U.S. patent application number 16/028160 was filed with the patent office on 2019-01-03 for chitosan-derived compositions.
The applicant listed for this patent is Immunophotonics, Inc.. Invention is credited to Luciano Alleruzzo, Raoul Carubelli, Wei R. Chen, Tomas Hode, Peter Jenkins, Robert E. Nordquist, Joseph Raker, Kristopher Waynant.
Application Number | 20190002594 16/028160 |
Document ID | / |
Family ID | 64734301 |
Filed Date | 2019-01-03 |
United States Patent
Application |
20190002594 |
Kind Code |
A1 |
Hode; Tomas ; et
al. |
January 3, 2019 |
Chitosan-Derived Compositions
Abstract
The present invention relates generally to therapeutic
compositions comprising chitosan-derived compositions used in
connection with methods for treating neoplasms, such as for
instance, malignant lung, thyroid and kidney neoplasms, and other
types of malignant neoplasms, and other medical disorders.
Inventors: |
Hode; Tomas; (St. Louis,
MO) ; Nordquist; Robert E.; (Oklahoma City, OK)
; Chen; Wei R.; (Edmond, OK) ; Carubelli;
Raoul; (Oklahoma City, OK) ; Alleruzzo; Luciano;
(Ballwin, MO) ; Jenkins; Peter; (Lawndale, NC)
; Waynant; Kristopher; (Olympia, WA) ; Raker;
Joseph; (Delmar, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Immunophotonics, Inc. |
|
|
|
|
|
Family ID: |
64734301 |
Appl. No.: |
16/028160 |
Filed: |
July 5, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14372586 |
Jul 16, 2014 |
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16028160 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/86 20180801;
A61K 47/6807 20170801; A61K 2039/884 20180801; A61K 41/0071
20130101; A61K 39/39558 20130101; A61K 2039/55583 20130101; A61N
5/0618 20130101; A61N 2005/067 20130101; A61B 18/20 20130101; A61K
39/39 20130101; A61N 5/062 20130101; A61N 5/0624 20130101; A61P
35/00 20180101; A61N 2005/0659 20130101; A61K 31/722 20130101; A61K
2039/585 20130101; A61K 2039/812 20180801; C08B 37/003 20130101;
A61K 41/0057 20130101 |
International
Class: |
C08B 37/08 20060101
C08B037/08; A61K 47/68 20060101 A61K047/68; A61K 39/395 20060101
A61K039/395; A61K 39/39 20060101 A61K039/39 |
Claims
1. A method of treating cancer in a subject, the method comprising:
administering to the subject in need thereof, a viscoelastic
glycated chitosan formulation of filter sterilized glycated
chitosan polymer and a substantially aqueous solution, the glycated
chitosan polymer having a molecular weight between about 50,000
Daltons to about 1,500,000 Daltons, the glycated chitosan polymer
having glycated amino groups present from about one tenth of one
percent to about thirty percent of available amino groups, the
degree of deacetylation of a chitosan parent of the viscoelastic
glycated chitosan is about 80%, wherein the substantially aqueous
solution having a pH from between about 5 to about 7, wherein about
one percent by weight of the glycated chitosan polymer is dissolved
in the substantially aqueous solution, and wherein the aqueous
solution has a viscosity from about one centistokes to about one
hundred centistokes measured at about 25 degrees Celsius.
2. The method, according to claim 1, in which the formulation is
administered by injection to the subject.
3. The method, according to claim 1, in which the glycated chitosan
polymer has a molecular weight less than 500,000 Daltons.
4. The method, according to claim 1, further includes treating the
cancer using a combination of the formulation and radiation
treatment.
5. The method, according to claim 1, further includes treating the
cancer with a combination of the formulation and an anti-neoplastic
drug.
6. The method, according to claim 1, in which the anti-neoplastic
drug is selected from the group consisting of: alkylating agents;
antimetabolites; plant alkaloids; antibiotics; nitrosoureas;
inorganic ions; enzymes; and hormones.
7. The method, according to claim 1, in which the subject is a
human subject.
8. A method of photophysically destroying a neoplasm and
concurrently generating an in situ autologous vaccine in a
tumor-bearing host, the method comprising: (a) irradiating the
neoplasm with a laser of a wavelength in the visible, near-infrared
or infrared range, at a power and for a duration sufficient to
produce a photothermal reaction inducing neoplastic cellular
destruction and generating fragmented neoplastic tissue and
cellular molecules; (b) introducing an immunoadjuvant into the
neoplasm by intratumor injection, wherein the immunoadjuvant
comprises a viscoelastic glycated chitosan formulation of filter
sterilized glycated chitosan polymer and a substantially aqueous
solution, the glycated chitosan polymer having a molecular weight
between about 50,000 Daltons to about 1,500,000 Daltons, the
glycated chitosan polymer having glycated amino groups present from
about one tenth of one percent to about thirty percent of available
amino groups, the degree of deacetylation of a chitosan parent of
the viscoelastic glycated chitosan is about 80%, wherein the
substantially aqueous solution having a pH from between about 5 to
about 7, wherein about one percent by weight of the glycated
chitosan polymer is dissolved in the substantially aqueous
solution, and wherein the aqueous solution has a viscosity from
about one centistokes to about one hundred centistokes measured at
about 25 degrees Celsius, and further wherein the in situ vaccine
comprises an amalgam of the fragmented neoplastic tissue and
cellular molecules and the immunoadjuvant; and (c) stimulating the
self-immunological defense system against neoplastic cellular
multiplication by having the vaccine presented locally to induce an
anti-tumor response systemically within the host.
9. The method, according to claim 8, in which the glycated chitosan
polymer has a molecular weight less than 500,000 Daltons.
10. The method, according to claim 8, in which the photothermal
reaction causes combined acute and chronic tumor destruction.
11. A method of photophysically destroying a neoplasm and
concurrently generating an in situ autologous vaccine in a
tumor-bearing host, the method comprising: (a) irradiating the
neoplasm with a laser of a wavelength in the visible, near-infrared
or infrared range, at a power and for a duration sufficient to
produce a photothermal reaction inducing neoplastic cellular
destruction and generating fragmented neoplastic tissue and
cellular molecules; (b) forming the in situ vaccine by introducing
an immunoadjuvant into the neoplasm by intratumor injection wherein
the in situ vaccine comprises an amalgam of the fragmented tissue
and cellular molecules and the immunoadjuvant, the immunoadjuvant
being a viscoelastic glycated chitosan formulation of filter
sterilized glycated chitosan polymer and a substantially aqueous
solution, the glycated chitosan polymer having a molecular weight
between about 50,000 Daltons to about 1,500,000 Daltons, the
glycated chitosan polymer having glycated amino groups present from
about one tenth of one percent to about thirty percent of available
amino groups, the degree of deacetylation of a chitosan parent of
the viscoelastic glycated chitosan is about 80%, wherein the
substantially aqueous solution having a pH from between about 5 to
about 7, wherein about one percent by weight of the glycated
chitosan polymer is dissolved in the substantially aqueous
solution, and wherein the aqueous solution has a viscosity from
about one centistokes to about one hundred centistokes measured at
about 25 degrees Celsius; and (c) stimulating the
self-immunological defense system against neoplastic cellular
multiplication by having the vaccine presented locally to induce an
anti-tumor response systemically within the host.
12. The method, according to claim 1, in which the glycated
chitosan polymer has a molecular weight less than 500,000
Daltons.
13. The method, according to claim 10, in which the photothermal
reaction causes combined acute and chronic tumor destruction.
14. A method for treating a neoplasm in a human or other animal
host, the method comprising: (a) introducing a chromophore and an
immunoadjuvant into the neoplasm to obtain a conditioned neoplasm,
the immunoadjuvant being a viscoelastic glycated chitosan
formulation of filter sterilized glycated chitosan polymer and a
substantially aqueous solution, the glycated chitosan polymer
having a molecular weight between about 50,000 Daltons to about
1,500,000 Daltons, the glycated chitosan polymer having glycated
amino groups present from about one tenth of one percent to about
thirty percent of available amino groups, the degree of
deacetylation of a chitosan parent of the viscoelastic glycated
chitosan is about 80%, wherein the substantially aqueous solution
having a pH from between about 5 to about 7, wherein about one
percent by weight of the glycated chitosan polymer is dissolved in
the substantially aqueous solution, and wherein the aqueous
solution has a viscosity from about one centistokes to about one
hundred centistokes measured at about 25 degrees Celsius; and (c)
irradiating the conditioned neoplasm whereby neoplastic cellular
destruction of the conditioned neoplasm is induced producing
fragmented neoplastic tissue and cellular molecules in the presence
of the immunoadjuvant which stimulates the self-immunological
defense system of the host against neoplastic cellular
multiplication, thereby treating the neoplasm.
15. The method, according to claim 14, further include conjugating
the immunoadjuvant to a tumor specific antibody, thereby forming a
conjugate, and administering the conjugate to the host.
16. The method, according to claim 14, further including
conjugating the immunoadjuvant to a tumor specific antigen, thereby
forming a conjugate, and administering the conjugate to the
host.
17. The method, according to claim 14, in which the chromophore is
selected from the group consisting of: indocyanine green; DHE;
m-THPP; AlPcS.sub.4; ZnET2; and Bchla.
18. The method, according to claim 12, further including
conjugating a combination of the chromophore and the immunoadjuvant
to a tumor specific antibody, thereby forming a conjugate, and
administering the conjugate to the host.
19. The method, according to claim 14, further including
conjugating the chromophore and the immunoadjuvant to a tumor
specific antigen, thereby forming a conjugate, and administering
the conjugate to the host.
20. The method, according to claim 14, in which the glycated
chitosan polymer has a molecular weight less than 500,000 Daltons
Description
PRIORITY CLAIM
[0001] This U.S. Nonprovisional patent application herein claims
priority to U.S. Nonprovisional patent application Ser. No.
14/372,586, entitled "Chitosan-Derived Compositions" filed on Jul.
16, 2014.
FIELD OF THE INVENTION
[0002] The present invention relates generally to therapeutic
compositions comprising chitosan-derived compositions used in
connection with methods for treating neoplasms, such as, malignant
lung, breast, prostate, skin, thyroid and kidney neoplasms, and
other types of malignant neoplasms, and other medical
disorders.
BACKGROUND OF THE INVENTION
[0003] Chitosan is a derivative of chitin, a compound usually
isolated from the shells of some crustaceans such as crab, lobster
and shrimp. Chitin is a linear homopolymer composed of
N-acetylglucosamine units joined by .beta. 1.fwdarw.4 glycosidic
bonds. Chitin, chitosan (partially deacetylated chitin) and their
derivatives are endowed with interesting chemical and biological
properties that have led to a varied and expanding number of
industrial and medical applications. Glycated chitosan, described
in U.S. Pat. No. 5,747,475 ("Chitosan-Derived Biomaterials"), which
is herein incorporated by reference, is one such chitosan
derivative.
[0004] Cancer can develop in any tissue of any organ at any age.
Once an unequivocal diagnosis of cancer is made, treatment
decisions become paramount. Though no single treatment approach is
applicable to all cancers, successful therapies must be focused on
both the primary tumor and its metastases. Historically, local and
regional therapy, such as surgery or radiation, have been used in
cancer treatment, along with systemic therapy, e.g., chemotherapy
drugs. Despite some success, conventional treatments are not
effective to the degree desired, and the search has continued for
more efficacious therapies. There is clearly a significant unmet
need for more efficient cancer therapies.
[0005] Conventional glycated chitosan preparations, as described in
U.S. Pat. No. 5,747,475 ("Chitosan-Derived Biomaterials"), have
shown significant efficacy as an immunoadjuvant in the treatment of
metastatic tumor models in animals.
[0006] However, conventional glycated chitosan preparations, when
dispersed, suspended or dissolved in aqueous solutions are often
very difficult to inject or dispense in the biomedical applications
to which they are put. Moreover, conventional glycated chitosan
preparations, as described in U.S. Pat. No. 5,747,475
("Chitosan-Derived Biomaterials"), are nearly impossible to sterile
filter, rendering them unsuitable for industrial manufacturing
according to Current Good Manufacturing Practices (cGMP), and
therefore unsuitable for human use. It is thus an object of the
present invention to provide improved viscoelastic glycated
chitosan preparations which are far less subject to the above-noted
disadvantages.
SUMMARY OF THE INVENTION
[0007] According to one embodiment, the present invention relates
generally to therapeutic formulations comprising chitosan-derived
compositions used in connection with methods for treating neoplasms
and other medical disorders. Additional aspects and/or advantages
of the invention will be set forth in part in the description which
follows and, in part, may be learned by practice of the
invention.
