.
US 5 968 488 (Henkel KgA) discloses deodorizing preparations containing
cationic
biopolymers, aluminium chlorohydrate and esterase inhibitors. It has
surprisingly been
found that cationic biopolymers, preferably of the chitosan type, inhibit the
activity of
esterase-producing bacteria and that a synergistic deodorizing effect is
obtained in
conjunction with the two components mentioned above. The biopolymers have a
bacteriostatic effect. At the same time, the use of the cationic biopolymers
leads to an
improvement in the dermatological compatibility of the products. Examples of
water-
based compositions are provided. US 5 968 488 further discloses use of
propellant
gases for spray applications. The formulations are preferably marketed as
rollers (roll-
on emulsion), sticks, deodorant sprays or pump sprays.
WO 2015/058935 discloses the use of chitosan or a salt thereof as the sole
anti-
perspirant ingredient in an anti-perspirant composition.
A number of products comprising, amongst other things, chitosan have been
launched.
Thus Laverana has launched a deodorant spray and roll-on under their Lavera
brand in
Germany. The product was also claimed as an anti-perspirant.
Jukona has launched a deodorant gel comprising, amongst other things,
chitosan,
under their Jukona Rose brand in Germany. It was claimed as free from
aluminium
salts.
Scholl has launched in Belgium an anti-perspirant foot spray comprising
chitosan and
aluminium chlorohydrate menthyl lactate.
Natura Cosmeticos has launched a roll-on anti-perspirant deodorant under their
Natura
Kaiak brand in Argentina comprising chitosan and aluminium chlorohydrate.
Further improvements in this area would be desirable.
CA 03001437 2018-04-09
WO 2017/080945
PCT/EP2016/076807
Summary of the invention
The present inventors have found that high molecular weight chitosans or salts
thereof
provide a synergistic anti-perspirant combination with aluminium, zinc,
magnesium and
5 copper salts.
Thus in a first aspect, the present invention relates to an anti-perspirant
composition
comprising:
(a) chitosan or a salt thereof, with a weight average molecular weight of
greater than 80 kDa; and
(b) an aluminium, zinc, magnesium or copper salt or mixtures thereof.
wherein the composition comprises less than 3 wt% of aluminium salt.
In a second aspect, the invention relates to a method of reducing perspiration
from the
surface of the human body comprising the topical application of a composition
as
described herein.
In a third aspect, the invention relates to the use of a composition described
herein, in
an anti-perspirant composition as an anti-perspirant ingredient.
Such compositions have been found to have a synergistically better performance
than
anti-perspirant formulations that comprise chitosan as sole anti-perspirant
active or an
aluminium, zinc, magnesium or copper salt as the sole anti-perspirant active.
Such combinations can therefore lead to more efficacious anti-perspirant
formulations
or to a reduction in the use of aluminium salts for a given anti-perspirant
effectiveness
or lead to formulations that use salts based on zinc, magnesium and/or copper.
For the purposes of this invention the zinc and magnesium salts are preferred,
with
magnesium being the most preferred.
For the purposes of this specification, the term "anti-perspirant composition"
means a
composition which prevents or reduces the appearance of perspiration or sweat
in
CA 03001437 2018-04-09
WO 2017/080945 PCT/EP2016/076807
6
humans. In a preferred embodiment, the composition will be packaged in a form
which
indicates to the consumer that the composition has an anti-perspirant effect,
and in
particular may contain the wording "antiperspirant" on the packaging.
For the purposes of this specification, the term "anti-perspirant ingredient"
means an
ingredient which prevents or reduces the appearance of perspiration or sweat
in
humans.
For the purpose of this specification, the degree of acetylation is as
measured using
the dye-binding method (Gummow et al., Makromol. Chem., 186, 1239-1244
(1985)).
For the purposes of this specification the weight average molecular weight of
chitosan
may be determined by size exclusion chromatography. An example method involves
dissolving 20 mg of chitosan in 1 % v/v aqueous formic acid. Polysaccharide
reference
standards are dissolved in the same diluent. Samples and standards are left to
stand
overnight to allow complete dissolution. Samples are prepared in duplicate.
The
analysis may be carried out on an Agilent 1200 series HPLC equipped with an
ELSD
detector. The chromatographic separation is achieved on an Agilent PL aquagel-
OH
MIXED H, 300 x 7.5 mm ID, 8 mm particle size GPO column, using a buffer of
0.01 M
aqueous ammonium formate (0.1 % formic acid) at pH 3.1 as mobile phase, at a
flow
rate of 1.0 ml.min-1.
