U.S. patent application number 11/514790 was filed with the patent office on 2006-12-28 for water-soluble chitosan having low endotoxin concentration and methods for making and using the same.
This patent application is currently assigned to Adjuvant Pharmaceuticals, LLC. Invention is credited to Katrina L. Bergbauer, William M. Hung, Kai C. Su, Sherry Wages, Guigui Wang.
Application Number | 20060293509 11/514790 |
Document ID | / |
Family ID | 34422308 |
Filed Date | 2006-12-28 |
United States Patent
Application |
20060293509 |
Kind Code |
A1 |
Hung; William M. ; et
al. |
December 28, 2006 |
Water-soluble chitosan having low endotoxin concentration and
methods for making and using the same
Abstract
Water-soluble chitosan having a low concentration of endotoxin
is disclosed. Products containing the water-soluble chitosan are
also disclosed. Methods of making and using water-soluble chitosan
having a low concentration of endotoxin are further disclosed.
Inventors: |
Hung; William M.;
(Alpharetta, GA) ; Bergbauer; Katrina L.;
(Decatur, GA) ; Su; Kai C.; (Alpharetta, GA)
; Wang; Guigui; (Cumming, GA) ; Wages; Sherry;
(Atlanta, GA) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
Adjuvant Pharmaceuticals,
LLC
Alpharetta
GA
30004
|
Family ID: |
34422308 |
Appl. No.: |
11/514790 |
Filed: |
September 1, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10681560 |
Oct 8, 2003 |
7125967 |
|
|
11514790 |
Sep 1, 2006 |
|
|
|
Current U.S.
Class: |
536/20 |
Current CPC
Class: |
C08B 37/003 20130101;
A61K 31/722 20130101 |
Class at
Publication: |
536/020 |
International
Class: |
C08B 37/08 20060101
C08B037/08 |
Claims
1. A water-soluble chitosan having low endotoxin content, wherein
the water-soluble chitosan has an_endotoxin content of less than
about 100 equivalent units (e.u.) of endotoxin per gram of dry
water-soluble chitosan, and wherein the water-soluble chitosan is
formed by: contacting water-insoluble chitosan with a basic
solution for a first period of time; rinsing the water-insoluble
chitosan to remove any residual basic solution; partially
acetylating the water-insoluble chitosan in a reaction solution
containing a phase transfer agent to form partially acetylated
water-soluble chitosan; dissolving the partially acetylated
water-soluble chitosan in an aqueous solution containing a
surfactant; adjusting a pH of the aqueous solution to a pH of at
least 7.0; adding a water-miscible solvent into the aqueous
solution having a pH of at least 7.0; further adjusting the pH of
the aqueous solution to a pH of at least 8.0 to cause precipitation
of water-soluble chitosan having low endotoxin content; separating
the water-soluble chitosan having low endotoxin content from the
aqueous solution; and washing the water-soluble chitosan having low
endotoxin content with the water-miscible solvent.
2. A water-soluble chitosan having low endotoxin content, wherein
the water-soluble chitosan has an endotoxin content of less than
about 50 equivalent units (e.u.) of endotoxin per gram of dry
water-soluble chitosan, and wherein the water-soluble chitosan is
formed by: contacting water-insoluble chitosan with a basic
solution for a first period of time; rinsing the water-insoluble
chitosan to remove any residual basic solution; partially
acetylating the water-insoluble chitosan in a reaction solution
containing a phase transfer agent to form partially acetylated
water-soluble chitosan; dissolving the partially acetylated
water-soluble chitosan in an aqueous solution containing a
surfactant; adjusting a pH of the aqueous solution to a pH of at
least 7.0; adding a water-miscible solvent into the aqueous
solution having a pH of at least 7.0; further adjusting the pH of
the aqueous solution to a pH of at least 8.0 to cause precipitation
of water-soluble chitosan having low endotoxin content; separating
the water-soluble chitosan having low endotoxin content from the
aqueous solution; and washing the water-soluble chitosan having low
endotoxin content with the water-miscible solvent.
3. A water-soluble chitosan having low endotoxin content, wherein
the water-soluble chitosan has an_endotoxin content of less than
about 20 equivalent units (e.u.) of endotoxin per gram of dry
water-soluble chitosan, and wherein the water-soluble chitosan is
formed by: contacting water-insoluble chitosan with a basic
solution for a first period of time; rinsing the water-insoluble
chitosan to remove any residual basic solution; partially
acetylating the water-insoluble chitosan in a reaction solution
containing a phase transfer agent to form partially acetylated
water-soluble chitosan; dissolving the partially acetylated
water-soluble chitosan in an aqueous solution containing a
surfactant; adjusting a pH of the aqueous solution to a pH of at
least 7.0; adding a water-miscible solvent into the aqueous
solution having a pH of at least 7.0; further adjusting the pH of
the aqueous solution to a pH of at least 8.0 to cause precipitation
of water-soluble chitosan having low endotoxin content; separating
the water-soluble chitosan having low endotoxin content from the
aqueous solution; and washing the water-soluble chitosan having low
endotoxin content with the water-miscible solvent.
4. A water-soluble chitosan having an endotoxin content of less
than about 100 equivalent units per gram of dry water soluble
chitosan, formed by: contacting water-insoluble chitosan with a
NaOH solution for a first period of time of greater than 1 hour;
partially acetylating the water-insoluble chitosan in a reaction
solution containing a phase transfer agent to form partially
acetylated water-soluble chitosan; dissolving the partially
acetylated water-soluble chitosan in an aqueous solution containing
a surfactant and having a pH of from about 7.0 at about 7.4; and
adding a water-miscible solvent into the aqueous solution and
further adjusting the pH of the aqueous solution to a pH of at
least 8.0 to cause precipitation of water-soluble chitosan having
low endotoxin content from the aqueous solution/water-miscible
solvent mixture.