BRIEF DESCRIPTION OF THE FIGURES
[0008] These and/or other aspects and advantages of the invention
will become apparent and more readily appreciated from the
following description of the embodiments, taken in conjunction with
the accompanying drawings of which:
[0009] FIG. 1 depicts one conventional example of glycated
chitosan, e.g., galactochitosan.
[0010] FIG. 2 depicts one exemplary structure of viscoelastic
glycated chitosan of the present invention, where the deacetylation
of the parent chitosan is 80%, and the glycation of total available
deacetylated amino groups is 12.5%.
[0011] FIG. 3 depicts a graph that shows viscosity (in Cp: y-axis)
vs. molecular weight (in kDa; x-axis) in samples of GC with
molecular weights ranging from 100 kDa to 1,500 kDa).
[0012] FIG. 4 depicts a graph that shows the percent of GC in
solution of the samples used in the viscosity experiment.
[0013] FIG. 5 depicts a graph that shows the survival rates
following interstitial laser-assisted immunotherapy with 0.2 ml of
GC.
[0014] FIG. 6 depicts a graph that shows the effect of
tumour-localized glycated chitosan treatment on the response to
mTHPC-based PDT in mouse Line 1 tumors. In this graph: GC=Glycated
chitosan; mTHPC=Meso-substituted tetra (meta-hydroxy-phenyl)
chlorin; and PDT=Photodynamic therapy.
[0015] FIGS. 7A-7C depict graphs that show rat survival rates
following treatment with one, two, or three components of the
laser-assisted immunotherapy system. In these graphs, GC=1.0%
glycated chitosan; and ICG=0.25% indocyanine green.
[0016] FIG. 8 depicts a graph that shows rat survival curves in the
adoptive immunity transfer experiments using rat splenocytes as
immune cells. In this graph, Group A=Results from tumor control
rats; group B=Results from rats injected with tumor cells admixed
with spleen cells from an untreated tumor-bearing rat; Group
C=Results from rats injected with tumor cells admixed with spleen
cells from laser-assisted immunotherapy successfully treated rat;
Group D=Results using spleen cells from a naive rate. Note: Data
collected from 2 separate experiments were combined and plotted
together.
[0017] Still other objects and advantages of preferred embodiments
of the present invention will become readily apparent to those
skilled in this art from the following detailed description,
wherein there is described certain preferred embodiments of the
invention, and examples for illustrative purposes.
DETAILED DESCRIPTION
[0018] The invention relates generally to therapeutic formulations
comprising chitosan-derived compositions used in connection with
methods for treating neoplasms and other medical disorders. It is
to be understood that all references cited herein are incorporated
by reference in their entirety.
[0019] Reference will now be made in detail to certain embodiments
of the present invention, examples of which are illustrated in the
accompanying drawings, wherein like reference numerals refer to the
like elements throughout. It is to be understood that the invention
is capable of modifications in various obvious respects, all
without departing from the spirit and scope of the invention.
Accordingly, the description should be regarded as illustrative in
nature, and not as restrictive.
[0020] Glycated Chitosan
[0021] Glycated chitosan is a product of the glycation (i.e.,
non-enzymatic glycosylation) of free amino groups of chitosan,
followed by stabilization by reduction. Glycation endows the
chitosan with advantageous solubility and viscosity characteristics
which facilitate the use of the derivative in conjunction with
laser-assisted immunotherapy and other applications of the
derivative. The glycation of chitosan also renders the chitosan
more hydrophilic whereby more water is absorbed and retained by the
polymer than would otherwise be the case.
[0022] In accordance with preferred embodiments of the present
invention, a chitosan-derived biomaterial comprises a linear
homopolymer of deacetylated chitin (chitosan), wherein the
deacetylated chitin has a number of otherwise free amino groups
bonded to a carbonyl group of a reducing monosaccharide or
oligosaccharide to form glycated chitosan. Glycated chitosan can
thus be obtained as the reaction product between the carbonyl group
of a reducing monosaccharide or oligosaccharide and the free amino
groups of deacetylated chitin. Thus, the term "glycated chitosan"
as used herein is intended to refer to a product of the glycation,
i.e., non-enzymatic glycosylation, of free amino groups of
chitosan, followed by stabilization by reduction. Generally
speaking, glycation (or non-enzymatic glycosylation) is intended to
refer to a process that occurs when a sugar molecule, such as
fructose or glucose, binds to a substrate, such as a protein or
lipid molecule, without the contributing action of an enzyme. One
such example is the non-enzymatic reaction of a sugar and an amine
group of a protein to form a glycoprotein.
[0023] Glycated chitosan, thus generally includes the products
resulting from the reaction between the free amino groups of
chitosan and the carbonyl groups of reducing monosaccharides and/or
oligosaccharides. The products of this reaction, which mainly are a
mixture of Schiff bases (i.e. the carbon atom from the carbonyl
group is now doubly bonded to the nitrogen from the free amine
releasing one molecule of water) and Amadori products (i.e. the
carbon atom of said carbonyl group is singly bonded to the nitrogen
atom of said amino group while an adjacent carbon atom is double
bonded to an oxygen atom) may be used as such or after
stabilization by reduction with hydrides, such as boron-hydride
reducing agents, for example NaBH4, NaBH3CN, NaBH(OAc).sub.3, etc,
or by exposure to hydrogen in the presence of suitable
catalysts.
[0024] The presence of primary and secondary alcohol groups, and of
primary amino groups in chitosan, facilitate a number of approaches
for chemical modifications designed mainly to achieve their
solubilization and to impart special properties for specific
applications.
[0025] Solubilization of chitin and chitosan can be achieved by
partial hydrolysis to oligosaccharides. For chitosan, treatment
with a variety of acids, both organic and inorganic, leads to the
formation of water soluble chitosonium salts by protonation of the
free amino groups. Additional modifications of the amino groups
include the introduction of chemical groups such as carboxymethyl,
glyceryl, N-hydroxybutyl and others. Glycation, i.e., non-enzymatic
glycosylation of the free amino groups of chitosan, followed by
stabilization by reduction, offers a preferred approach for the
preparation of various pharmaceutical formulations utilized in the
present invention. For illustrative purposes, one conventional
example of glycated chitosan, e.g., galactochitosan, is shown in
FIG. 1, which is also described and illustrated in U.S. Pat. No.
5,747,475. FIG. 1 is an exemplary structure of conventional
glycated chitosan, where the molecular weight is approximately
1,500,000 Daltons and all amino groups are glycated.
[0026] U.S. Pat. No. 5,747,475 is very limited in its description
and describes only one specific galactochitosan in terms of
molecular weight; specifically, U.S. Pat. No. 5,747,475 only
describes galactochitosan with a molecular weight of 1500 kDa.
[0027] Unlike the conventional 1500 kDa galactochitosan described
in U.S. Pat. No. 5,747,475, it is to be clearly understood that the
glycated chitosan of the present invention as described herein is
intended to include glycated chitosan having a molecular weight
less than 1500 kDa. Moreover, unlike conventional chitosans, the
glycated chitosans of the present invention is a completely
different and novel composition of matter with a number of
surprisingly unexpected properties, benefits and advantages,
including unexpectedly beneficial viscoelastic properties.
[0028] The glycated chitosan of the present invention is in the
form of a Schiff base, an Amadori product, or preferably, in their
reduced secondary amine or alcohol, respectively. In another
embodiment, the glycated chitosan includes a carbonyl reactive
group. It is preferred that glycated chitosan of the present
invention is obtained by reacting chitosan with a monosaccharide
and/or oligosaccharide, preferably in the presence of an acidifying
agent, for a time sufficient to accomplish Schiff base formation
between the carbonyl group of the sugar and the primary amino
groups of chitosan (also referred to herein as glycation of the
amino group) to a degree whereby about 0.1% to about 30% (and most
preferably above 2%) glycation of the amino groups of the chitosan
polymer is achieved. This is preferably followed by stabilization
by reduction of Schiff bases and of their rearranged derivatives
(Amadori products) to the secondary amines or alcohols.
[0029] The present invention is the first demonstration whereby
about 0.1% to about 30% (and most preferably above 2%) glycation of
the chitosan polymer is achieved. Contrary to the present
invention, others have failed to achieve or recognize this
significant result. Thus according to a preferred embodiment, the
present invention provides a viscoelastic glycated chitosan
formulation, consisting essentially of glycated chitosan polymer,
wherein the glycated chitosan polymer has a molecular weight
between about 50,000 Daltons to about 1,500,000 Daltons, and
further wherein the glycated chitosan polymer possesses from about
one tenth of a percent to about thirty percent glycation of its
otherwise free amino groups.
[0030] The products resulting from the non-enzymatic glycosylation
of free amino groups of chitosan are thus mainly a mixture of
Schiff bases, i.e. the carbon atom of the initial carbonyl group
double bonded to the nitrogen atom of the amino group (also known
as the imine functional group), and Amadori products, i.e. the
carbon atom of the initial carbonyl group bonded to the nitrogen
atom of said amino group by a single bond while an adjacent carbon
atom is double bonded to an oxygen atom forming a ketone group.
These products (resulting from the non-enzymatic glycosylation
process) may be used as such, or after stabilization by reduction
with hydrides, such as boron-hydride reducing agents, for example
NaBH4, NaBH3CN, NaBH(OAc).sub.3, etc, or by exposure to hydrogen in
the presence of suitable catalysts.
[0031] Chitosan deamination with nitrous acid can be used to
generate reducing aldoses and oligosaccharides suitable for the
glycation of chitosan. Deamination of the deacetylated glucosaminyl
residues by nitrous acid results in the selective cleavage of their
glycosidic bonds with the formation of 2,5-anhydro-D-mannose
residues. Depending on the composition of specific areas of the
chitosan chain, the anhydro hexose could be released as the
monosaccharide, or occupy the reducing end of an oligosaccharide.
Release of free N-acetylglucosamine could also occur from some
regions of the chitosan chain. Similar treatment of N-deacetylated
glycoproteins and glycolipids can be utilized to obtain
oligosaccharides of defined chemical composition and biological
activity for special preparations of glycated chitosan.
[0032] Various products obtained by chitosan glycation will be
utilized as such or reacted with other natural or synthetic
materials, e.g., reaction of aldehyde-containing derivatives of
glycated chitosan with substances containing two or more free amino
groups, such as on the side chains of amino acids rich in lysine
residues as in collagen, on hexosamine residues as in chitosan and
deacetylated glycoconjugates, or on natural and synthetic diamines
and polyamines. This is expected to generate crosslinking through
Schiff base formation and subsequent rearrangements, condensation,
dehydration, etc. Stabilization of modified glycated chitosan
materials can be made by chemical reduction or by curing involving
rearrangements, condensation or dehydration, either spontaneous or
by incubation under various conditions of temperature, humidity and
pressure. The chemistry of Amadori rearrangements, Schiff bases and
the Leukart-Wallach reaction is detailed in The Merck Index, Ninth
Edition (1976) pp. ONR-3, ONR-55 and ONR-80, Library of Congress
Card No. 76-27231, the same being incorporated herein by reference.
The chemistry of nucleophilic addition reactions as applicable to
the present invention is detailed in Chapter 19 of Morrison and
Boyd, Organic Chemistry, Second Edition (eighth printing 1970),
Library of Congress Card No. 66-25695, the same being incorporated
herein by reference.
[0033] As further described herein, particular types (e.g.,
particular types of reducing sugars) and degrees of glycation have
surprisingly been found to endow the chitosan with unexpected and
advantageous solubility characteristics which facilitate the use of
the glycated chitosan in conjunction with laser-assisted
immunotherapy and other therapeutic applications. The glycation of
chitosan also advantageously renders the chitosan more hydrophilic
whereby more water is absorbed and retained by the polymer than
would otherwise be the case. The D-galactose derivative of chitosan
is particularly preferred insofar as D-galactose has a relatively
higher naturally occurring incidence of its open chain form. The
glycated chitosan may be prepared in any number of suitable
formulations including, for example, a powder form, as a viscous
formulation, or in any other suitable form.
[0034] In accordance with other preferred embodiments of the
invention, chitosan may be non-enzymatically glycated utilizing any
of a number of the same or different reducing sugars, e.g., the
same or different monosaccharides and/or oligosaccharides. Exemples
of such monosaccharide glycosylation agents are the more naturally
occurring D-trioses, D-tetroses, D-pentoses, D-hexoses, D-heptoses,
and the like, such as D-glucose, D-galactose, D-fructose,
D-mannose, D-allose, D-altrose, D-idose, D-talose, D-fucose,
D-arabinose, D-gulose, D-hammelose, D-lyxose, D-ribose, D-rhamnose,
D-threose, D-xylose, D-psicose, D-sorbose, D-tagatose,
D-glyceraldehyde, dihydroxyacetone, D-erythrose, D-threose,
D-erythrulose, D-mannoheptulose, D-sedoheptulose and the like.