Chitosan is a partially deacetylated form of the arthropod shell material
chitin and is
soluble in water at a pH of no more than 6Ø As well as from arthropods,
chitosan and
its precursor, chitin, are produced by fungi and bacteria, thus potentially
providing a
non-animal source for chitosan from a by-product of the fermentation industry.
Without being bound by theory, it is thought that when chitosan or a salt
thereof is
applied to the skin, it can diffuse into pores where it comes into contact
with sweat,
which has a pH of approximately 6.2 to 7.7, and precipitates forming a gel
blocking the
pores and reducing sweat flow. The gel formed is not permanent as it is
hydrolysed
over time. However the presence of the aluminium, zinc, magnesium or copper
salt or
mixture thereof is believed to greatly strengthen the formation of the gel in
a synergistic
CA 03001437 2018-04-09
WO 2017/080945 PCT/EP2016/076807
7
manner or is involved in precipitation, or a chelation/bridging phenomenon
owing to the
metal ion.
Preferably the chitosan or salt thereof has a weight average molecular weight
of
greater than 100 kDa, preferably greater than 300 kDa, more preferably greater
than
500 kDa, most preferably greater than 1000 kDa.
Preferably the chitosan or salt thereof has a degree of acetylation of 0-40 %.
Preferred salts of chitosan are selected from the group consisting of acetate,
chloride,
citrate, formate, fumarate, gluconate, glycolate, lactate, maleate, malate,
phosphate,
propionate, succinate, sulphate, tartrate and mixtures thereof, preferably
selected from
the group consisting of formate, glycolate, lactate and mixtures thereof.
Preferred salts are chlorides, sulphates and nitrates. Particular examples of
suitable
salts are aluminium chloride, aluminium chlorohydrate, zinc chloride, zinc
sulphate,
zinc nitrate, magnesium sulphate, magnesium chloride and copper chloride.
Preferably the anti-perspirant composition comprises 0.01-5, preferably 0.01-
2, most
preferably 0.01-1 % w/w chitosan or chitosan salt.
Preferably the anti-perspirant composition comprises 0.01-5, preferably 0.01-
2, most
preferably 0.01-1 % w/w aluminium, zinc, copper salt or mixture thereof.
Preferably the anti-perspirant composition comprises less than 2 wt% aluminium
salt,
preferably less than 1 wt%, and most preferably is substantially or completely
free of
aluminium salt.
The chitosan or salt thereof is preferably either in an anhydrous form or
dissolved in
water at a pH of no more than 6.0, preferably no more than 5.5, most
preferably no
more than 5Ø The chitosan or salt thereof can be dissolved in water at a pH
of at least
3.5, preferably at least 4.0, more preferably 4.5.
CA 03001437 2018-04-09
WO 2017/080945
PCT/EP2016/076807
8
Preferably a 0.5% wt/vol aqueous solution of the chitosan or salt thereof has
an
apparent viscosity of greater than 6.0, more preferably greater than 10.0 most
preferably greater than 15.0 mPas at a shear rate of 100s-1 at room
temperature.
The composition may additionally comprise auxiliary ingredients selected from
the
group consisting of a fragrance, a bactericidal agent, a bacteriostatic agent,
a
perspiration absorber, an esterase inhibitor, a surfactant, a thickener, a
chelator and a
preservative.
The composition preferably has a pH of from 3.0 to 6.0, preferably from 3.0 to
4.5,
more preferably from 3.5 to 4.5.
Suitable bactericides include chlorinated aromatics such as biguanide
derivatives of
which triclosan (e.g. lrgasan DP300 or Triclorban), and chlorhexidine warrant
specific
mention. Another class of effective bactericide comprises polyaminopropyl
biguanide
salts such as are available under the trade mark Cosmosil.
Chelators that can sequester iron retard bacterial growth and thereby inhibit
malodour
formation. Examples include aminopolycarboxylates such as ethylenediamine
tetraacetic acid (EDTA) or higher homologues such as diethylenetriamine
pentaacetic
acid (DTPA).
Bactericides and chelators are commonly employed at a concentration of from
0.1 to 5,
and particularly 0.1 to 2 % w/w.