5. A pharmaceutically acceptable solution comprising the
water-soluble chitosan of claim 4 and at least one buffer
material.
6. The pharmaceutically acceptable solution of claim 5, where the
pharmaceutically acceptable solution is a contact lens cleaning
solution.
7. A pharmaceutically acceptable solution comprising: at least one
buffer material; and a water-soluble chitosan made by: contacting
water-insoluble chitosan with a basic solution for a first period
of time; rinsing the water-insoluble chitosan to remove any
residual basic solution; partially acetylating the water-insoluble
chitosan in a reaction solution containing a phase transfer agent
to form partially acetylated water-soluble chitosan; dissolving the
partially acetylated water-soluble chitosan in an aqueous solution
containing a surfactant; adjusting a pH of the aqueous solution to
a pH of at least 7.0; adding a water-miscible solvent into the
aqueous solution having a pH of at least 7.0; further adjusting the
pH of the aqueous solution to a pH of at least 8.0 to cause
precipitation of water-soluble chitosan having low endotoxin
content; separating the water-soluble chitosan having low endotoxin
content from the aqueous solution; and washing the water-soluble
chitosan having low endotoxin content with the water-miscible
solvent.
8. The pharmaceutically acceptable solution of claim 7, where the
pharmaceutically acceptable solution is a contact lens cleaning
solution.
9. A pharmaceutically acceptable solution comprising: at least one
buffer material; and a water-soluble chitosan made by: contacting
water-insoluble chitosan with a NaOH solution for a first period of
time of greater than 1 hour; partially acetylating the
water-insoluble chitosan in a reaction solution containing a phase
transfer agent to form partially acetylated water-soluble chitosan;
dissolving the partially acetylated water-soluble chitosan in an
aqueous solution containing a surfactant and having a pH of from
about 7.0 at about 7.4; and adding a water-miscible solvent into
the aqueous solution and further adjusting the pH of the aqueous
solution to a pH of at least 8.0 to cause precipitation of
water-soluble chitosan having low endotoxin content from the
aqueous solution/water-miscible solvent mixture.
10. The pharmaceutically acceptable solution of claim 9, where the
pharmaceutically acceptable solution is a contact lens cleaning
solution.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/681,560 filed on Oct. 8, 2003, which is incorporated
herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to water-soluble chitosan
having a low concentration of endotoxin and products containing the
same. The present invention further relates to methods for making
and using the water-soluble chitosan having a low concentration of
endotoxin.
BACKGROUND OF THE INVENTION
[0003] Endotoxin is a lipopolysaccharide existing on the surface of
the outer membrane of gram-negative bacteria and exhibits
pyrogenicity. Endotoxin must be removed from pharmaceutically
acceptable products.
[0004] Lipopolysaccharide endotoxin is not living bacteria and
cannot be deactivated by common sterilization techniques, such as
autoclaving. While gamma irradiation and dry heat sterilization
techniques destroy endotoxin, these techniques may also destroy or
damage many other components in a given composition. Therefore,
many sterile products can contain significant levels of endotoxin
unless the endotoxin is specifically removed or deactivated.
[0005] Current techniques employed in the pharmaceutical industry
to remove endotoxin from biomaterials have one or more
shortcomings. Size-separation techniques, such as gel permeation
chromatography or ultrafiltration, provide less than acceptable
results if the size of biomaterial is close to the size of
endotoxin present in the biomaterial. Other techniques, such as
absorption techniques, absorb endotoxin into one or more
absorbents. However, if the endotoxin has a greater affinity for
the biomaterial than the one or more absorbents, unacceptable
separation results.
[0006] There exists a need in the art of effective methods of
reducing endotoxins in chitosan and products containing
chitosan.
SUMMARY OF THE INVENTION
[0007] The present invention addresses some of the difficulties and
problems discussed above by the discovery of a method for making
water-soluble chitosan or chitosan derivatives having a low
concentration of endotoxin. In one exemplary embodiment of the
present invention, the method for making water-soluble chitosan
comprises contacting water-insoluble chitosan with a NaOH solution
for a first period of time of greater than about one hour;
partially acetylating the water-insoluble chitosan in a reaction
solution containing a phase transfer agent to form partially
acetylated water-soluble chitosan; dissolving the partially
acetylated water-soluble chitosan in an aqueous solution containing
a surfactant and having a pH of greater than 7.0; and adding a
water-miscible solvent into the aqueous solution to cause
precipitation of water-soluble chitosan having low endotoxin
content from the aqueous solution/water-miscible solvent mixture.
The exemplary method may further comprise rinsing steps, as well
as, other product processing steps.
[0008] The present invention is further directed to water-soluble
chitosan having a low concentration of endotoxin, and products made
therefrom. One exemplary product containing water-soluble chitosan
having a low concentration of endotoxin is a pharmaceutically
acceptable solution, such as a preserving solution for contact
lens. In one exemplary embodiment of the present invention, the
water-soluble chitosan comprises partially acetylated water-soluble
chitosan having a degree of N-- acetylation of from about 24% to
about 55%, and a degree of O-- acetylation of from about 1% to
about 60%, wherein the partially acetylated water-soluble chitosan
comprises less than about 100 equivalent units (e.u.) of endotoxin
per gram of dry water-soluble chitosan.
[0009] These and other features and advantages of the present
invention will become apparent after a review of the following
detailed description of the disclosed embodiments and the appended
claims.