Suitable oligosaccharides include the fructo-oligosaccharides
(FOS), the galacto-oligosaccharides (GOS), the
mannan-oligosaccharides (MOS) and the like.
[0035] Preferred Viscoelastic Properties
[0036] Conventionally produced chitosan products, when dispersed,
suspended or dissolved in aqueous solutions are very difficult to
produce according to GMP standards, and have a number of
disadvantages in terms of administration and other uses.
[0037] Preferred embodiments of the present invention overcome the
long unmet needs for improved therapeutic chitosan products by
providing improved viscoelastic glycated chitosan preparations
which are not subject to the disadvantages of conventional
approaches.
[0038] The term "viscoelastic" as used herein refers to the
viscosity of a particular composition, preparation or formulation.
Viscosity is well understood as a measure of the resistance of a
fluid which is being deformed by either shear stress or tensile
stress. In other words, viscosity describes a fluid's internal
resistance to flow and may be thought of as a measure of fluid
friction.
[0039] a. Unexpected Improvements in Injectability of GC
Preparations
[0040] It has been surprisingly and unexpectedly discovered that
the injectability of formulations of glycated chitosan (GC), for
instance solutions or suspensions, is nonobviously dependent upon
the viscosity and rheological properties of the GC. These
properties are, in turn, highly dependent upon the molecular weight
of the GC, the degree of polymerization of the chitin parent to the
chitosan, the degree of deacetylation of the chitin parent, and the
degree of glycation of the chitosan. These latter properties
determine the degree of entanglement of the polymer chains of the
GC as well as the degree of intramolecular hydrogen bonding
occasioned by the number and nature of the substituents present on
the GC molecule (i.e., acetyl and saccharide), both of which
contribute significantly to the viscosity and other rheological
properties of solutions prepared therefrom.
[0041] It has been surprisingly and unexpectedly discovered that
the improved viscoelastic glycated chitosan preparations of the
present invention possess numerous advantages, for instance, (i)
administration of a non-toxic preparation for treatment of neoplasm
in a patient; (ii) far superior injectability (e.g., through
different gauge needles) in a clinical setting as compared to
conventional treatments; (iii) improved sterile filtration of the
viscoelastic preparations; and (iv) a less painful and thus an
improved treatment option for patients. The term "injectability" as
used herein refers to the ease with which a formulation or
preparation, for instance, a formulation comprising glycated
chitosan (GC), is injected into a subject.
[0042] According to one preferred embodiment, the invention
provides an injectable viscoelastic preparation comprising
approximately 1 percent by weight of the above-described glycated
chitosan dispersed, suspended or dissolved in an aqueous
solution.
[0043] Preferred embodiments of the invention include preparations
of glycated chitosan, including for instance solutions or
suspensions, that have a viscosity that renders the preparations
readily injectable via a needle with a relatively large needle
gauge (G), thus reducing pain and discomfort for the subject.
Preferred examples of relatively large gauge needles include
needles that have the following dimensions: a nominal inner
diameter of from about 0.337 mm (23 G) to about 0.210 mm (27
G).
[0044] According to one example, a viscoelastic glycated chitosan
preparation is administered via injection using an injection needle
having a diameter of about 20 G to about 22 G and an effective
length of a tube of the injection needle is about 1,000 mm or more
such that the inflow rate of the injectable preparation, when
injected at a pressure of about two to about three atmospheres
through said injection needle, ranges from about 0.05 ml/second to
0.1 ml/second. According to another example, a viscoelastic
glycated chitosan preparation can also be administered via
injection using an injection needle having a diameter of about 25G
to about 27G. It is also to be understood that a viscoelastic
glycated chitosan preparation according to the present invention
can also be administered using any other suitable gauge needle or
instrument.
[0045] It has been surprisingly found that the viscoelastic
glycated chitosan preparations of the present invention, for
instance, solutions or suspensions, are injectable at a relatively
wide range of concentrations through catheters or needles of the
most commonly used gauges.
[0046] It has also been discovered that these improved viscoelastic
glycated chitosan preparations (i.e., by improving the viscosity
and rheological properties of the glycated chitosan compositions)
also unexpectedly improve the overall ease of administration of the
preparation to a subject; the efficiency of administration by the
individual administering the formulation (for example, nurse,
physician, or other healthcare practitioner), and the compliance
and efficacy of the glycated chitosan formulations may also be
enhanced.
[0047] b. Unexpected Improvements in Manufacturing and
Filtration
[0048] It has also been surprisingly found that sterile filtration
is unexpectedly improved using the improved viscoelastic glycated
chitosan preparations of the present invention. Conventional
glycated chitosan preparations, as described in U.S. Pat. No.
5,747,475 ("Chitosan-Derived Biomaterials"), was shown to be very
difficult to sterile filter through a 0.22 um sterile filter, which
renders it unsuitable for commercial cGMP manufacturing. In
contrast, the improved viscoelastic glycated chitosan, which was
discovered to have nonobvious rheological properties, was shown to
be highly suitable for sterile filtration, cGMP manufacturing, and
human use.
[0049] Furthermore, it has been surprisingly found that
diafiltration and ultrafiltration is unexpectedly improved using
the improved viscoelastic glycated chitosan preparations of the
present invention. Conventional glycated chitosan preparations were
difficult to diafilter and ultrafilter, causing the filter to clog,
thus rendering it unsuitable for commercial cGMP manufacturing. The
improved viscoelastic glycated chitosan, on the other hand, was
highly suitable for diafiltration and ultrafiltration, thus
significantly improving the manufacturing process.
[0050] Exemplary Methods for Determination of Viscosity
[0051] Any number of suitable techniques in the chemical arts can
be used to reliably and accurately determine viscosity of a
glycated chitosan formulation.
[0052] It is to be understood that viscosity can be reliably
measured with various types of instruments, e.g., viscometers and
rheometers. A rheometer is used for those fluids which cannot be
defined by a single value of viscosity and therefore require more
parameters to be set and measured than is the case for a
viscometer. Close temperature control of the fluid is essential to
accurate measurements, particularly in materials like lubricants,
whose viscosity can double with a change of only 5.degree. C.
[0053] In accordance with the present invention, the viscosity of a
glycated chitosan preparation can be determined according to any
suitable method known in the art.
[0054] For instance, viscosity can be reliably measured in units of
centipoise. The poise is a unit of dynamic viscosity in the
centimeter gram second system of units. A centipoise is one
one-hundredth of a poise, and one millipascal-second (mPas) in SI
units (1 cP=10.sup.-2 P=10.sup.-3 Pa's). Centipoise is properly
abbreviated cP, but the alternative abbreviations cps, cp, and cPs
are also commonly seen. A viscometer can be used to measure
centipoise. When determining centipoise, it is typical that all
other fluids are calibrated to the viscosity of water.
[0055] Exemplary Determination of Viscosity of Glycated Chitosan
Preparations
[0056] There are numerous factors that affect the viscosity of
solutions and, in particular, solutions of polymers, other than
molecular weight. In the case of glycated chitosan (GC) the
injectability of solutions of GC is highly dependent upon the
viscosity and rheological properties of the GC in solution. These
properties are, in turn, highly dependent upon the molecular weight
of the GC, the degree of polymerization of the chitin parent to the
chitosan, the degree of deacetylation of the chitin parent, and the
degree of glycation of the chitosan. These latter properties
determine the degree of entanglement of the polymer chains of the
GC as well as the degree of intramolecular hydrogen bonding
occasioned by the number and nature of the substituents present on
the GC molecule (i.e., acetyl and saccharide), both of which
contribute significantly to the viscosity of solutions prepared
therefrom.
[0057] It has been surprisingly discovered that the improved
viscosity and rheological properties of glycated chitosan
preparations are, in turn, highly dependent upon particular
physiochemical properties of the glycated chitosan. The term
"physiochemical property" as used herein is intended to include,
but is not limited to, any physical, chemical or physical-chemical
property of a molecular structure, such as glycated chitosan. As
described further herein, a few examples of these physiochemical
properties are: [0058] (i) the molecular weight of the glycated
chitosan; [0059] (ii) the degree of polymerization of the chitin
parent to the chitosan; [0060] (iii) the degree of deacetylation of
the chitin parent; and [0061] (iv) the degree of glycation of the
chitosan.
[0062] FIG. 2 shows one example of a viscoelastic glycated chitosan
of the present invention, where the molecular weight is
approximately 250 kDa, the degree of deacetylation of the chitin
parent is about 80%, and the degree of glycation of the free amino
groups on the chitosan is about 12.5%.
[0063] FIG. 2 is an exemplary structure of viscoelastic glycated
chitosan of the present invention, where the deacetylation of the
parent chitosan is about 80%, and the glycation of total available
deacetylated amino groups is about 12.5%.
[0064] (i) Molecular Weight of the Glycated Chitosan
[0065] Any number of suitable techniques in the chemical arts can
be used to reliably and accurately determine the molecular weight
(MW) of the glycated chitosan.
[0066] It is preferred that a viscoelastic glycated chitosan
preparation is prepared as an injectable formulation comprising
glycated chitosan with a molecular weight (MW) less than about 1500
kDa. Examples of preferred viscoelastic glycated chitosan
preparations comprise glycated chitosan with a molecular weight
(MW) of between about 50 Kilodaltons (kDa) and about 1500 kDa.
[0067] In certain embodiments, a viscoelastic glycated chitosan
preparation comprises glycated chitosan with a molecular weight
(MW) of between about 100 kDa and about 1000 kDa; and more
preferably, between about 100 kDa and about 300 kDa. Various
techniques can be used to accurately determine molecular
weight.
[0068] The invention encompasses chitosan-derived compositions
comprising derivatives of chitosan which are water-soluble or
water-dispersible. In accordance with the present invention, it has
also been surprisingly found that in certain embodiments, with
increasing molecular weight (MW), more water is required to
solubilize the glycated chitosan (GC). This in turn means less
amount of the water is "free", i.e. not hydrogen-bonded to the GC
(assuming no additional water is added to the solution), which in
itself contributes to higher viscosity. As shown in example 3
below, this result has been unexpectedly found to add to the
viscosity increase that is given by the increasing size of the
molecule, giving an exponential (or something similar), rather than
a linear relationship between viscosity and MW (when concentration
is compensated for).
[0069] (ii) Degree of Polymerization (DP) of the Chitin Parent to
the Chitosan
[0070] The degree of polymerization (DP) of the chitin parent to
the chitosan can be reliably and accurately determined according to
any number of suitable methods or techniques known in the chemical
arts.
[0071] In one approach, it is preferred that the degree of
polymerization (DP) is determined by dividing the molecular weight
of the chitosan by the molecular weight of the glucosamine
link.
[0072] (iii) Degree of Deacetylation of the Chitin Parent
[0073] Another physiochemical property is the degree of
deacetylation of the chitin parent. Any number of suitable
techniques in the chemical arts can be used to reliably and
accurately determine the degree of deacetylation of the chitin
parent.
[0074] NMR is one technique that can be used to determine the
degree of deacetylation of chitin or chitosan.
[0075] (iv) Degree of Glycation of the Chitosan
[0076] Any number of suitable techniques in the chemical arts can
be used to reliably and accurately determine the degree of
glycation of the chitosan.
[0077] NMR is one technique that can be used to detect and measure
the bonding of monosaccharides and/or oligosaccharides to the
chitosan polymer.
[0078] C/N elemental combustion analysis is another technique that
can be used to determine the percent glycation of the glycated
chitosan by means of comparing the C/N ratio of glycated chitosan
vs. the parent chitosan.
[0079] Enzymatic digestion coupled with HPLC is yet another
technique that can be used to determine percent glycation.
[0080] It is to be understood that other suitable analytical
methods and instrumentation can also be used for simultaneous
detection, measurement and identification of multiple components in
a sample, e.g., for simultaneous detection, measurement and
identification of glycated and non-glycated chitosan in a
sample.
[0081] Colorimetric measurement of chemicals bound to remaining
free amino groups, such as via a ninhydrin reaction, can be used to
assess the degree of glycation.
[0082] It has thus been found that glycated chitosans having
preferred molecular weights, degrees of polymerization of the
chitin parent to the chitosan, degrees of deacetylation of the
chitin parent, and degrees of glycation of the chitosan enable
improved preparation of glycated chitosan solutions which are
injectable at a relatively wide range of concentrations of the
glycated chitosan through catheters or needles of commonly used
gauges.