The composition can be in the form of a gel, or suitable for spray
application, or
suitable for application by aerosol, or suitable for application with a stick
applicator.
The method for their manufacture is well known to those skilled in the art.
One preferred format is that the anti-perspirant composition is an aerosol
composition
comprising a volatile propellant.
As the composition is intended for use as an anti-perspirant composition, it
is a leave-
on composition, which means that the product is applied to the body without
washing
CA 03001437 2018-04-09
WO 2017/080945 PCT/EP2016/076807
9
off with water at the time of application so that it is left on the body
surface for a
substantial period of time of e.g. a few or several hours at least.
The composition typically comprises less than 10 wt% surfactant and preferably
less
than 5 wt% surfactant. If any surfactant is present then it is preferably only
of the non-
ionic type and is substantially free of any other types of surfactants.
Preferably the composition comprises less than 10 wt% dipropyleneglycol,
preferably
less than 5 wt%. Preferably the composition comprises less than 10 wt%
triglycerides,
preferably less than 5 wt%. Such materials can provide the composition with an
oily
nature, which is undesirable in an anti-perspirant leave-on composition which
is often in
contact with clothing of a user.
Examples
Methods
This utilised 0.5 pl TLC dropper pipettes, manufactured by Camag and
obtainable
through VWR International, Lutterworth, UK. From the known volume (0.5 pl) and
length of the capillary (3.2 cm) it was possible to calculate the internal
diameter as 141
pm.
Artificial sweat was drawn into a glass capillary (141 pm aperture) under
capillary
action for one hour. The artificial sweat was of the following composition and
adjusted
to the appropriate pH with sodium hydroxide (default pH was 7.7 unless
stated).
160 mg.I-1 Potassium chloride
1180 mg.I-1 Sodium bicarbonate
840 mg.I-1 Sodium chloride
212 mg.I-1 Ammonium chloride
892 mg.I-1 L-(+)-lactic acid
540 mg.I-1 L-Methionine
52 mg.I-1 Mucic acid
180 mg.I-1 Urea
CA 03001437 2018-04-09
WO 2017/080945 PCT/EP2016/076807
Artificial sweat of known pH (range 6.0-7.7) was drawn up the 141 pm capillary
by
capillary action and the capillary was noted to be full within 5 seconds. The
capillary
was then suspended in a solution of the active to be tested at the
concentration and pH
5 desired for a period of 1 hour. The capillary was then removed from the
active solution
and allowed to dry for approximately 15 minutes before the break pressure
measurement was made. This permitted the observation of sweat breakthrough
that
would otherwise be masked by residual active solution on the outside of the
capillary.
The use of tissue to dry the capillary was avoided as this may have drawn out
material
10 from within the capillary.
The capillary to be measured for break pressure was inserted into the break
pressure
rig using the correct size adapter for the 141 pm capillary. The hydrostatic
pressure
applied to the capillary was increased gradually at a rate of 0.05 ml/min
until sweat was
seen to emerge from the tip of the capillary. The pressure at which this
occurred was
noted and recorded.
After immersion in the active solution, the glass capillary was attached to
the pressure
sensor rig using the correct adapter for the 10 pm capillaries. The
hydrostatic pressure
applied to the capillary was increased very gradually using a syringe pump set
to
dispense 0.2 ml/min of water. The pressure increase was monitored and recorded
by a
pressure sensor (OmegaDyne Inc., OH, USA, model PXM409, maximum of 1 Bar),
with an instantaneous readout available on a computer screen using the
software
supplied by the sensor manufacturer (TRH Control, OmegaDyne Inc., OH, USA).
However in practice the maximum readable pressure is 800mBar due to the
equipment
attached to it. The pressure at which a visual breakthrough of water from the
tip of the
capillary is achieved is noted.
CA 03001437 2018-04-09
WO 2017/080945
PCT/EP2016/076807
11
Table 1: Summary of Chitosan Sources
Company Origin Name/ Mw (kDa) Viscosity Degree of
Degree of
Code of 0.5% Acetylation
Acetylation
tCompany solution (Dye binding)
(UV spectrum)
data (mPa.s at n = 3, SD, n = 2, range,
a shear except 740179
except 740179
* RSSL rate of 100 (single (single
data _1
s ) determination)
determination)
2796*/
Sigma Crab 41865 t 141.4 0.0 0.0 18.4
4.1
600
Shrimp C3646 1373* 29.54 10.1 0.8 23.7
4.4
Clariant/
Aspergillus Zenvivo 50-80t 5.02 1.1 1.9 14.6
3.2
Kitozyme Aqua
Error calculations
For all measurements of breakthrough pressures with 141 pm capillaries, the
error
calculations have used the Standard Error of the Mean:
SE 2 =
where
s is the sample standard deviation (i.e., the sample-based estimate of the
standard deviation of the population), and
n is the size (number of observations) of the sample.