DETAILED DESCRIPTION OF THE INVENTION
[0010] To promote an understanding of the principles of the present
invention, descriptions of specific embodiments of the invention
follow and specific language is used to describe the specific
embodiments. It will nevertheless be understood that no limitation
of the scope of the invention is intended by the use of specific
language. Alterations, further modifications, and such further
applications of the principles of the present invention discussed
are contemplated as would normally occur to one ordinarily skilled
in the art to which the invention pertains.
[0011] The present invention is directed to methods for making
water-soluble chitosans or chitosan derivatives having a low
concentration of endotoxin, as well as, products containing the
water-soluble chitosans or chitosan derivatives. The products are
desirably "pharmaceutically acceptable" products. As used herein,
the phrase "pharmaceutically acceptable" is used to describe a
material that is not biologically or otherwise undesirable, i.e.,
the material may be administered to an individual without causing
any undesirable biological effects or interacting in a deleterious
manner with any of the other components of the pharmaceutical
composition in which it is contained.
[0012] The resulting chitosans or chitosan derivatives are also
"water-soluble." As used herein, the phrase "water-soluble" is used
to include chitosans or derivatives thereof having a water
solubility of at least about 0.2% as measured by the Water
Solubility Test described below. The water-soluble chitosans or
chitosan derivatives of the present invention may have water
solubility of at least 0.2%, and in some cases, greater than
2%.
[0013] The Water Solubility Test comprises preparing a mixture of
0.200 g of a sample chitosan in 10 ml of deionized water, and
stirring at room temperature for approximately 18 hours. The
mixture is filtered through #1 qualitative filter paper. The
container is washed with a small amount of deionized water. The
filtrate is then placed in a weighed aluminum weighing dish and
dried in a vacuum oven at around 60.degree. C. The observed weight
difference is the weight of soluble chitosan. Percent water
solubility is expressed in terms of [(grams of soluble
chitosan)/(grams of water)].times.(100), so the maximum measurable
solubility is 2.0%. As discussed above, in some cases, the
water-soluble chitosans or chitosan derivatives of the present
invention may have water solubility greater than 2%; however, to
check the actual water solubility of these water-soluble chitosans
or chitosan derivatives, more than 0.200 g of chitosan or chitosan
derivative must be used in the above-described test method.
[0014] The water-soluble chitosans or chitosan derivatives are also
referred to herein as being "randomly substituted partial N-,
partial O-acetylated chitosans or chitosan derivatives." As used
herein, the phrase "randomly substituted" is used to describe
random substitution of acetyl groups on the chitosan main chain.
The random substitution of acetyl groups contributes to the water
solubility or hydrophilicity of the resultant chitosan polymer. As
used herein, the phrase "partial N-, partial O-acetylated chitosan"
or derivative thereof refers to a poly(N-,
O-acetylated-D-glucosamine).
[0015] As used herein, the term "degree of deacetylation" refers to
the percentage of free amino groups on the water-soluble, chitosan
or chitosan derivative. The percent of N-acetylation may be
calculated from the deacetylation value. The terms "percent
N-acetylation" or "percent O-acetylation" also refer to the degree
of --C(O)CH.sub.3 substitution on either the N or O of the chitosan
or chitosan derivative.
[0016] It is to be understood that although greater than 50%
N-acetylation is sometimes described in the art as a chitin, in the
present invention, the term "chitosan" include chitosan having a
percent N-acetylation of greater than 50% (i.e., includes
chitins).
[0017] Examples of chitosans or chitosan derivatives include, but
are not limited to, chitosan salts; water-soluble chitosan;
water-soluble, randomly substituted partial N-, partial
O-acetylation chitosan; chitosan oligosaccharide; carboxymethyl
chitosan; and hydroxyalkyl chitosan. The hydroxyalkyl substituents
of hydroxyalkyl chitosans and the carboxymethyl substituents of
carboxymethyl chitosans may be attached to any of the pendant
nitrogen or oxygen groups on the chitin or chitosan ring. Specific
hydroxyalkyl chitosans include, but are not limited to,
hydroxyethyl chitosan (also known as glycol chitosan);
hydroxypropyl chitosan; dihydroxypropyl chitosan; hydroxybutyl
chitosan; and dihydroxybutyl chitosan.
[0018] In a desired embodiment of the present invention, the
water-soluble, randomly substituted partial N-, partial
O-acetylated chitosan or derivative thereof is represented by the
following formula: ##STR1##
[0019] wherein R.sub.1, R.sub.2 and R.sub.3 are each independently
--H or --C(O)CH.sub.3, and wherein the chitosan or derivative
thereof is partially acetylated such that R.sub.1 has a degree of
substitution of --C(O)CH.sub.3 of from about 24 to about 55%, and
R.sub.2 has a degree of substitution of --C(O)CH.sub.3 of from
about 1 to about 60%; m is greater than 25; and wherein the partial
N-, partial O-acetylated chitosan or derivative thereof is randomly
substituted and is water-soluble.
[0020] The term "m" is the number of repeat units in the
water-soluble, chitosan or polymer chain. In one exemplary
embodiment of the present invention, m is about 100,000 or higher.
The molecular weight range of the water-soluble chitosan or polymer
chain herein refers to the weight average molecular weight. The
weight average molecular weight of the water-soluble chitosan or
polymer is typically at least about 5,000. In some embodiments of
the present invention, the weight average molecular weight may be
up to about 3,000,000 or higher.
[0021] The present invention is directed to methods for making any
of the above-described water-soluble chitosans or chitosan
derivatives having a low concentration of endotoxin. Methods for
making water-soluble chitosans or chitosan derivatives having a low
concentration of endotoxin are described below.