[0083] Preferred Methods of Preparing Glycated Chitosan
[0084] Still other embodiments of the invention relate to methods
for the preparation of glycated chitosan formulations. Glycated
chitosan is preferably obtained by reacting chitosan with a
monosaccharide and/or oligosaccharide, preferably in the presence
of an acidifying agent, for a time sufficient to accomplish Schiff
base formation between the carbonyl group of the sugar and the
primary amino groups of chitosan (also referred to herein as
glycation of the amino group) to a degree whereby at least some
percentage (for example, two percent or higher) glycation of the
chitosan polymer is achieved. This is preferably followed by
stabilization by reduction of Schiff bases and of their rearranged
derivatives (Amadori products) to their secondary amines or
alcohols, respectively. NMR tracings can be used to verify the
bonding of the monosaccharides and/or oligosaccharides to the
chitosan polymer, whereas chemical measurement of remaining free
amino groups, such as via a ninhydrin reaction, can be used to
assess the degree of glycation.
[0085] In preferred embodiments, conditions can be adjusted as
needed to improve desired results during the manufacture of
glycated chitosan. For instance, it has been unexpectedly
discovered, in accordance with the present invention, that
improvements in the manufacture of glycated chitosan can be
achieved by controlling the pH conditions, as described for
instance in Example 4.
[0086] According to one example of preparation of glycated chitosan
for use in the present invention, approximately three grams of a
reducing monosaccharide (e.g., glucose, galactose, ribose), or an
equivalent amount of a reducing oligosaccharide, is dissolved in
100 ml of distilled water under gentle magnetic stirring in an
Erlenmeyer flask. Then approximately one gram of chitosan is added,
and thereafter suitable process steps can then be performed to
yield the glycated chitosan preparation with desired viscoelastic
properties and with desired purity characteristics.
[0087] One exemplary method for industrial-scale production of
chitosan involves the following four steps: demineralization (DM),
deproteinization (DP), decoloration (DC) and deacetylation (DA).
Chitin extraction from, e.g., crustacean shells is carried out by
an alkali-acid treatment. Samples are deproteinized by treating
with alkaline formulation, demineralized with acid and decolorized
with organic solvent (e.g., acetone), followed by bleaching (with,
e.g., sodium hypochlorite). Chitin deacetylation is carried out
using, e.g., sodium hydroxide formulation. The degree of
polymerization of chitosan is adjusted by depolymerization; the
most convenient procedures being (1) nitrous acid degradation in
deuterated water. The reaction is selective, stoichiometric with
respect to GlcN, rapid, and easily controlled, (2) depolymerization
by acid hydrolysis, or (3) enzymatic degradation with a commercial
preparation (Pectinex Ultra Spl). The enzymatic method yields
shorter fragments with a higher proportion of fully deacetylated
chitooligomers. Conversely, acid hydrolysis of the starting
chitosan results in fragments with degrees of polymerization up to
sixteen and more monoacetylated residues than with the enzymatic
procedure.
[0088] Polymerized Glycated Amino-Sugars
[0089] As described herein, chitosan (partially deacetylated
chitin) is a derivative of chitin (a linear homopolymer composed of
N-acetylglucosamine units joined by .beta. 1.fwdarw.4 glycosidic
bonds). Chitosan-derived compositions thus comprise a homopolymer
of partially deacetylated chitin, wherein the partially
deacetylated chitin has a number of otherwise free amino groups
bonded to a carbonyl group of a reducing monosaccharide or
oligosaccharide creating an imine bond (Schiff Base) or related
product (Amadori Rearrangement) and releasing one molecule of
water.
[0090] Since chitin and chitosan are polymers of glucosamine, the
present invention also contemplates non-enzymatically glycated
glucosamine, e.g., glycated glucosamine monomers, or glycated
glucosamine units. In other words, the present invention also
contemplates non-enzymatic glycation of amino-sugar monomers in
general.
[0091] For instance, one example is a glycated glucosamine wherein
the N-substituent is a galactose. It is preferred that glycation of
glucosamine monomers is performed after at least a percentage of
the glucosamine monomers are initially deacetylated.
[0092] Moreover, the present invention also contemplates (1)
polymers of glycated glucosamine units (polymerized glycated
amino-sugars), (2) combination polymers of glycated and
non-glycated glucosamines, and (3) combinations of glycated and
non-glycated glucosamine polymers wherein: [0093] (i) the
percentage of non-deacetylated glucosamine monomers is from about
1% to about 30%; [0094] (ii) the degree of polymerization (of the
various combinations of deacetylated, non-deacetylated, glycated
and non-glycated glucosamine units) is from about x.sub.n=300 to
about x.sub.n=8000, most preferably about x.sub.n=1500; and/or
[0095] (iii) the percent glycation of the free amino groups of the
deacetylated polymerized glucosamine is from about 0.1% to about
30%.
[0096] The present invention also contemplates uses of polymerized
glycated glucosamine polymers that are the same or similar to uses
of glycated chitosan. These include, for instance, immunoadjuvant
properties and uses, e.g., in the context of in situ cancer
vaccines (inCVAX) such as laser-assisted immunotherapy (LIT).
[0097] Exemplary Formulations and Applications
[0098] Examples of various types of pharmaceutically acceptable
formulations or preparations that can be used in accordance with
the present invention include, for instance, solutions,
suspensions, and other types of liquid or semi-liquid formulations
for injectability of the viscoelastic glycated chitosan
preparations. For instance, the pharmaceutically acceptable
formulations or preparations may include glycated chitosan
dispersed, suspended or dissolved in substantially aqueous
formulations. By use of the term "substantially aqueous" it is to
be understood that the formulations or preparations, in certain
embodiments, may include some percentage of one or more non-aqueous
components, and one or more pharmaceutically acceptable
excipients.
[0099] According to one example, a viscoelastic preparation is
preferably formulated as an aqueous solution possessing a pH from
between about 5.0 to about 7.
[0100] A viscoelastic preparation can also be formulated as an
aqueous solution comprising a buffered physiological saline
solution consisting essentially of glycated chitosan.
[0101] A viscoelastic preparation can also be formulated consisting
essentially of glycated chitosan polymer, wherein the glycated
chitosan polymer possesses from about one tenth (0.1) of a percent
to about thirty (30) percent glycation of its otherwise free amino
groups.
[0102] In another embodiment, a viscoelastic preparation can be
formulated consisting essentially of glycated chitosan (GC)
polymer, wherein the glycated chitosan polymer possesses about two
(2) percent glycation of its otherwise free amino groups.
[0103] In another embodiment, a viscoelastic preparation can be
formulated consisting essentially of glycated chitosan polymer,
wherein the glycated chitosan polymer has a molecular weight
between about 50,000 to about 1,500,000 Daltons.
[0104] Another example includes a viscoelastic GC preparation
comprising about one (1) percent by weight of a glycated chitosan
polymer dispersed in an aqueous solution, said aqueous solution
having a viscosity of between about one (1) to about one hundred
(100) centistokes measured at about 25 degrees Celsius.
[0105] Yet another example includes an aqueous solution having
about one percent by weight of glycated chitosan and from about one
tenth (0.1) of a percent to about thirty (30) percent glycation of
otherwise free amino groups of said glycated chitosan, wherein the
aqueous solution has a viscosity from about one (1) centistokes to
approximately one hundred (100) centistokes.
[0106] In yet another embodiment, a viscoelastic preparation can be
formulated consisting essentially of glycated chitosan polymer,
comprising about or above one percent by weight of the glycated
chitosan polymer dispersed in an aqueous solution, wherein the
glycated chitosan polymer possesses about two (2) percent glycation
of its otherwise free amino groups, and wherein the aqueous
solution has a viscosity suitable for ease of injectability and
administration to a subject.
[0107] In yet another embodiment, a viscoelastic preparation can be
formulated consisting essentially of glycated chitosan polymer,
additionally containing one or more different viscoelastic
materials miscible in an aqueous solution. Examples of suitable
viscoelastic materials include, but are not limited to, hyaluronic
acid, chondroitin sulfate and carboxymethylcellulose.
[0108] The viscoelastic preparation can include glycated chitosan
polymer comprising a monosaccharide bonded to an otherwise free
amino group. The glycated chitosan polymer can take any suitable
form, such as a Schiff base, an Amadori product or mixtures
thereof. The glycated chitosan polymer can also be in the form of a
reduced Schiff base (secondary amine), a reduced Amadori product
(alcohol) or mixtures thereof.
[0109] The viscoelastic preparation can also be formulated wherein
the glycated chitosan polymer possesses a number of chemically
modified monosaccharide or oligosaccharide substituents. In one
embodiment, the monosaccharide comprises galactose.
[0110] The inventive formulations or preparations preferably also
contain glycated chitosan in a physiologically compatible carrier.
"Physiologically compatible" as used herein is to be understood to
refer to materials which, when in contact with tissues in the body,
are not harmful thereto. The term is intended in this context to
include, but is not limited to, aqueous formulations (e.g.,
solutions) which are approximately isotonic with the physiological
environment of interest. Non-isotonic formulations (e.g.,
solutions) sometimes may also be clinically useful such as, for
example dehydrating agents. Additional components of the inventive
solutions may include various salts such as, for instance, NaCl,
KCl, CaCl.sub.2, MgCl.sub.2 and Na based buffers.
[0111] The above and other objects are realized by the present
invention, certain preferred embodiments of which relate to
glycated chitosan preparations having particular physiochemical
properties that confer unexpected and surprisingly beneficial
properties.
[0112] The present invention also encompasses a wide range of uses
of viscoelastic glycated chitosan preparations that have surprising
and unexpected properties as immunoadjuvants, for instance, in
connection with in situ autologous cancer vaccines, such as
laser-assisted immunotherapy for cancer, as described further
herein.
[0113] Preferred embodiments of the invention provide
immunoadjuvants comprising an injectable viscoelastic preparation.
It is thus another object of the present invention to provide
improved viscoelastic glycated chitosan preparations for other
therapeutic applications, including therapeutic use as an
immunoadjuvant and immunomodulator.
[0114] The present invention also encompasses various routes of
administering the viscoelastic glycated chitosan immunoadjuvant
formulations, such as via injection. In a preferred approach, the
immunoadjuvant is preferably prepared as a formulation for
injection into or around the tumor mass. It should be recognized
however that other methods may be sufficient for localizing the
immunoadjuvant in the tumor site. One such alternative delivery
means is conjugation of the immunoadjuvant to a tissue specific
antibody or tissue specific antigen, such that delivery to the
tumor site is enhanced. Any one method, or a combination of varying
methods, of localizing the immunoadjuvant in the tumor site is
acceptable so long as the delivery mechanism insures sufficient
concentration of the immunoadjuvant in the neoplasm.
[0115] According to certain preferred embodiments, the present
invention provides for various pharmaceutical formulations
comprising viscoelastic glycated chitosan used in connection with
in situ autologous cancer vaccines (inCVAX), such as laser-assisted
immunotherapy, photodynamic cancer therapy (PDT) and/or other tumor
immunotherapy methods, as described in further detail herein. It is
been observed that it is desirable to utilize glycated chitosan
preparations having a suitable viscosity that enables their use as
an injectable or other formulation as an immunoadjuvant in
applications such as inCVAX and/or PDT and/or tumor immunotherapy
methods. Such applications typically involve injection of the
viscoelastic glycated chitosan formulation into the corpus of a
patient. The term "immunoadjuvant" as used herein is intended to
refer to any molecule, composition or substance that acts to
enhance the immune system's response to an antigen; for instance,
glycated chitosan which acts to enhance the immune system's
response to a tumor antigen.
[0116] The immunoadjuvant composition can further include a tumor
specific antibody conjugated to the glycated chitosan. The
immunoadjuvant composition can also include a tumor specific
antigen conjugated to the glycated chitosan. The glycated chitosan
can further include a carbonyl reactive group.
[0117] According to one preferred embodiment, the present invention
provides an immunoadjuvant formulation that includes a suspension
or a solution of viscoelastic glycated chitosan. The viscoelastic
glycated chitosan is in this preferred embodiment used in
connection with photothermal treatment of a neoplasm without the
use of a chromophore, where the light energy is delivered directly
to the neoplasm. The light energy can be delivered topically if the
neoplasm is accessible on the tissue surface (for example
melanoma), or is exposed by means of surgery. The light energy can
also be delivered to the neoplasm by means of fiberoptics, for
example if the neoplasm is present below the tissue surface (for
example breast cancer) and is not exposed through surgery.
[0118] According to another embodiment, and as described in further
detail herein, the immunoadjuvant formulations of the present
invention can further include a suitable chromophore. The selection
of an appropriate chromophore is largely a matter of coordination
with an acceptable laser wavelength of radiation. The wavelength of
radiation used must, of course, be complementary to the
photoproperties (i.e., absorption peak) of the chromophore. Other
chromophore selection criteria include ability to create thermal
energy, to evolve singlet oxygen and other active molecules, or to
be toxic in their own right such as cis-platinin. In the present
invention, a preferred wavelength of radiation is 805.+/-0.10 nm.