Example 1: Effect of aluminium and chitosans as sole actives
Three commercially used antiperspirant actives based on aluminium have been
tested
with the capillary set up: aluminium chloride, aluminium chlorohydrate and
AZAG
(activated zirconium aluminium glycine).
CA 03001437 2018-04-09
WO 2017/080945
PCT/EP2016/076807
12
Aluminium chloride solutions were pH 4.6 (any higher causes precipitation),
aluminium
chlorohydrate and AZAG solutions were all pH 5Ø All were tested against pH
7.7
artificial sweat. The results are shown in Table 2.
Table 2: Effect of Aluminium active (as sole active)
water 0.1% AlC13 0.2% AlC13 0.5% AlC13 1% AlC13
mean 18.7 25.9 26.9 25.8 26.5
se
1.0 0.6 1.9 0.9 0.5
4 3 4 3 2
0.1% 0.2% 0.5% 1% 0.05% 0.1% 0.2% 0.5% 1%
AICH AICH AICH AICH AZAG
AZAG AZAG AZAG AZAG
mean 17.5 21.5 26.0 25.2 16.1 26.3 32.7
29.3 31.5
se
0.8 2.0 3.2 0.6 1.4 3.1 1.3 0.9 2.6
3 3 3 2 6 9 2 3 3
No real pore blocking effect was observed with these aluminium materials with
the 141
pm capillaries. Unbuffered higher concentrations up to 20% were evaluated,
with no
difference to the data but the pH of these solutions was as low as pH 1 and
therefore
unlikely to have been gelled by a weak buffer artificial sweat.
The three chitosans were tested for their pore-blocking capability. The
results are
shown in Table 3.
25
CA 03001437 2018-04-09
WO 2017/080945 PCT/EP2016/076807
13
Table 3: Effect of Chitosans as sole active
Crab Chitosan
Chitosan 0 0.01% 0.05% 0.10%
Concentration
mean 11.3 22.6 93.9 111.4
8 14 8
se 0.7 5.7 44.9 10.9
Shrimp Chitosan
Chitosan 0 0.01% 0.05% 0.10%
Concentration
10 mean 11.3 14.6 86.7 104.1
15 22 22 20
se 0.7 1.6 27.3 35.7
Zenvivo Aqua Chitosan
Chitosan 0 0.01% 0.05% 0.10%
Concentration
15 mean 11.3 15.5 12.6 13.6
15 4 5 5
se 0.7 1.7 1.5 0.8
Example 2: Effect of Combinations of Chitosans and Metal Salts to show a
possible synergistic effect
The solutions of actives were made up by mixing chitosan and metal salt
solutions so
that the final concentrations of the individual components were 0, 0.01, 0.5
or 0.1% w/v
and ph was 5.0 (in the case of Aluminium chloride combinations it was only
possible to
adjust to pH4.6 without precipitation).
Table 4: Various Chitosans or EDTA in combination with Aluminium Chloride
(AIC13)
c. 0c. 0 m m
0 .1 o 0 .1 o c 7) '= 1 8
0 .1
Metal AlC13 0% AlC13 0.01% AlC13 0.05%
AlC13 0.1% 0
Concn
n.)
o
%
--.1
Crab 0.0% 0.01% 0.05% 0.10% 0.0% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05%
0.10% 0.0% 0.01% 0.05% 0.10% o
oe
Chitosan
o
mean 12 20 34.25 99.33 14.4 12 15.5 718 13.75 15.25 15.25 800 11.75 14.5 24.75
800 .6.
un
n 4 4 4 3 4 4 4 4 4 4
4 2 4 4 4 2
se 1.732 1.633 4.767 12.73 0.758 0.707 1.190 82 1.97 1.79 4.49 0
3.04 1.323 2.287 0
AlC13 0% AlC13 0.01% AlC13 0.05%
AlC13 0.1%
Shrimp 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
0.00% 0.01% 0.05% 0.10%
Chitosan
mean 12 14.4 32.75 81.75 11.8 13.5 21.5 15.25 13.75 13.5 34.5
17.75 11.75 17 606.33 688.66
P
n 4 5 4 4 4 4 4 4 4 4
4 4 4 4 4 3 .
i,
.
se 1.732 0.678 6.725 52.3 3.305 2.102 4.349 3.09 1.974 1.041 14.57
2.13 3.04 4.378 167.7 111.3 .