[0022] I. Methods for Making Water-Soluble Chitosan Having Low
Endotoxin Concentration
[0023] The present invention is directed to methods for making
water-soluble chitosan or chitosan derivatives (collectively
referred to herein as "chitosan" or "chitosans"). The methods of
the present invention comprise a combination of steps, wherein the
method comprises at least the following steps: contacting
water-insoluble chitosan with a basic solution (i.e., a solution
having a pH of greater than 7.0) for a period of time; and
partially acetylating the water-insoluble chitosan in a reaction
solution containing a phase transfer reagent to form partially
acetylated water-soluble chitosan. Typically, the water-insoluble
chitosan is placed in contact with a basic solution for a period of
time, wherein the period of time is greater than or equal to about
one hour. Desirably, the period of time is greater than or equal to
about 2 hours, more desirably, from about 2 hours to about 6 hours;
however, the period of time may even be greater than 6 hours if so
desired.
[0024] The basic solution used to contact the water-insoluble
chitosan may be a variety of basic solutions. Suitable basic
solutions include, but are not limited to, alkaline hydroxides,
such as potassium hydroxide or sodium hydroxide; alkaline
carbonates, such as sodium carbonate, or trisodium phosphate; and
combination thereof. Desirably, the basic solution used to contact
the water-insoluble chitosan comprises a NaOH solution having a
molar concentration ranging from 0.25M NaOH to about 1.0M NaOH,
more desirably a 1.0M NaOH solution.
[0025] The basic solution treated water-insoluble chitosan is
placed in a reaction solution to partially acetylate the chitosan.
Desirably, the reaction solution comprises an acetylating agent and
at least one phase transfer reagent. In one desired embodiment, the
reaction solution has a solution pH of less than about 6.0, more
desirably, from about 1.0 to about 4.0, and even more desirably,
from about 2.0 to about 3.0.
[0026] Suitable acetylating agents for use in the present invention
include any known acetylating agent. Exemplary acetylating agents
include, but are not limited to, acetyl halides, acetic anhydride,
and combinations thereof. Desirably, the acetylating agent is
acetic anhydride.
[0027] Suitable phase transfer reagents for use in the present
invention include any known phase transfer reagent. Suitable phase
transfer reagents include, but are not limited to, those described
in "Phase-Transfer Catalysis," Starks, C., et al. Chapman &
Hall, 1994, which is incorporated by reference is its entirety.
Example phase transfer reagents include, but are not limited to,
quaternary ammonium salts having a structure as shown in Equation I
below; quaternary phosphonium salts having a structure as shown in
Equation II below; crown ethers having structures as shown in
Equations IIIa-IIIc below; and pyridinium salts having a structure
as shown in Equation IV below:
[A].sub.w[B].sub.x[C.sub.].sub.y[D].sub.zN+Q Equation (I)
[A].sub.w[B].sub.x[C].sub.y[D].sub.zP.sup.+Q.sup.- Equation (II)
[0028] wherein: [0029] each of w, x, y, and z is independently an
integer from 0 to 4 and w+x+y+z=4; [0030] Q is a counter-ion
selected from F.sup.-, Cl.sup.-, Br.sup.-, I.sup.-,
CH.sub.3COO.sup.-, OH.sup.-, HSO.sub.4.sup.-, NO.sub.3.sup.-,
PF.sub.6.sup.-, BF.sub.4.sup.-, HCOO.sup.- and
H.sub.2PO.sub.4.sup.-; and [0031] A, B, C and D are each
independently selected from C.sub.1-C.sub.18 alkyl, phenyl in which
the phenyl ring is unsubstituted or substituted by C.sub.1-C.sub.8
alkyl, C.sub.1-C.sub.8 alkoxy, halo, hydroxy, phenoxy, nitro,
carboxy, acetamido, or aryl; benzyl; and cycloalkyl have 5-6 ring
member of heterocyclic ring system.
[0032] In one desired embodiment, quaternary ammonium salts (I) and
quaternary phosphonium salts (II) include, but are not limited to,
tetra C.sub.1-C.sub.4 alkyl ammonium halides, such as
tetrabutylammonium bromide ("TBABr"), tetramethylammonium chloride
("TMAC1"), tetrabutylammonium dihydrogen phosphate ("TBADHP"), and
tetrabutyl ammonium iodide ("TBAI"); benzyl tri C.sub.1-C.sub.4
alkylammonium halides, such as benzyltriethylammonium chloride
("BTEACI"); and tetra C.sub.1-C.sub.18 phosphonium halides, such as
tetrabutyl phosphonium bromide ("TMPBr") and hexadecyltributyl
phosphonium bromide ("HDTRPBr").
[0033] In a further embodiment, the phase transfer reagent
comprises at least one crown ether having a structure as shown in
Equations IIIa-IIIc below: ##STR2## [0034] wherein each X
independently represents O or S; [0035] R.sub.3 and R.sub.4 each
independently represent --H, C.sub.1 to C.sub.4 alkyl, or a
halogen; [0036] W1 and W2 each independently represent a
cycloaliphatic ring or an aromatic ring; and [0037] k, m, n and p
each independently represent integers ranging from 1 to 3.
[0038] Suitable crown ethers encompassed by Equation IIIa include,
but are not limited to, 12-crown-4,15-crown-5,18-crown-6 and
1,4,7,10,13,16-hexathiacyclooctadecane. Suitable crown ethers
encompassed by Equation IIIb include, but are not limited to,
benzo-12-crown-4, benzo-15-crown-5, and benzo-18-crown-6. Suitable
crown ethers encompassed by Equation Ilic include, but are not
limited to, dicylohexano-18-crown-6, dicyclohexano-24-crown-8,
dibenzo-18-crown-6, dibenzo-21-crown-7, dibenzo-24-crown-8,
dibenzo-30-crown-10, di-tere-butyl-di-benzo-18-crown-6 and
`4-bromoenzo-18-crown-6.