The desired chromophores have strong absorption in the red and
near-infrared spectral region for which tissue is relatively
transparent. Another advantage of this wavelength is that the
potential mutagenic effects encountered with UV-excited sensitizers
are avoided. Nevertheless, wavelengths of between 150 and 2000 nm
may prove effective in individual cases. The preferred chromophore
is indocyanine green. Other chromophores may be used, however,
their selection being based on desired photophysical and
photochemical properties upon which photosensitization efficiency
and photocytotoxicity are dependent. Examples of alternative
chromophores include, but are not limited to, single walled carbon
nanotubes (SWNT), buckminsterfullerenes (C.sub.60), indocyanine
green, methylene blue, DHE (polyhaematoporphrin ester/ether),
mm-THPP (tetra(meta-hydroxyphenyl)porphyrin), AlPcS.sub.4
(aluminium phthalocyanine tetrasulphonate), ZnET2 (zinc
aetio-purpurin), and Bchla (bacterio-chlorophyll .alpha.).
[0119] In one embodiment, the immunoadjuvant composition is
formulated as a solution or suspension. The solution or suspension
can include, for instance, about 0.25% by weight of a chromophore
and about 1% by weight of the glycated chitosan.
[0120] According to another preferred embodiment, the present
invention provides a composition for use in conditioning a neoplasm
for tandem photophysical and immunological treatment, comprising an
immunoadjuvant, wherein the immunoadjuvant is conjugated to a tumor
specific antigen, and wherein the immunoadjuvant is glycated
chitosan.
[0121] According to still another embodiment, the present invention
provides a composition for use in conditioning a neoplasm for
tandem photophysical and immunological treatment, comprising a
combination of a chromophore and an immunoadjuvant, wherein the
chromophore and the immunoadjuvant are conjugated to a tumor
specific antigen, and wherein the immunoadjuvant is glycated
chitosan.
[0122] According to another preferred embodiment, the present
invention provides a composition for use in conditioning a neoplasm
for tandem photophysical and immunological treatment, comprising an
immunoadjuvant, wherein the immunoadjuvant is conjugated to a tumor
specific antibody, and wherein the immunoadjuvant is glycated
chitosan. The immunoadjuvant can, in certain instances, consist
essentially of glycated chitosan. The glycated chitosan can also
further include a carbonyl reactive group.
[0123] According to another embodiment, the present invention
provides a composition for use in conditioning a neoplasm for
tandem photophysical and immunological treatment, comprising a
combination of a chromophore and an immunoadjuvant, wherein the
chromophore and the immunoadjuvant are conjugated to a tumor
specific antibody, and wherein the immunoadjuvant is glycated
chitosan. The immunoadjuvant can, in certain instances, consist
essentially of glycated chitosan. The glycated chitosan can also
further include a carbonyl reactive group.
[0124] The present invention thus provides injectable formulations
for conditioning a neoplasm for tandem photophysical and
immunological treatment, that in certain instances may include a
combination of, or a mixture of, a chromophore and an
immunoadjuvant, wherein the immunoadjuvant is glycated
chitosan.
[0125] A composition may furthermore be prepared for use in
conditioning a neoplasm for tandem photophysical and immunological
treatment, comprising an immunoadjuvant, wherein the immunoadjuvant
is conjugated to a tumor specific antigen, and wherein the
immunoadjuvant is viscoelastic glycated chitosan with a molecular
weight (MW) of between about 100 kDa and about 1000 kDa; and more
preferably, between about 100 kDa and about 300 kDa.
[0126] A composition may also be prepared for use in conditioning a
neoplasm for tandem photophysical and immunological treatment,
comprising a combination of a chromophore and an immunoadjuvant,
wherein the chromophore and the immunoadjuvant are conjugated to a
tumor specific antigen, and wherein the immunoadjuvant is
viscoelastic glycated chitosan with a molecular weight (MW) of
between about 100 kDa and about 1000 kDa; and more preferably,
between about 100 kDa and about 300 kDa.
[0127] Furthermore, an injectable solution may be prepared for
conditioning a neoplasm for tandem photophysical and immunological
treatment comprising an immunoadjuvant wherein the immunoadjuvant
is viscoelastic glycated chitosan with a molecular weight (MW) of
between about 100 KDa and about 1000 kDa; and more preferably,
between about 100 kDa and about 300 kDa.
[0128] An injectable solution may also be prepared for conditioning
a neoplasm for tandem photophysical and immunological treatment
comprising a mixture of a chromophore and an immunoadjuvant wherein
the immunoadjuvant is viscoelastic glycated chitosan with a
molecular weight (MW) of between about 100 kDa and about 1000 kDa;
and more preferably, between about 100 kDa and about 300 kDa.
[0129] In one example, the viscoelastic glycated chitosan
compositions of the present invention is used as an immunoadjuvant
in a novel cancer treatment. Photothermal and immunological
therapies are combined by irradiating the neoplasm directly to the
tumor without the use of a chromophore, and subsequently
introducing the chitosan-derived immunoadjuvant into or around the
irradiated neoplasm. Following the application of a laser with
irradiance sufficient to induce neoplastic cellular destruction,
cell-mediated and humoral immune responses to the neoplastic
antigens thus released are stimulated (enhanced) by the
immunoadjuvant component.
[0130] In another example, photodynamic and immunological therapies
are combined by introducing both a chromophore and a
chitosan-derived immunoadjuvant (also called immuno-modulator or
immunopotentiator) into a neoplasm. Upon application of a laser
with irradiance sufficient to induce neoplastic cellular
destruction, cell-mediated and humoral immune responses to the
neoplastic antigens thus released are stimulated (enhanced) by the
immunoadjuvant component.
[0131] The chromophore and immunoadjuvant may be combined into a
solution for injection into the center of the tumor mass, or
injected separately into the tumor mass. It should be recognized
however that other methods may be sufficient for localizing the
chromophore and immunoadjuvant in the tumor site. One such
alternative delivery means is conjugation of the chromophore or
immunoadjuvant or both to a tissue specific antibody or tissue
specific antigen, such that delivery to the tumor site is enhanced.
Any one method, or a combination of varying methods, of localizing
the chromophore and immunoadjuvant in the tumor site is acceptable
so long as the delivery mechanism insures sufficient concentration
of the components in the neoplasm.
[0132] According to another embodiment, a method for treating a
neoplasm in a human or other animal host, comprises: (a) selecting
an immunoadjuvant, wherein the immunoadjuvant comprises
viscoelastic glycated chitosan; (b) irradiating the conditioned
neoplasm whereby neoplastic cellular destruction of the conditioned
neoplasm is induced producing fragmented neoplastic tissue and
cellular molecules; and (c) introducing the immunoadjuvant into or
around the neoplasm, which stimulates the self-immunological
defense system of the host to process the fragmented neoplastic
tissue and cellular molecules, such as tumor antigens, and thus
create an immunity against neoplastic cellular multiplication.
[0133] According to yet another embodiment, a method for treating a
neoplasm in a human or other animal host, comprises: (a) selecting
a chromophore and an immunoadjuvant, wherein the immunoadjuvant
comprises viscoelastic glycated chitosan; (b) introducing the
chromophore and the immunoadjuvant into the neoplasm to obtain a
conditioned neoplasm; and (c) irradiating the conditioned neoplasm
whereby neoplastic cellular destruction of the conditioned neoplasm
is induced producing fragmented neoplastic tissue and cellular
molecules in the presence of the immunoadjuvant which stimulates
the self-immunological defense system of the host against
neoplastic cellular multiplication.
[0134] In yet another embodiment, a method of producing tumor
specific antibodies in a tumor-bearing host, includes irradiating a
tumor with a laser of a wavelength in the visible, near-infrared or
infrared range, to a degree sufficient to induce neoplastic
cellular destruction and generating fragmented neoplastic tissue
and cellular molecules, followed by the introduction of an
immunoadjuvant into or around a neoplasm by means of injection so
that the host's immune system is stimulated to interact with and
process fragmented neoplastic tissue and cellular molecules, upon
which a systemic anti-tumor response is induced.
[0135] In another embodiment, a method of producing tumor specific
antibodies in a tumor-bearing host, includes simultaneously
introducing a chromophore and an immunoadjuvant into a neoplasm by
intratumor injection to obtain a conditioned neoplasm, the
chromophore being suitable to generate thermal energy upon
activation in the near-infrared or infrared wavelength range; and
activating the chromophore with a laser of a wavelength in the
near-infrared or infrared range to a degree sufficient to activate
the chromophore to produce a photothermal reaction inducing
neoplastic cellular destruction and generating fragmented
neoplastic tissue and cellular molecules.
[0136] An exemplary method of photophysically destroying a neoplasm
and concurrently generating an in situ autologous vaccine in a
tumor-bearing host, includes: (a) selecting an immunoadjuvant; (b)
irradiating the neoplasm with a laser of a wavelength in the
visible, near-infrared or infrared range, at a power and for a
duration sufficient to produce a photothermal reaction inducing
neoplastic cellular destruction and generating fragmented
neoplastic tissue and cellular molecules; (c) forming the in situ
vaccine by introducing the immunoadjuvant into the neoplasm by
intratumor injection wherein the in situ vaccine comprises an
amalgam of the fragmented tissue and cellular molecules and the
immunoadjuvant; and (d) stimulating the self-immunological defense
system against neoplastic cellular multiplication by having the
vaccine presented locally to induce an anti-tumor response
systemically within the host.
[0137] Another exemplary method of photophysically destroying a
neoplasm and concurrently generating an in situ autologous vaccine
in a tumor-bearing host, includes: (a) selecting a chromophore and
an immunoadjuvant, the chromophore being suitable to generate
thermal energy upon activation in the near-infrared or infrared
wavelength range; (b) introducing the chromophore into the neoplasm
by intratumor injection; (c) irradiating the neoplasm with a laser
of a wavelength in the visible, near-infrared or infrared range, at
a power and for a duration sufficient to activate the chromophore
to produce a photothermal reaction inducing neoplastic cellular
destruction and generating fragmented neoplastic tissue and
cellular molecules; (d) forming the in situ vaccine by introducing
the immunoadjuvant into the neoplasm by intratumor injection
wherein the in situ vaccine comprising an amalgam of the fragmented
tissue and cellular molecules and the immunoadjuvant; and (e)
stimulating the self-immunological defense system against
neoplastic cellular multiplication by having the vaccine presented
locally to induce an anti-tumor response systemically within the
host.
[0138] Yet another exemplary method of photophysically destroying a
neoplasm and concurrently generating an in situ autologous vaccine
in a tumor-bearing host, includes: (a) selecting a chromophore and
an immunoadjuvant, the chromophore being suitable to generate
thermal energy upon activation in the near-infrared or infrared
wavelength range; (b) simultaneously or separately introducing the
chromophore and the immunoadjuvant into the neoplasm by intratumor
injection to obtain a conditioned neoplasm; (c) forming the in situ
vaccine by irradiating the conditioned neoplasm with a laser of a
wavelength in the near-infrared or infrared range at a power and
for a duration sufficient to activate the chromophore to produce a
photothermal reaction inducing neoplastic cellular destruction and
generating fragmented neoplastic tissue and cellular molecules,
wherein the in situ vaccine comprising an amalgam of the fragmented
tissue and cellular molecules and the immunoadjuvant; and (d)
stimulating the self-immunological defense system against
neoplastic cellular multiplication by having the vaccine presented
locally and by allowing the vaccine to be dispersed systemically
within the host.
[0139] As described elsewhere herein, the method can further
include conjugating the immunoadjuvant to a tumor specific
antibody, thereby forming a conjugate, and administering the
conjugate to the host. Alternatively, the method can further
include conjugating the immunoadjuvant to a tumor specific antigen,
thereby forming a conjugate, and administering the conjugate to the
host. Any number of suitable chromophores can be used, for
instance, indocyanine green, DHE, m-THPP, APcS.sub.4, ZnET2, and
Bchla.
[0140] Furthermore, the method can include conjugating a
combination of the chromophore and the immunoadjuvant to a tumor
specific antibody, thereby forming a conjugate, and administering
the conjugate to the host. Alternatively, the method can further
include conjugating the chromophore and the immunoadjuvant to a
tumor specific antigen, thereby forming a conjugate, and
administering the conjugate to the host. Any number of suitable
chromophores can be used, for instance, indocyanine green, DHE,
m-THPP, APcS.sub.4, ZnET2, and Bchla.
[0141] The preparations and formulations of the present invention,
including the viscoelastic glycated chitosan (GC) preparations, can
also be used in conjunction with photodynamic therapy (PDT).