,
1-,
L.
4=,
-,
Iv
o
AlC13 0% AlC13 0.01% AlC13 0.05%
AlC13 0.1% ,
00
i
Aqua 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10% 0.00% 0.01%
0.05% 0.10% 0.00% 0.01% 0.05% 0.10% .
i
Chitosan
'
mean 13.33 16 13.33 13.75 8.667 12 31.25 21 16.5 22.2 24
21.25 14.33 14 21.25 17
n 3 2 3 4 3 3 4 4 2 5
4 4 3 5 4 5
se 0.882 4 2.603 1.031 1.201 1.732 7.273 0.408 1.5 1.2 5.033
1.702 2.028 2.49 0.629 1.48
AlC13 0% AlC13 0.01% AlC13 0.05%
AlC13 0.1%
EDTA 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
0.00% 0.01% 0.05% 0.10% IV
n
,-i
mean 12.33 12.5 13.25 11 12 11 9 13 8 8 12
12 14.5 12.25 13 14 t=1
IV
n 3 4 4 2 1 1 1 4 1 1 1
4 4 4 4 4 n.)
o
1-,
se 0.33 0.288 0.853 1 1.732
0.707 1.5 0.946 0.408 0.707 c:
-1
--.1
o
oe
o
-4
CA 03001437 2018-04-09
WO 2017/080945 PCT/EP2016/076807
Table 5: Shrimp Chitosan + Aluminium Chlorohydrate (AICH)
5
AICH 0% AICH 0.01%
Shrimp 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
Chitosan
mean 13.5 14.7 39.8 298.0 16.8 54.7 39.4
188.3
4 3 4 3 4 3 5 4
se 1.2 1.2 6.0 251.3 1.3 36.7 12.6 27.2
AICH 0.05% AICH 0.1%
Shrimp 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
Chitosan
mean 11.6 22.0 74.0 170.3 25.3 125.5 57.8
345.0
5 4 4 4 4 4 4 3
se 0.9 1.9 40.9 23.8 4.6 42.2 12.2 227.8
10
15
CA 03001437 2018-04-09
WO 2017/080945 PCT/EP2016/076807
16
Table 6: Various Chitosans or EDTA + AZAG (Activated Zirconium Aluminium
Glycine
complex)
AZAG 0% AZAG 0.01%
Crab 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
Chitosan
mean 13.8 25.3 30.0 127.0 11.0 23.0 237.8
800.0
n 4 4 4 4 13 4 4 2
se 0.9 12.0 5.2 17.2 0.5 3.3 188.1 0.0
AZAG 0.05% AZAG 0.1%
Crab 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
Chitosan
mean 16.8 32.3 228.5 800.0 33.9 130.3 604.0
800.0
n 12 4 4 1 12 3 4 1
se 0.6 10.0 190.5 0.0 9.2 35.0 196.0 0.0
AZAG 0% AZAG 0.01%
Shrimp 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
Chitosan
mean 13.8 11.0 23.7 29.5 11.0 16.3 16.0
800.0
n 4 4 3 4 13 4 4 2
se 0.9 0.9 1.2 2.2 0.5 3.6 1.7 0.0
AZAG 0.05% AZAG 0.1%
Shrimp 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
Chitosan
mean 16.8 15.8 23.3 800.0 33.9 618.7 800.0
800.0
n 12 4 4 1 12 3 1 1
se 0.6 1.7 2.9 0.0 9.2 181.3 0.0 0.0
AZAG 0% AZAG 0.01%
EDTA 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
mean 12.3 12.5 13.3 11.0 13.0 14.0 11.0
15.3
n 3 4 4 2 1 1 1 4
se 0.3 0.3 0.9 1.0 0.0 0.0 0.0 1.9
AZAG 0.05% AZAG 0.1%
EDTA 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
mean 8.0 8.0 8.0 12.0 11.0 32.3 11.0 11.8
n 1 1 1 4 4 4 5 4
se 0.0 0.0 0.0 0.6 1.4 22.3 0.4 0.5
10
CA 03001437 2018-04-09
WO 2017/080945 PCT/EP2016/076807
17
Table 7: Various Chitosans + Zinc Chloride ZnCl2
ZnCl2 0% ZnCl2 0.01%
I I I I I I
Shrimp Chitosan 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
mean 8.8 16.8 31.3 84.5 13.5 245.0 256.5 542.0
n 4 4 4 4 4 4 4 3