[0039] In yet a further embodiment, the phase transfer reagent
comprises at least one pyridinium salt having a structure as shown
in Equation IV below. ##STR3## [0040] wherein: [0041] R.sub.1
represents C.sub.1 to C.sub.18 alkyl, benzyl, or carboxymethyl;
[0042] R.sub.2 represents C.sub.1 to C.sub.4 alkyl, chloro, fluoro,
bromo, hydroxy, C.sub.1 to C.sub.4 alkoxyl or alkoxylcarbonyl; and
[0043] X represents a counterion of F, Cl, Br, I or p-toluene
sulfonate.
[0044] Suitable pyridinium salts encompassed by Equation IV
include, but are not limited to, C.sub.1 to C.sub.18 alkyl
pyridinium halides, such as 1-dodecylpyridinium chloride and
1-cetylpyridinium bromide, 1-benzyl pyridinium halides, and
1-benzyl-3-hydroxypyridinium chloride.
[0045] In one desired embodiment of the present invention, the
phase transfer reagent comprises a quaternary ammonium salt having
a structure as shown in Equation I, more desirably,
tetrabutylammonium bromide.
[0046] The solvent used in the reaction solution may be any solvent
suitable for use in an acetylating reaction system. Suitable
solvents include, but are not limited to, water; alcohols, such as
methanol, ethanol, and isopropanol; ethers such as diethyl ether
and tetrahydrofuran; polar solvents, such as dimethylormamide,
dimethyl sulfoxide and N-methylpyrrolidinone; ketones such as
acetone and 2-butanone; and combinations thereof. Desirably, the
solvent comprises an acidic aqueous solution (i.e., an aqueous
solution having a pH of less than 7.0).
[0047] The method for making water-soluble chitosan or chitosan
derivatives may further comprise one or more of the following
steps: [0048] agitating/mixing the water-insoluble chitosan in the
basic solution during the basic treatment step; [0049] rinsing the
water-insoluble chitosan to remove any residual basic solution
after the basic treatment step; [0050] dissolving the partially
acetylated water-soluble chitosan in an aqueous solution containing
a surfactant; [0051] adjusting a pH of the aqueous solution
(containing the partially acetylated water-soluble chitosan) to a
pH of about 7.0 desirably, slightly higher than 7.0, more
desirably, about 7.2; [0052] adding a water-miscible solvent into
the above aqueous solution; [0053] further adjusting the pH of the
aqueous solution (containing the partially acetylated water-soluble
chitosan and the water-miscible solvent) to a pH of at least 8.0 to
cause precipitation of water-soluble chitosan having low endotoxin
content; [0054] separating the water-soluble chitosan having low
endotoxin content from the aqueous solution; and [0055] washing the
water-soluble chitosan having low endotoxin content with a
water-miscible solvent.
[0056] Desirably, any rinsing step of the above-described method
for making water-soluble chitosan, wherein the water-insoluble
chitosan is rinsed comprises rinsing the water-insoluble chitosan
with endotoxin-free water.
[0057] During the dissolving step, one or more surfactants may be
added to the aqueous mixture containing dissolved partially
acetylated water-soluble chitosan. Suitable surfactants include,
but are not limited to, surfactants, which are block copolymers of
ethylene oxide and propylene oxide and commercially available under
the trade designation PLURONIC.RTM. from BASF Corporation (Mount
Olive, N.J.); surfactants, which are block copolymers resulting
from addition or ethylene oxide and propylene oxide to ethylene
diamine and commercially available under the trade designation
TETRONIC.RTM. from BASF Corporation (Mount Olive, N.J.); and
polyoxyethylene sorbitan fatty acid esters commercially available
under the trade designation TWEEN.RTM. from Uniqema Corporation
(New Castle, Del.). Desirably, the surfactant comprises TWEEN.TM.
20, a polyoxyethylene sorbitan monolaurate.
[0058] Adjustments to the pH of solutions used during the
above-described method for making water-soluble chitosan may be
made using a variety of pH control agents. Suitable pH control
agents for use in the present invention include, but are not
limited to, acids, such as acetic acid, and hydrochloric acid; and
bases such as those described above. Desirably, pH control agents
used in the present invention to increase solution pH comprise NaOH
solutions, more desirably, NaOH solutions having a NaOH molar
concentration of from about 0.25M to about 1M NaOH. Desirably, pH
control agents used in the present invention to decrease solution
pH comprise diluted hydrochloric acid solutions.
[0059] A variety of water-miscible solvents may be used in the
precipitation and washing steps of the above-described method.
Suitable water-miscible solvents include, but are not limited to,
isopropanol, methanol, and acetone. Desirably, the water-miscible
solvent used in the present invention comprises isopropanol.
[0060] II. Water-Soluble Chitosan Having Low Endotoxin
Concentration
[0061] The present invention is further directed to water-soluble
chitosan having a low concentration of endotoxin, and products made
therefrom. The above-described method for making water-soluble
chitosan produces water-soluble chitosan having an extremely low
concentration of endotoxin. The water-soluble chitosan may have a
concentration of endotoxin of less than about 100 eu/gram,
desirably, less than about 50 eu/gram, and more desirably, less
than about 20 eu/gram of dry chitosan.