Photosensitizing compounds show a photochemical reaction when
exposed to light. Photodynamic therapy (PDT) uses such
photosensitizing compounds and lasers to produce tumor necrosis.
Treatment of solid tumors by PDT usually involves the systemic
administration of tumor localizing photosensitizing compounds and
their subsequent activation by laser. Upon absorbing light of the
appropriate wavelength the sensitizer is converted from a stable
atomic structure to an excited state. Cytotoxicity and eventual
tumor destruction are mediated by the interaction between the
sensitizer and molecular oxygen within the treated tissue to
generate cytotoxic singlet oxygen.
[0142] Two good general references pertaining to PDT, biomedical
lasers and photosensitizing compounds, including light delivery and
dosage parameters, are Photosensitizing Compounds: Their Chemistry,
Biology and Clinical Use, published in 1989 by John Wiley and Sons
Ltd., Chichester, U.K., ISBN 0 471 92308 7, and Photodynamic
Therapy and Biomedical Lasers: Proceedings of the International
Conference on Photodynamic Therapy and Medical Laser Applications,
Milan, 24-27 Jun. 1992, published by Elsevier Science Publishers
B.V., Amsterdam, The Netherlands, ISBN 0 444 81430 2, both
incorporated herein by reference.
[0143] United States patents related to PDT include U.S. Pat. Nos.
5,095,030 and 5,283,225 to Levy et al.; U.S. Pat. No. 5,314,905 to
Pandey et al.; U.S. Pat. No. 5,214,036 to Allison et al; and U.S.
Pat. No. 5,258,453 to Kopecek et al., all of which are incorporated
herein by reference. The Levy patents disclose the use of
photosensitizers affected by a wavelength of between 670-780 nm
conjugated to tumor specific antibodies, such as receptor-specific
ligands, immunoglobulins or immunospecific portions of
immunoglobulins. The Pandey patents are directed to
pyropheophorbide compounds for use in standard photodynamic
therapy. Pandey also discloses conjugating his compositions with
ligands and antibodies. The Allison patent is similar to the Levy
patents in that green porphyrins are conjugated to lipocomplexes to
increase the specificity of the porphyrin compounds for the
targeted tumor cells. The Kopecek patent also discloses
compositions for treating cancerous tissues. These compositions
consist of two drugs, an anti-cancer drug and a photoactivatable
drug, attached to a copolymeric carrier. The compositions enter
targeted cells by pinocytosis. The anti-cancer drug acts after the
targeted cell has been invaded. After a period of time, a light
source is used to activate the photosensitized substituent.
[0144] Further Applications for Tumor Immunotherapy
[0145] The preparations and formulations of the present invention,
including the viscoelastic glycated chitosan (GC) preparations, can
be used, e.g., as immunoadjuvants, in the context of tumor
immunotherapy.
[0146] The major functions of the immune system are to develop the
concept of "self" and eliminate what is "nonself". Although
microorganisms are the principal non-self entities encountered
every day, the immune system also works to eliminate neoplasms and
transplants.
[0147] There are several distinct types of immunity. Nonspecific,
or innate, immunity refers to the inherent resistance manifested by
a species that has not been immunized (sensitized or allergized) by
previous infection or vaccination. Its major cellular component is
the phagocytic system, whose function is to ingest and digest
invading microorganisms. Phagocytes include neutrophils and
monocytes in the blood and macrophages in the tissues. Complement
proteins are the major soluble component of nonspecific immunity.
Acute phase reactants and cytokines, such as interferon, are also
part of innate immunity.
[0148] Specific immunity is an immune status in which there is an
altered reactivity directed solely against the antigenic
determinants (infectious agent or other) that stimulated it. It is
sometimes referred to as acquired immunity. It may be active and
specific, as a result of naturally acquired (apparent or
unapparent) infection or intentional vaccination; or it may be
passive, being acquired from a transfer of antibodies from another
person or animal. Specific immunity has the hallmarks of learning,
adaptability, and memory. The cellular component is the lymphocyte
(e.g., T-cells, B-cells, natural killer (NK) cells), and
immunoglobulins are the soluble component.
[0149] The action of T-cells and NK-cells in recognizing and
destroying parasitized or foreign cells is termed cell-mediated
immunity. In contradistinction to cell-mediated immunity, humoral
immunity is associated with circulating antibodies produced, after
a complex recognition process, by B-cells.
[0150] As regards tumor immunology, the importance of lymphoid
cells in tumor immunity has been repeatedly shown. A cell-mediated
host response to tumors includes the concept of immunological
surveillance, by which cellular mechanisms associated with
cell-mediated immunity destroy newly transformed tumor cells after
recognizing tumor-associated antigens (antigens associated with
tumor cells that are not apparent on normal cells). This is
analogous to the process of rejection of transplanted tissues from
a nonidentical donor. In humans, the growth of tumor nodules has
been inhibited in vivo by mixing suspensions of a patient's
peripheral blood lymphocytes and of tumor cells, suggesting a
cell-mediated reaction to the tumor. In vitro studies have shown
that lymphoid cells from patients with certain neoplasms show
cytotoxicity against corresponding human tumor cells in culture.
These cytotoxic cells, which are generally T-cells, have been found
with neuroblastoma, malignant melanomas, sarcomas, and carcinomas
of the colon, breast, cervix, endometrium, ovary, testis,
nasopharynx, and kidney. Macrophages may also be involved in the
cell-mediated host's response to tumors when in the presence of
tumor-associated antigens, lymphokines or interferon.
[0151] Humoral antibodies that react with tumor cells in vitro have
been produced in response to a variety of animal tumors induced by
chemical carcinogens or viruses. Hydridoma technology in vitro
permits the detection and production of monoclonal antitumor
antibodies directed against a variety of animal and human
neoplasms. Antibody-mediated protection against tumor growth in
vivo, however, has been demonstrable only in certain animal
leukemias and lymphomas. By contrast, lymphoid cell-mediated
protection in vivo occurs in a broad variety of animal tumor
systems.
[0152] Immunotherapy for cancer is best thought of as part of a
broader subject, namely biologic therapy, or the administration of
biologic-response modifiers. These agents act through one or more
of a variety of mechanisms (1) to stimulate the host's antitumor
response by increasing the number of effector cells or producing
one or more soluble mediators; (2) to serve as an effector or
mediator; (3) to decrease host suppressor mechanisms; (4) to alter
tumor cells to increase their immunogenicity or make them more
likely to be damaged by immunological processes; or (5) to improve
the host's tolerance to cytotoxics or radiation therapy. Heretofore
the focus of cell-mediated tumor immunotherapy has been on
reinfusion of the patient's lymphocytes after expansion in vitro by
exposure to interleukin-2. One variation includes isolating and
expanding populations of lymphocytes that have infiltrated tumors
in vivo, so-called tumor-infiltrating lymphocytes. Another is the
concurrent use of interferon, which is thought to enhance the
expression of histocompatibility antigens and tumor-associated
antigens on tumor cells, thereby augmenting the killing of tumor
cells by the infused effector cells.
[0153] Humoral therapy has long concentrated on the use of
antitumor antibodies as a form of passive immunotherapy, in
contrast to active stimulation of the host's own immune system.
Another variation is the conjugation of monoclonal antitumor
antibodies with toxins, such as ricin or diphtheria, or with
radioisotopes, so the antibodies will deliver these toxic agents
specifically to the tumor cells. Active immunization with a host's
own tumor cells, after irradiation, neuraminidase treatment, hapten
conjugation, or hybridization has also been tried. Clinical
improvement has been seen in a minority of patients so treated.
Tumor cells from others have been used after their irradiation in
conjunction with adjuvants in acute lymphoblastic leukemia and
acute myeloblastic leukemia after remission. Prolongation of
remissions or improved reinduction rates have been reported in some
series, but not in most. Interferons, tumor necrosis factor and
lymphotoxins have also been used to affect immunologically mediated
mechanisms. A recent approach, using both cellular and humoral
mechanisms, is the development of "heterocross-linked antibodies,"
including one antibody reacting with the tumor cell linked to a
second antibody reacting with a cytotoxic effector cell, making the
latter more specifically targeted to the tumor. Host immune cell
infiltration into a PDT treated murine tumor has been reported.
[0154] Combined PDT and Immunotherapy
[0155] In accordance with the present invention, it is desirable to
utilize glycated chitosan (GC) preparations having a suitable
viscosity that enables their use as an injectable material in
additional applications, such as combined photodynamic cancer
therapy (PDT) and tumor immunotherapy methods.
[0156] The potential for combining PDT with immunotherapy was
explored by Korbelik, Krosl, Dougherty and Chaplin. See
Photodynamic Therapy and Biomedical Lasers, supra, at pp. 518-520.
In their study, they investigated a possibility of amplification of
an immune reaction to PDT and its direction towards more pervasive
destruction of treated tumors. The tumor, a squamous cell carcinoma
SCCVII, was grown on female C3H mice. An immunoactivating agent SPG
(a high molecular weight B-glucan that stimulates macrophages and
lymphoid cells to become much more responsive to stimuli from
cytokines and other immune signals) was administered
intramuscularly in 7 daily doses either ending one day before PDT
or commencing immediately after PDT. Photofrin based PDT was
employed; photofrin having been administered intravenously 24 hours
before the light treatment. The SPG immunotherapy was shown to
enhance the direct killing effect of the PDT The indirect killing
effect (seen as a decrease in survival of tumor cells left in situ)
was, however, much more pronounced in tumors of animal not
receiving SPG The difference in the effectiveness of SPG
immunotherapy when performed before and after PDT suggested that
maximal interaction is achieved when immune activation peaks at the
time of the light delivery or immediately thereafter. With SPG
starting after PDT (and attaining an optimal immune activation 5-7
days later), it is evidently too late for a beneficial
reaction.
[0157] In another study the use of PDT to potentiate the effect of
bioreactive drugs that are cytotoxic under hypoxic conditions was
investigated. See Photodynamic Therapy and Biomedical Lasers,
supra, at pp. 698-701. It was found that the antitumor activity of
such drugs could be enhanced in vivo when they were used in
combination with treatments that increase tumor hypoxia.
[0158] Cancer Treatment by Photodynamic Therapy, in Combination
with an Immunoadjuvant
[0159] In accordance with the present invention, it is desirable to
utilize glycated chitosan (GC) preparations having a suitable
viscosity as injectable materials for use in the treatment of
cancer. This can be achieved in any suitable manner, for instance,
in conjunction with applications such as combined photothermal or
photodynamic cancer therapy (PDT) and tumor immunotherapy methods.
The term cancer, as used herein, is a general term that is intended
to include any of a number of various types of malignant neoplasms,
most of which invade surrounding tissues, may metastasize to
several sites, and are likely to recur after attempted removal and
to cause death of the patient unless adequately treated. A
neoplasm, as used herein, refers to an abnormal tissue that grows
by cellular proliferation more rapidly than normal. It continues to
grow even after the stimulus that initiated its growth dissipates.
Neoplasms show a partial or complete lack of structural
organization and functional coordination with the normal tissue and
usually form a distinct mass which may be either benign or
malignant.
[0160] In accordance with the present invention, certain examples
of cancers that may be treated with glycated chitosan (GC)
preparations having a suitable viscosity as injectable materials
include, but are not limited to, those of the cervix, breast,
bladder, colon, prostate, larynx, endometrium, ovary, oral cavity,
kidney, testis (nonseminomatous) and lung (non-small cell).
[0161] Moreover, in accordance with the present invention,
treatment may also be administered in a suitable manner in
conjunction with other types of cancer treatment, for instance,
radiation treatment. Radiation plays a key role, for example, in
the remediation of Hodgkin's disease, nodular and diffuse
non-Hodgkin's lymphomas, squamous cell carcinoma of the head and
neck, mediastinal germ-cell tumors, seminoma, prostate cancer,
early stage breast cancer, early stage non-small cell lung cancer,
and medulloblastoma. Radiation can also be used as palliative
therapy in prostate cancer and breast cancer when bone metastases
are present, in multiple myeloma, advanced stage lung and
esophagopharyngeal cancer, gastric cancer, and sarcomas, and in
brain metastases. Cancers that may be treated include, for
instance, Hodgkin's disease, early-stage non-Hodgkin's lymphomas,
cancers of the testis (seminomal), prostate, larynx, cervix, and,
to a lesser extent, cancers of the nasopharynx, nasal sinuses,
breast, esophagus, and lung.
[0162] Treatment may also be administered in a suitable manner in
conjunction with other types of antineoplastic drugs.
Antineoplastic drugs include those that prevent cell division
(mitosis), development, maturation, or spread of neoplastic cells.