se 1.4 6.4 7.2 16.1 5.8 125.1 183.1
258.0
ZnCl2 0.05% ZnCl2 0.1%
Shrimp Chitosan 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
mean 13.0 800.0 800.0 800.0 12.0 231.3 701.0 800.0
n 4 3 1 1 4 4 2 1
se 0.8 1.1 190.0 99.0
ZnCl2 0% ZnCl2 0.01%
i I I I
Crab 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
mean 8.8 16.8 31.3 84.5 nd 6.0 5.5 259.3
n 4 4 4 4 1 2 4
se 1.4 6.4 7.2 16.1 0.0 1.5 137.2
ZnCl2 0.05% ZnCl2 0.1%
Crab 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
mean nd nd 4.5 1036.0 13.0 13.3 703.8
n 2 2 2 4 4
se 0.5 36.0 4.0 2.9 209.2
10
20
CA 03001437 2018-04-09
WO 2017/080945 PCT/EP2016/076807
18
Table 8: Shrimp Chitosans + Zinc Sulphate ZnSat
ZnSO4 0% ZnSO4 0.01%
Shrimp 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
mean 11.75 21.33333 12.75 14.25 15 15 321 127.5
n 4 4 4 4 1 1 4 4
se 0.853 3.6 0.946 0.946 00 , , 186.5
70.62
I I I I
Zn504 0.05% Zn504 0.1%
Shrimp 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
mean 19 16 34 141.25 15.75 18 609 272.75
n 1 1 4 4 4 5 4 4
se 0 0 10.29 80.26 0.478 2.5 114.78 39.70
Table 9: Shrimp Chitosan + Copper (II) Chloride CuCl2
CuCl2 0% CuCl2 0.01%
Shrimp 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
Chitosan
mean 13.3 12.0 349.0 732.0 13.0 14.8 800.0 nd
n 4 4 4 4 4 4 4 3
se 1.3 0.9 22.9 1.7 1.8
CuCl2 0.05% CuCl2 0.1%
Shrimp 0.00% I 0.01% I 0.05% I 0.10% 0.00% I 0.01% I 0.05% I 0.10%
Chitosan
mean 13.3 15.5 800.0 800.0 14.3 22.3 800.0 800.0
n 4 3 1 1 4 4 1 1
se 0.3 , 0.7 , 0.6 , 6.2 ,
i .
20
CA 03001437 2018-04-09
WO 2017/080945
PCT/EP2016/076807
19
Table 10: Shrimp Chitosan + Calcium Chloride (CaCl2)
CaCl2 0% CaCl2 0.01%
Shrimp 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
mean 11.8 21.3 12.8 14.3 nd nd nd 14.8
n 4 4 4 4 4
se 0.9 3.6 0.9 0.9 0.5
CaCl2 0.05% CaCl2 0.1%
Shrimp 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
mean nd nd nd 13.5 10.0 6.5 13.0 13.9
n 4 5 4 8 12
se 1.0 1.3 1.3 2.8 1.0
Table 11: Shrimp Chitosan + Glycine
Glycine 0% Glycine 0.01%
Shrimp 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
Chitosan
mean 8.8 16.8 31.3 84.5 13.0 16.3 42.8
141.5
n 4 4 4 4 4 4 4 2
se 1.4 6.4 7.2 16.1 3.2 2.3 3.4 14.5
Glycine 0.05% Glycine 0.1%
Shrimp 0.00% 0.01% 0.05% 0.10% 0.00% 0.01% 0.05% 0.10%
Chitosan
mean 12.5 6.5 nd nd 10.7 15.5 20.3 17.8
n 4 4 4 4 3 5
se 2.1 1.0 0.8 1.0 4.7 2.1
CA 03001437 2018-04-09
WO 2017/080945
PCT/EP2016/076807
Table 12: 0.1% Shrimp Chitosan with various metal salts
Metal MgC12 MgSO4 Mg(NO3)2
Salt
0.05% 0.10% 0.20% 0.05% 0.10% 0.20% 0.05% 0.10% 0.20%
mean 457.3
483.0 410.5 1055.8 1072.2 1072.3 257.7 582.5 1072.5
n 4 4 4 5 5 9 3 2 2
se 216.8 152.2 169.5 10.4 0.2 0.2 10.9 142.5
0.5
Metal KCI FeCl2 FeCI3
Salt
0.05% 0.10% 0.20% 0.05% 0.10% 0.20% 0.05% 0.10% 0.20%
mean 25.4 26.1 47.1 27.5 23.5 30.3 25.5
25.0 28.6
n 9 10 10 2 2 3 2 2 7