[0062] In one exemplary embodiment of the present invention, the
water-soluble chitosan comprises partially acetylated water-soluble
chitosan having a degree of N-- acetylation of from about 24% to
about 55%, and a degree of O-acetylation of from about 1% to about
60%, wherein the partially acetylated water-soluble chitosan
comprises less than about 100 equivalent units (e.u.) of endotoxin
per gram of dry water-soluble chitosan. Desirably, the partially
acetylated water-soluble chitosan comprises less than about 50
equivalent units (e.u.) of endotoxin per gram of dry water-soluble
chitosan, more desirably, less than about 20 equivalent units
(e.u.) of endotoxin per gram of dry water-soluble chitosan.
[0063] III. Products Containing Water-Soluble Chitosan Having Low
Endotoxin Concentration
[0064] The present invention is further directed to products
containing water-soluble chitosan having a low concentration of
endotoxin. The water-soluble chitosan produced by the
above-described method may be combined with one or more components
to form a variety of products. Suitable products include, but are
not limited to, solutions and compositions disclosed in U.S. patent
application Ser. No. 10/045,959 filed on Oct. 19, 2001 and entitled
"WATER-SOLUBLE, RANDOMLY SUBSTITUTED PARTIAL N-PARTIAL O-ACETYLATED
CHITOSAN, PRESERVING COMPOSITIONS CONTAINING CHITOSAN, AND
PROCESSES FOR MAKING THEREOF," the subject matter of which is
incorporated herein in its entirety.
[0065] In one desired embodiment of the present invention, the
product comprises a pharmaceutically acceptable solution, such as a
preserving solution for contact lens, wherein the solution contains
(i) water-soluble chitosan having a low concentration of endotoxin
produced by the above-described method, and (ii) at least one
buffer material. The preserving composition can be used in various
ophthalmic products such as contact lens rinsing, lubricating,
cleaning and storage solutions, artificial tear solutions and
ophthalmic drugs. The preserving composition may also be used to
preserve otic and nasal solutions.
[0066] Contact lens solutions in particular present a special
preservative challenge because lens wearers are usually exposed to
the preserving agents for many years on a daily basis. The
possibility that the lens wearer can experience discomfort or
develop sensitivity to the preservative is even higher than would
be the case in short-term exposure. Typical contact lens solution
preserving agents used in the prior art are sorbic acid,
thimerosal, or DYMED.TM. (polyaminopropyl biguanide).
[0067] The compositions of the present invention comprise at least
one chitosan or chitosan derivative having a low concentration of
endotoxin, and at least one buffer solution. The compositions of
the present invention may further comprise at least one biocidal
adjuvant. Compositions of the present invention contain these
components in amounts to be effective as pharmaceutical preserving
compositions useful for preserving pharmaceutical products,
including ophthalmic, nasal and otic preparations.
[0068] In one exemplary embodiment of the present invention, the
composition is used as a contact lens solution preservative. In
another exemplary embodiment, the composition is used as a contact
lens disinfection regiment. The compositions of the present
invention may be incorporated into existing contact lens solutions.
When the composition of the present invention is used in a contact
lens disinfection regimen, the contact lens is rinsed and rubbed
with the composition, and the contact lens then soaks in the
composition for a suitable period of time, such as not less than 15
minutes, more desirably for not less than 1 hour, even more
desirably not less than four hours. Desirably, the soaking occurs
at room temperature; however, any suitable temperature may be
employed.
[0069] The compositions of the present invention containing
water-soluble chitosan and chitosan derivatives having a low
concentration of endotoxin have the additional advantage of being
capable of performing several functions normally requiring other
ingredients. For instance, in an exemplary embodiment, the chitosan
or chitosan derivative may, in addition to its preserving role, act
as a natural surfactant, and aid in lens cleaning by emulsifying
lens proteins and lipids away from the lens surface into solution.
Furthermore, chitosan, as a polymeric saccharide, may be used in an
exemplary embodiment as a solution-thickening agent and lens
lubricant thereby enhancing lens comfort by reducing lens drying
rate. As such, the compositions of the present invention containing
water-soluble chitosan and chitosan derivatives having a low
concentration of endotoxin have a demulcent effect so as to enhance
lens wearer comfort.
[0070] In a further embodiment of the present invention,
compositions containing water-soluble chitosan and chitosan
derivatives having a low concentration of endotoxin may be combined
with certain buffer solutions, such as borate or phosphate buffers,
to exhibit exceptional antimicrobial activity. Thus, in one
embodiment of the present invention, the buffer solution may
comprise a borate buffer. Suitable borate buffers include, but are
not limited to, boric acid, sodium borate, potassium tetraborate,
potassium metaborate, and mixtures of the same. In yet another
embodiment, the buffer solution may comprise a phosphate buffer.
Suitable phosphate buffers include, but are not limited to, sodium
dihydrogen phosphate, disodium hydrogen phosphate, and mixtures of
the same.
[0071] The compositions of the present invention may also include a
biocidal adjuvant. The biocidal adjuvant may be used against, for
example, bacteria, fungi and viruses. One advantage of the
compositions of the present invention is the surprising synergistic
preservative effect. Suitable biocidal adjuvants include, but are
not limited to, disodium ethylenediaminetetracetic acid (EDTA),
nitrilotriacetic acid, and
ethyleneglyco-bis(.beta.-amino-ethylether)-N,N-tetraacetic
acid.
[0072] The compositions of the present invention may contain other
ingredients to perform a desired function. One possible additional
component may be used to allow the composition to have an osmotic
pressure near that of normal lachrymal fluids. Such a function may
be achieved, for instance, by a tonicity agent, such as sodium
chloride, potassium chloride or glycerol.