The ideal antineoplastic drug would destroy cancer cells without
adverse effects or toxicities on normal cells, but no such drug
exists. Despite the narrow therapeutic index of many drugs,
however, treatment and even cure are possible in some patients.
Certain stages of choriocarcinoma, Hodgkin's disease, diffuse large
cell lymphoma, Burkitt's lymphoma and leukemia have been found to
be susceptible to antineoplastics, as have been cancers of the
testis (nonseminomatous) and lung (small cell). Common classes of
antineoplastic drugs include, but are not limited to, alkylating
agents, antimetabolites, plant alkaloids, antibiotics,
nitrosoureas, inorganic ions, enzymes, and hormones.
[0163] In Situ Autologous Cancer Vaccines, Such as Laser-Assisted
Immunotherapy
[0164] The chitosan-derived compositions and, in particular, the
viscoelastic glycated chitosan preparations of the present
invention, are effective in treating neoplasms and other medical
disorders. Additional uses of glycated chitosan, alone or in
combination with other drugs, include use as an immunostimulant in
the treatment of immuno-compromised patients including but not
limited to cancer and acquired immunodeficiency syndrome.
[0165] The chitosan-derived compositions of the present invention
are thus useful in a myriad of applications, including for instance
as an immunoadjuvant or as a component of an immunoadjuvant, as
described in detail herein. Notwithstanding other uses, a principal
use of the chitosan-derived compositions is as an immunoadjuvant in
connection with in situ autologous cancer vaccines (inCVAX), such
as laser-assisted immunotherapy (LIT), and it is in this context
that the chitosan-derived compositions are described in detail
herein.
[0166] As described further herein, additional embodiments of the
present invention are directed to uses of the glycated chitosan
preparations of the present invention as immunoadjuvants in
conjunction with inCVAX in general, and LIT in particular, for
cancer treatment. Laser-assisted immunotherapy utilizing the
present invention preferably encompasses introducing into or around
a neoplasm an immunoadjuvant comprising viscoelastic
chitosan-derived compositions following photothermal irradiation of
the same tumor. The photothermal action is performed at an
irradiance sufficient to induce neoplastic cellular destruction,
which can be performed with or without intratumoral injection of,
or by other means delivered, a chromophore, and combined with
injection of, or by other means delivered, the viscoelastic
glycated chitosan preparations of the present invention,
cell-mediated and humoral anti-tumor immune responses are
induced.
[0167] In preferred embodiments, improved LIT is provided wherein
the improvement comprises the use of the herein-described
injectable viscoelastic glycated chitosan preparations of the
present invention. The present invention also contemplates methods
of in vivo activation of specific components of the immune system
in conjunction with inCVAX in general, or LIT in particular,
comprising treatment with a viscoelastic glycated chitosan
preparation.
[0168] As described further herein, it has been determined that LIT
provides an in situ autologous cancer vaccine (inCVAX) that
overcomes limitations of current immunotherapies and cancer
vaccines. In general, the two principles underlying LIT are (1)
local heating of the primary tumor with a laser to devitalize the
tumor and liberate tumor antigens, and (2) local injection of a
potent and nontoxic immunoadjuvant comprising glycated chitosan
(GC), which interacts with liberated tumor antigens to induce an
immune response against the cancer. Thus, LIT effectively functions
as an in situ autologous cancer vaccine that uses whole tumor cells
as the sources of tumor antigens from each individual patient
without pre-selection of tumor antigens or ex vivo preparation.
[0169] In accordance with the present invention, another advantage
of using the herein-described injectable viscoelastic glycated
chitosan preparations of the present invention, in conjunction with
LIT, is that by using this LIT approach, there is activation of
dendritic cells (DC), and subsequently exposure of the activated DC
to tumor antigens in vivo. LIT thus represents an advantageous
approach to other whole-cell cancer vaccinations, by eliminating
the need of ex vivo preparations, and by using LIT in conjunction
with the viscoelastic glycated chitosan preparations as
immunoadjuvants.
[0170] One exemplary formulation of a glycated chitosan preparation
was manufactured under the name PROTECTIN. It has been observed
that PROTECTIN in conjunction with LIT stimulates the immune system
and induces tumor-specific immunity by 1) activating dendritic
cells, 2) increasing the interaction between tumor cells and
dendritic cells, and 3) increasing the tumor antigen presentation
to the immune system.
[0171] Other viscoelastic glycated chitosan preparations of the
present invention also function to stimulate the immune system and
induce tumor-specific immunity by 1) activating dendritic cells, 2)
increasing the interaction between tumor cells and dendritic cells,
and 3) increasing the tumor antigen presentation to the immune
system.
[0172] Thus, in accordance with a preferred embodiment of the
invention, formulations of viscoelastic glycated chitosan activate
one or more components of the immune system, mediating desired
therapeutic effects.
[0173] As described further herein, certain components of the
immune system that are activated include components of nonspecific,
or innate, immunity, namely the phagocytic system including
neutrophils and monocytes in the blood and macrophages in the
tissues; complement proteins, the major soluble component of
nonspecific immunity; and acute phase reactants and cytokines, such
as interferon, also part of innate immunity. There are many
different components of specific immunity, for example, the
lymphocyte (e.g., T-cells, B-cells, natural killer (NK) cells), and
immunoglobulins. The glycated chitosan formulations of the
invention also interact with lymphoid cells to promote tumor
immunity. Macrophages may also be involved in the cell-mediated
host's response to tumors when in the presence of tumor-associated
antigens, lymphokines or interferon.
[0174] Specific components of the immune system are activated after
"photothermal" treatment. When photothermal destruction occurs, the
fragmented tissue and cellular molecules are disbursed within the
host in the presence of the immunologically potentiating material,
such as chitosan. In effect, an in situ vaccine is formed. This
mixture of materials then circulates in the host and is detected by
the immunological surveillance system. There follows an immediate
mobilization of cell-mediated immunity which encompasses NK-cells
and recruited killer T-cells. These cells migrate to the sites of
similar antigens or chemicals. In time, the cell-mediated immunity
shifts to a humoral immunity with the production of cytotoxic
antibodies. These antibodies freely circulate about the body and
attach to cells and materials for which they have been encoded. If
this attachment occurs in the presence of complement factors, the
result is cellular death.
[0175] The injectable viscoelastic glycated chitosan preparations
of the present invention have unexpected utility in "in situ cancer
vaccines", which are based on an in situ activation of
antigen-presenting cells (e.g., dendritic cells and macrophages),
and the subsequent exposure of tumor antigens to the
antigen-presenting cells. The injectable viscoelastic glycated
chitosan preparations of the present invention also activate other
cellular mediators including, but not limited to, tumor necrosis
factor (e.g., TNFa) and nitric oxide which contribute to the
therapeutic effects.
[0176] Another advantage of using the herein-described injectable
viscoelastic glycated chitosan preparations of the present
invention, in conjunction with LIT, is that by using this approach,
this method independently triggers the immune response in each
individual, and it does not depend upon cross reactivity in the
expression of tumor-specific antigen between hosts (as is required
in conventional antibody immunotherapy and vaccination).
Histochemical studies have revealed that sera from LIT-cured
tumor-bearing rats contained antibodies that bound to the plasma
membrane of both living and preserved tumor cells. Western blot
analysis of tumor cell proteins using sera (from rats successfully
treated by LIT) as the source of primary antibodies showed distinct
bands, indicating induction of tumor-selective antibodies. It was
also shown that successfully treated rats could acquire long-term
resistance to tumor re-challenge, and adoptive immunity could be
transferred using spleen cells from successfully treated rats,
indicating tumor-specific immunity.
[0177] Thus, using the herein-described injectable viscoelastic
glycated chitosan preparations of the present invention, there are
several advantages that meet critical needs in providing effective
cancer treatment. This is particularly advantageous for cancer
patients, since the present invention also provides surprisingly
and unexpectedly beneficial preparations that are easy to
administer by injection, and therefore increase compliance and
provide effective treatment alternatives to conventional approaches
that do not provide (1) effective, (2) nontoxic, and (3) practical
treatments for late-stage metastatic cancer. A critical issue in
breast cancer therapy is that not all patients are treatable with
current, conventional methodologies and those diagnosed at late
stages have a poor prognosis, with even fewer valid options for
treatment. And, while there have been many advances and
developments in breast cancer treatment in recent years, crucial
problems remain. The injectable viscoelastic glycated chitosan
preparations of the present invention, as described herein, provide
several advantages that meet critical needs in providing effective
cancer treatment.
[0178] LIT has been shown to induce maturation of dendritic cells
(assessed by CD80 expression), enhance T-cell proliferation,
increase IFN-.gamma. secretion and increase HSP70 expression.
Furthermore, the combined effects of LIT (for instance, tumor
heating with a laser and injection of glycated chitosan
preparations in accordance with the present invention) has been
shown to induce tumor-specific immunity, with an infiltration of
tumor-specific cytotoxic CD4 and CD8 cells into the tumors
following the treatment.
[0179] As described in further detail herein, LIT thus provides
numerous advantages including, but not limited to: [0180]
Eliminates treated primary tumors [0181] Eliminates untreated
metastases [0182] Induces long-term immunity and survival [0183]
Creates resistance to tumor rechallenges [0184] Is non-toxic and
safe to use in humans at therapeutic doses
[0185] In accordance with one aspect of the invention, a neoplasm,
such as a malignant tumor, is irradiated with visible,
near-infrared or infrared light with a power and a duration
sufficient to elevate the temperature of the neoplasm to a level
that induces neoplastic cellular destruction and stimulates the
self-immunological defense system against neoplastic cellular
multiplication. To facilitate the heating of the tumor, a
chromophore with absorption peaks corresponding to the wavelength
of the applied light, may be injected prior to applying the light
treatment. Following the light irradiation, a viscoelastic glycated
chitosan-derived immunoadjuvant is administered, for example by
injection, into the tumor or the tissue immediately surrounding the
tumor.
[0186] In accordance with another aspect of the invention, a
solution of indocyanine green (ICG) and glycated chitosan is
prepared at a concentration of 0.1 to 2% of ICG to chitosan. The
solution is injected into the neoplasm, and the neoplasm is then
irradiated using a laser having a power of about 5 watts and a
wavelength of radiation capable of readily penetrating normal
cellular tissues without significant disruption. The irradiation
continues for a duration of from about one to about ten minutes,
which is sufficient to elevate the temperature of the neoplasm to a
level that induces neoplastic cellular destruction and stimulates
cell-mediated and humoral immune responses.
[0187] As described further herein, the present invention has
several advantages over other conventional and unconventional
treatment modalities. The combination of tumor destruction and
immune-stimulation adjuvant is the key. The most significant
advantage is combined acute and chronic tumor destruction. The
acute tumor loss is caused by photovaporization, photoablation or
thermal killing of the neoplastic tissue, on a large and controlled
scale, in the immediate area, reducing the tumor burden and hence
the base of multiplication so that the self-defense system can
fight a weaker "enemy". When local tumor destruction occurs, the
fragmented tissue and cellular molecules are locally disbursed
within the host in the presence of the immunologically potentiating
material, such as glycated chitosan. In effect, an in situ vaccine
is formed. There follows an immediate mobilization of cell-mediated
immunity which encompasses NK-cells and recruited killer T-cells.
These cells migrate to the sites of similar antigens or chemicals.
In time, the cell-mediated immunity shifts to a humoral immunity
with the production of cytotoxic antibodies. These antibodies
freely circulate about the body and attach to cells and materials
for which they have been encoded. If this attachment occurs in the
presence of complement factors, the result is cellular death. The
time frames for these two immunological modes of action are 0 to 2
weeks for the cell-mediated response, while the humoral arm matures
at approximately 30 days and should persist for long periods, up to
the life span of the host.
[0188] In summary, long-term survival with total cancer eradication
can be achieved by using the viscoelastic glycated chitosan
preparations of the present invention. It is a combined result of
reduced tumor burden due to ablative (for example photothermal)
interactions and an enhanced immune system response due to the
presence of glycated chitosan or other immunomodulators.
[0189] According to other embodiments, the glycated chitosan
preparations of the present invention may also be used for
antimicrobial and/or hemostatic applications. Thus the glycated
chitosan (GC) preparations can be formulated, for instance, as an
antimicrobial hemostatic spray, wherein the GC formulation has a
viscosity and exhibits rheological properties that enable it to be
sprayed from conventional containers. Moreover, GC can be included
in other formulations provided that it is applied in antimicrobial
and/or hemostatic effective concentrations and with
viscosities/rheological properties that enable its ability to be
dispensed from containers suitable for the purpose.
[0190] The present invention is further illustrated by the
following examples. These examples are provided by way of
illustration and are not intended in any way to limit the scope of
the invention. The examples should therefore not be construed as
limitations on the scope of the invention, but rather should be
viewed as exemplifications of preferred embodiments thereof. Many
other variations are possible.