se 1.9 3.5 10.0 1.5 4.5 6.3 1.5 4.0
2.8
Metal NiCl2 CoCl2 GaCI3
Salt
I I I I I I
0.05% 0.10% 0.20% 0.05% 0.10% 0.20% 0.05% 0.10% 0.20%
mean 21.3 28.3 28.8 25.0 22.5 22.2 25.0
23.6 113.5
n 4 3 5 4 4 5 11 11 12
se 3.1 , 1.3 , 1.5 2.3 , 1.8 , 3.2
1.6 , 2.4 6.0
Metal MnCl2 AgNO3 Zn(NO3)2
Salt
0.05% 0.10% 0.20% 0.05% 0.10% 0.20% 0.05% 0.10% 0.20%
mean 22.3 80.5 91.6 29.3 48.2 46.1 813.5 -
1073.0
n 12 11 11 4 5 8 4 - 3
se 1.7 26.1 23.7 4.8 23.1 28.4 259.5 -
0.0
5
Table 13: 0.5% Aqua Chitosan with magnesium salts
Mg504 MgC12
Metal salt 0.05% 0.10% 0.20% 0.05% 0.10% 0.20%
mean 12.4 21.5 14.4 8.6 23.0 1419
n 5 4 5 5 5 5
se 1.3 5.1 1.3 1.0 6.3 0.4
CA 03001437 2018-04-09
WO 2017/080945 PCT/EP2016/076807
21
An effect has been observed with chitosans of high molecular weight (e.g. crab
and
shrimp chitosan) and aluminium, zinc, magnesium and copper salts whereby the
pore
blocking capability of the combination is greater than the sum of the pore
blocking
capability of the individual components at the same concentration.
The synergistic effect is not shown to be present between chitosan of high
molecular
weight (e.g. shrimp chitosan) and calcium chloride.
EDTA is a known chelating agent as it was considered that the chitosan may be
chelating the divalent and trivalent salts to create the synergistic pore
blocking effect.
Glycine was also evaluated as a simple amino acid that was present as part of
the
AZAG complex.
The synergistic effect is also not shown to be present between chitosans of
high
molecular weight (e.g. shrimp chitosan) and glycine.
The synergistic effect is also not shown to be present between EDTA and
aluminium
salts.
Example 3: Speed of Gelation Assay
0.5 ml aliquots of the test chitosans in 100 mM acetic acid were carefully
pipetted into a
2 ml Eppendorf. A weighed filter paper disc was pushed onto the surface with a
plastic
rod and 0.5 ml aliquots of 100 mM sodium hydroxide carefully pipetted on top.
For the
control tubes, the sodium hydroxide solution was removed and the filter paper
taken
out with forceps and dipped three times in distilled water. The washed paper
was then
left to dry at 50 C in a forced-air oven for one hour on a weighing boat,
then weighed.
The same procedure was followed for the remaining tubes at known times. The
initial
rate of gelation was determined using the quadratic equation function in
Microsoft'
ExcelTM.
The initial rates of gelation on the filter papers were:
0.50 % Shrimp Chitosan = 4 pg.min-1
CA 03001437 2018-04-09
WO 2017/080945
PCT/EP2016/076807
22
0.50 % ZnCl2 = 1 ug.min-1
0.50 % Chitosan + 0.25 % ZnCl2 = 7 ug.min-1
0.50 % Chitosan + 0.50 % ZnCl2 = 10 ug.min-1
At two concentration levels the speed of gelation is greater than the sum of
the speeds
of gelation of chitosan and zinc chloride separately.