[0073] In one desired contact lens solution of the present
invention, the water-soluble chitosan having a low concentration of
endotoxin acts to stabilize proteins against denaturing when
compared to commercial multi-purpose contact lens solutions. This
effect may be accomplished by adding at least one surfactant to the
composition. The surfactant may also aid in the cleaning of the
lens. Typical surfactants include, but are not limited to,
surfactants, which are block copolymers of ethylene oxide and
propylene oxide and commercially available under the trade
designation PLURONIC.RTM. from BASF Corporation (Mount Olive,
N.J.); surfactants, which are block copolymers resulting from
addition or ethylene oxide and propylene oxide to ethylene diamine
and commercially available under the trade designation
TETRONIC.RTM. from BASF Corporation (Mount Olive, N.J.);
polyoxyethylene sorbitan fatty acid esters commercially available
under the trade designation TWEEN.RTM. from Uniqema Corporation
(New Castle, Del.).
[0074] The contact lens solutions of the present invention may also
contain viscosity agents to provide lubrication to the eye.
Suitable viscosity agents include, but are not limited to,
polymeric saccharides such as dextran; cellulose derivatives such
as carboxymethyl cellulose and hydroxypropyl methylcellulose;
polyvinyl alcohol; polyvinylpyrrolidinone; polyethylene glycol;
glycerin; and combinations thereof.
[0075] The compositions of the present invention have at least a
minimal preserving activity. In one embodiment, the biocidal
activity of the compositions of the present invention is sufficient
to meet the performance criteria of the Preservative Efficacy Test
("PET") of the USP (United States Pharmacopoeia) as modified by the
FDA. As such, the compositions of the present invention reduce 0
day challenge inocula and 14 day re-challenge inocula of the
bacteria Staphylococcus aureus (ATCC No. 6538), Pseudomonas
aeruginosa (ATCC No. 9027) and Escherichia coli (ATCC No. 8739) by
at least 99.99% (3 logs) within 14 days after the challenge and
re-challenge dates, each. In the fungal challenge portion of the
PET, the compositions of the present invention do not allow any
growth of Aspergillus niger (ATCC No. 16404) and Candida albicans
(ATCC No. 10231) within 14 days following a 0 day challenge and a
14 day re-challenge.
[0076] In one exemplary embodiment, the compositions of the present
invention have a near neutral pH. A neutral pH is desired for
compatibility with an organism, such as the human eye. Desirably,
the compositions of the present invention have a pH of from about 6
to about 8, more desirably about 6.6 to about 7.8, and even more
desirably about 6.8 to about 7.2. Insofar as the antimicrobial
activity alone of the compositions of the present invention is
concerned, the lowest pH in the above range is desired.
[0077] The present invention is further illustrated by the
following examples, which are not to be construed in any way as
imposing limitations upon the scope thereof. On the contrary, it is
to be clearly understood that resort may be had to various other
embodiments, modifications, and equivalents thereof which, after
reading the description herein, may suggest themselves to those
skilled in the art without departing from the spirit of the present
invention and/or the scope of the appended claims.
EXAMPLE 1
Determining the Concentration of Endotoxin in Water-Soluble
Chitosan Solutions
[0078] The presence of endotoxins was determined in water-soluble
chitosan using a Limulus Amebocyte Lysate (LAL) gel-clot assay
commercially available under the trade designation ENDOSAFE.RTM.
KTA (Kinetic Turbidimetric Assay) from Charles River Laboratories,
Inc. (Charleston, S.C.). Sample solutions of water-soluble chitosan
were prepared by dissolving a known amount of one selected batch of
water-soluble chitosan sample into endotoxin-free water. The
samples were agitated until completely dissolved. The pH of the
solutions was about 5.
[0079] After dissolution of the water-soluble chitosan, the pH of
each solution was adjusted using 1M NaOH. Each solution was then
stirred for 30 to 60 minutes. The samples were then diluted
serially and assayed for the presence of endotoxins. Table 1 below
provides results. TABLE-US-00001 TABLE 1 Sample Endotoxin
Concentrations Average Amount of Endotoxin Detected Sample No.
Sample pH (range of endotoxin) 1 5.3 45 eu/gram (from 30-60
eu/gram) 2 6.8 45 eu/gram (from 30-60 eu/gram) 3 7.0 45 eu/gram
(from 30-60 eu/gram) 4 7.2 45 eu/gram (from 30-60 eu/gram) 5 7.7 45
eu/gram (from 30-60 eu/gram)
[0080] As shown in Table 1, adjustment of pH did not have an effect
on the concentration of endotoxin detected in water-soluble
chitosan solutions using the above procedure.
EXAMPLE 2
Determining the Concentration of Endotoxin in Water-Soluble
Chitosan Solutions Containing a Surfactant
[0081] The effect of the addition of a surfactant to the
water-soluble chitosan solutions following pH adjustment was
determined. Water-soluble chitosan solutions were prepared from the
same batch of water-soluble chitosan sample as used in Example 1
and the pH adjusted as above in Example 1. Following pH adjustment,
a surfactant, TWEEN.TM. 20 (polyoxyethylene sorbitan monolaurate)
commercially available from Uniqema Corporation (New Castle, Del.),
was added at a known concentration. The concentration of TWEEN.TM.
20 in the final samples was 0.008 wt %, based on a total weight of
the sample. Each surfactant-containing solution was stirred for 30
to 60 minutes. Each solution was then diluted serially and assayed
for the presence of endotoxins. Table 1 below provides results.
TABLE-US-00002 TABLE 2 Sample Endotoxin Concentrations Average
Amount of Endotoxin Detected Sample No. Sample pH (range of
endotoxin) 1 5.3 90 eu/gram (from 60-120 eu/gram) 2 6.8 180 eu/gram
(from 120-240 eu/gram) 3 7.0 180 eu/gram (from 120-240 eu/gram) 4
7.2 180 eu/gram (from 120-240 eu/gram) 5 7.7 180 eu/gram (from
120-240 eu/gram)
[0082] As shown in Table 2, adjustment of pH along with the
presence of a surfactant did have an effect on the concentration of
endotoxin detected in water-soluble chitosan solutions using the
above procedure. Endotoxin assay was enhanced by the adjustment of
pH along with the addition of surfactant to the chitosan
solution.
EXAMPLE 3
Preparation of Water-Soluble Chitosan Having Low Endotoxin
Concentration
[0083] All process equipment was treated with a 1M NaOH solution
and washed thoroughly with endotoxin-free water prior to use.
[0084] Basic Treatment Step A
[0085] 30 g of water-insoluble chitosan having a deacetylation
degree of about 84% was slurried in 1380 g of 1M NaOH solution for
sixty hours. The solid was collected and washed with endotoxin-free
water until the filtrate had a pH of 11 or lower.
[0086] Acetylating Step B
[0087] 30 g of the above-treated water-insoluble chitosan was
combined with 1000 ml of a 4 wt % acetic acid solution, 0.3 g of
tetrabutylammonium bromide, and 7.5 g of acetic anhydride to
partially acetylate the chitosan. 29.8 g of water-insoluble
chitosan having a degree of deacetylation (DD) of about 71% was
produced. The partially acetylated water-soluble chitosan was
tested for endotoxin levels using the Limulus Amebocyte Lysate
(LAL) gel-clot assay described in Example 1. The results are
provided in Table 3 below as Example 3B.
[0088] pH/Surfactant Treatment Step C
[0089] An aqueous solution was prepared by dissolving 2.0 g of the
above partially acetylated water-soluble chitosan (i.e., product
from step 3B) in 70 g of endotoxin-free water and 1.0 g of 0.8 wt %
TWEEN.TM. 20 aqueous solution. The pH was adjusted to 7.04 by
adding 0.025 M NaOH solution. The solution was then diluted to a
total weight of 100 g with endotoxin-free water. 25 g of
isopropanol was slowly added to the solution. The pH of the
solution was further adjusting to a pH of at least about 8.0 to
cause precipitation of partially acetylated water-soluble chitosan
from the solution by the addition of 0.025 M NaOH solution.
[0090] The precipitate was collected and washed twice with 50 g of
isopropanol. The product was dried in a vacuum oven at 60.degree.
C. over night to obtain 1.5 g of solid material. The sample was
tested for endotoxin levels as described above. The result are
provided below in Table 3 as Example 3C.
COMPARATIVE EXAMPLE 3
Preparation of Water-Soluble Chitosan
[0091] The procedure as described in Example 3 was followed except
Basic Treatment Step A was omitted. The resulting chitosan sample
had a very high level of endotoxin as shown in Table 3 below
compared to product of Example 3B, which was exposed to Basic
Treatment Step A.
EXAMPLE 4
Preparation of Water-Soluble Chitosan Having Low Endotoxin
Concentration
[0092] The procedure as described in Example 3 was followed except
the final pH of the solution was raised to 9.03 in pH/Surfactant
Treatment Step C. The resulting chitosan had a low level of
endotoxin as shown in Table 3 below.
EXAMPLE 5
Preparation of Water-Soluble Chitosan Having Low Endotoxin
Concentration
[0093] The procedure as described in Example 3 was followed except
the final pH of the solution was raised to 9.61 in pH/Surfactant
Treatment Step C. The resulting chitosan had a low level of
endotoxin as shown in Table 3 below.
EXAMPLE 6
Preparation of Water-Soluble Chitosan Having Low Endotoxin
Concentration
[0094] The procedure as described in Example 3 was followed except
the final pH of the solution was raised to 10.2 in pH/Surfactant
Treatment Step C. The resulting chitosan had a low level of
endotoxin as shown in Table 3 below.
EXAMPLE 7
Preparation of Water-Soluble Chitosan Having Low Endotoxin
Concentration
[0095] The procedure as described in Example 3 was followed except
the pH/Surfactant Treatment Step C was repeated a second time. The
resulting chitosan had a low level of endotoxin as shown in Table 3
below. TABLE-US-00003 TABLE 3 Endotoxin Levels In Water-Soluble
Chitosan Example No. Endotoxin levels (eu/g) 3B 48-96 3C 12-24
Comparative - 3 6000-12000 4 12-24 5 12-24 6 24-48 7 6-12
EXAMPLE 8
Time Study for the Preparation of Water-Soluble Chitosan Having Low
Endotoxin Concentration
[0096] The procedure as described in Example 3 was followed except
Basic Treatment Step A was carried out for two hours. The resulting
chitosan sample had a low level of endotoxin as shown in Table 4
below.
EXAMPLE 9
Time Study for the Preparation of Water-Soluble Chitosan Having Low
Endotoxin Concentration
[0097] The procedure as described in Example 3 was followed except
Basic Treatment Step A was carried out for six hours. The resulting
chitosan sample had a low level of endotoxin as shown in Table 4
below. TABLE-US-00004 TABLE 4 Endotoxin Levels in Water-Soluble
Chitosan Example No. Endotoxin levels (eu/g) 8 48-96 9 12-24
[0098] While the specification has been described in detail with
respect to specific embodiments thereof, it will be appreciated
that those skilled in the art, upon attaining an understanding of
the foregoing, may readily conceive of alterations to, variations
of, and equivalents to these embodiments. Accordingly, the scope of
the present invention should be assessed as that of the appended
claims and any equivalents thereto.
* * * * *