EXAMPLES
Example 1
[0191] Exemplary Process for the Preparation of Glycated Chitosan
(GC)
[0192] Glycated chitosan is obtained by reacting chitosan with a
monosaccharide and/or oligosaccharide, preferably in the presence
of an acidifying agent, for a time sufficient to accomplish Schiff
base formation between the carbonyl group of the sugar and the
primary amino groups of chitosan (also referred to herein as
glycation of the amino group) to a predetermined degree whereby a
predetermined percent (%) glycation of the chitosan polymer is
achieved. This is followed by stabilization by reduction of Schiff
bases and of their rearranged derivatives (Amadori products). NMR
tracings are used to verify the bonding of the monosaccharides
and/or oligosaccharides to the chitosan polymer, whereas chemical
measurement of remaining free amino groups, such as via a
ninhydrine reaction, is used to assess the degree of glycation.
Example 2
[0193] Sterile Filtration
[0194] While conventional 1500 kDa galactochitosan, described in
U.S. Pat. No. 5,747,475, is relatively simple to synthesize, the
sterilization with, for example a 0.22 micron filter, is impossible
without compromising the integrity of the filter, thus rendering
the conventional glycated chitosan unsuitable for GMP production
and human use. In contrast, the new viscoelastic glycated chitosan
described herein has significant advantages with regard to GMP
production and sterile filtration due to unexpected and beneficial
physiochemical properties. For example, at a molecular weight
(M.W.) of 250,000 Da (250 kDa), sterile filtration with a 0.22
micron filter is highly feasible, with a flow rate of 100 ml/min
without loss of material during filtration.
Example 3
[0195] Viscosity of Glycated Chitosan (GC)
[0196] GC Preparations of Higher Molecular Weight Display Higher
Viscosities (Measured in Cp):
TABLE-US-00001 kDa of GC Cp 100 0.914 250 7.68 500 20.79 1500
84.7
[0197] FIG. 3 shows viscosity (in Cp; y-axis) vs. molecular weight
(in kDa; x-axis) in samples of GC with molecular weights ranging
from 100 kDa to 1,500 kDa. The concentration of GC in solution in
this experiment decreased with increasing molecular weight, ranging
from 0.6% (100 kDa) to 0.11% (1,500 kDa).
[0198] Very surprisingly, it was found viscosity increases linearly
with increasing molecular weight only if the concentration of GC in
the sample is reduced with increasing molecular weight. The table
and FIG. 4 shows the percent of GC in solution of the samples used
in the viscosity experiment above.
[0199] The results clearly show that 1) GC preparations of higher
molecular weight correlate with higher viscosities (measured in
Cp), and 2) the correlation between viscosity and molecular weight
is not linear if the concentration is kept constant. In other
words, the viscosity increases disproportionally with increasing
molecular weight, which renders the higher molecular weight
glycated chitosans (such as those disclosed in U.S. Pat. No.
5,747,475) unsuitable for injection or sterile filtration.
[0200] Viscoelastic glycated chitosan preparations comprising lower
molecular weight (i.e. below .about.400 kDa) glycated chitosan thus
provide improved injectability; these preparations are useful, for
instance, for cancer treatments utilizing photodynamic therapy and
laser-assisted immunotherapy to induce neoplastic cellular
destruction and to stimulate the self-immunological defense system
against neoplastic cells.
Example 4
[0201] Improvement of Manufacturing
[0202] In this exemplary study, it was determined that experimental
conditions could be adjusted as needed to improve overall yield
during the manufacture of glycated chitosan. It was unexpectedly
discovered that manufacturing of GC could be improved by
controlling the pH conditions, and thus controlling the percent
glycation. Specifically, it was determined that because the
half-life of sodium borohydride (NaBH4) is proportional to pH,
meaning that at lower pH the half-life of NaBH4 is extremely short,
and only at higher pH is the NaBH4 somewhat more stable. It was
thus determined that NaBH4 was not as effective in stabilizing the
glycated chitosan by reduction of the Schiff bases and Amidori
products at lower pH. For instance, when the pH was kept below five
(pH<5), the half-life of NaBH4 is extremely short, and thus the
reduction of the Schiff bases and Amadori products was less
efficient, and percent glycation of GC thus went down.
[0203] It was determined, however, that with a higher pH, the
formulation "gels" and becomes non-newtonian. For instance, when
the pH was kept above six (pH >6), the formulation was observed
to gel and thus the batch had to be discarded. In other words, to
achieve the goal of efficient GC manufacturing, the pH was not kept
so high that the formulation would "gel", but the pH was also not
kept so low that the percent glycation was minimized due to the
short half life of NaBH4.
Example 5
[0204] Laser-Assisted Immunotherapy (LIT) Treatment in a Human
Trial
[0205] An investigator-driven breast cancer trial was performed on
10 patients with advanced breast cancer (5 stage IV, 5 stage III).
Most of the patients had responded poorly, or not at all, to
conventional modalities, and received at least one Laser-Assisted
Immunotherapy (LIT) treatment in which viscoelastic glycated
chitosan was used as the immunoadjuvant. Two (2) patients withdrew
prematurely due to unrelated reasons, leaving 8 evaluable patients.
The independent investigators acquired IRB and government approvals
prior to the trials. Biopsies and medical imaging (CT scans, etc.)
were used for the evaluation of the primary lesions and
metastasis.
[0206] The primary efficacy parameter was the best overall response
by the investigators' assessments using Response Evaluation
Criteria in Solid Tumors (RECIST). Complete response (CR) was
defined as disappearance or lack of qualifying metabolic activity
of all target lesions. Partial response (PR) was defined as a
.gtoreq.30% decrease from baseline in activity or in the sum of the
longest diameter of target lesions. Progressive disease (PD) is
defined as a .gtoreq.20% increase in the sum of the longest
diameter of target lesions or the appearance of 1 or more new
lesions. Stable disease (SD) was defined as neither sufficient
reduction to qualify for PR nor sufficient increase to qualify for
PD. Of the 8 breast cancer patients available for evaluation, CR
was observed in 1 patient, PR in 4 patients and SD in 1 patient. In
patients available for evaluation, the objective response rate
(CR+PR) was 62.5%, and the clinically beneficial response rate
(CR+PR+SD) was 75%. PD was observed in 2 patients. All local
lesions irradiated by laser responded to LIT. In addition, most of
the distant metastases of these patients responded to LIT. The
diameters and activity of the metastases in lymph node, lung and
liver in several patients decreased dramatically.
[0207] Local and systemic toxicity was graded according to National
Cancer Institute Common Toxicity Criteria, version 3.0. Laboratory
assessment and physical examinations were performed periodically.
Adverse events were closely monitored and recorded throughout the
study period. LIT only induced local reactions within the treatment
area in breast cancer patients, most of which were related to the
thermal effects of the topical laser treatment. Redness, pain,
edema and ulceration of the treatment area were the common adverse
events (AEs). No grade 3 or 4 adverse events were observed. In
patients who had not received prior radiation therapy the swelling
was minor. For the patients who have received prior radiation
therapy, the swelling was more substantial with longer
duration.
Example 6
[0208] Laser-Assisted Immunotherapy with Glycated Chitosan
Demonstrates Antitumor Immunity Against B16 Melanoma Tumors in
Mice
[0209] Female C57BC/6 mice (8 weeks of age; 12 mice/group) were
subcutaneously inoculated with the B16-F1 melanoma tumor (10.sup.6
viable tumor cells) into the back area. The tumors reached
treatment size (7 to 8 mm in diameter) around 7 days after
implantation. Five treatment groups (12 female mice/group) were
included in the study: an untreated control; laser-assisted
immunotherapy treatment control; and laser-assisted immunotherapy
treatment with 0.2 mL of 1% glycated chitosan peritumorally
injected 24 h prior, immediately following, or 24 h after laser
treatment. The 805 nm diode laser was used for laser irradiation,
with parameter settings of 2 W for 10 min in duration. The laser
was directed through an optical fiber with a diffuser lens at the
end to the treatment site and the laser tip was maintained at a
distance of 4 mm from the skin.
[0210] Animal survival was evaluated. Darkening and hardening of
the mouse skin at the treatment site was observed after laser
treatment. Tumor reoccurrence usually occurred several days after
treatment. Thermal treatment in combination with glycated chitosan
application resulted in a significant improvement in animal
survival with glycated chitosan administered 24 h before laser
irradiation showing the most significant improvements (see table
below).
TABLE-US-00002 Effect of Laser and Glycated Chitosan Treatment in
B16 Melanoma Tumor-Bearing Mice Long-Term (>90 Days) Treatment
Injection.sup.a Survival Rate (%).sup.b Untreated Control 0.0 Laser
Only 16.7 Laser + 0.2 mL 1% GC 24 h After Laser 16.7 Laser + 0.2 mL
1% GC Immediately 25.05 After Laser Laser + 0.2 mL 1% GC 24 h
Before Laser 41.7 .sup.a= 805 nm diode laser with the energy (2 W,
10 min) directed through an optical fiber with a diffuser lens that
was maintained at a distance of 4 mm from the skin. .sup.b=
Long-term survival was defined as >90 days after inoculation
without tumor recurrence. GC = Glycated chitosan. n = 12 female
C57BC/6 mice.
Example 7
[0211] Interstitial Laser-Assisted Immunotherapy in a Metastatic
Mammary Model Using 805 nm Laser and Glycated Chitosan
[0212] A study was conducted to determine the optimal interstitial
laser dose and the optimal glycated chitosan dose. Female Wistar
Furth rats (5 to 6 weeks of age, 100 to 125 g) were subcutaneously
injected with the transplantable, metastatic mammary tumor, DMBA-4,
(10.sup.5 viable tumor cells) into the back area. DMBA-4 tumors,
originally induced chemically, are highly metastatic and poorly
immunogenic. The tumors metastasize along the lymphatics and
rapidly form multiple metastases at distant sites, killing all the
rats 30 to 40 days after tumor implantation. When the primary tumor
was 0.2 to 0.5 cm.sup.3, the hair overlying the tumor was clipped
and laser-assisted immunotherapy was performed on anesthetized
animals (2% isofluorane). An 805 nm diode laser was used to deliver
near-infrared light for target tumors. Continuous laser power was
delivered through an optical fiber with an active cylindrical tip.
An active tip of 1.0 cm was used, with a transparent plastic sheath
to protect the active tip. For the insertion of the active fiber
tip, either needle-guided or puncture-assisted insertion methods
were used. The intratumoral position of the fiber was verified by a
digital camera, which can capture the infrared light from the
805-nm laser. The rats were observed daily and the tumors were
measured twice a week for a period of at least 100 days. The
criterion for successful treatment was a 100-day survival after
tumor implantation. The optimal interstitial laser dose was
determined by evaluating effects in a control (9 rats, no
treatment); interstitial laser powers of 1, 1.5, 2, 2.5, and 3
W/cm.sup.2 for 10 min (14 rats/group); and interstitial laser power
of 2 W/cm.sup.2 for 30 min (14 rats/group). The rats in the 3 W at
10 min and 2 W at 30 min appeared to have average survival rates
higher than other groups. The optimal glycated chitosan dose was
determined by evaluating survival following administration of 0.1,
0.2, 0.4, and 0.6 mL of 1% glycated chitosan following interstitial
laser-assisted immunotherapy at 2.5 W for 20 min. A group of rats
that received no treatment was included as a control. The best
survival, at 42%, was observed following a 0.2 mL glycated chitosan
dose (see FIG. 5).
Example 8
[0213] Induced Antitumor Immunity Against DMBA-4 Metastatic Mammary
Tumors in Rats Using Laser-Assisted Immunotherapy
[0214] Female Wistar Furth rats (6 to 7 weeks of age, 110 to 130 g)
were inoculated with the DMBA-4 transplantable, metastatic mammary
tumor (10.sup.5 viable tumor cells) into the inguinal area. The
primary tumor generally appeared 7 to 10 days after inoculation and
was approximately 1 to 5 g within 3 weeks. The tumor metastasized
through the lymphatics to inguinal and axillary lymph nodes.
Treatment was initiated when the primary tumor was 0.2 to 0.5
cm.sup.-1, generally 10 to 15 days after inoculation. Rats were
administered a 0.25% indocyanine green and 1% Glycated Chitosan
Solution (0.20 mL) injected directly into the center of the tumor
prior to irradiation. An 805 nm diode laser was used for laser
irradiation, with parameter settings of 2 W for 10 min in duration.
The laser was directed through an optical fiber to the treatment
site. Following irradiation, animals were housed individually and
observations and tumor measurements were recorded twice weekly.
Rats which were successfully treated (cured rats) were rechallenged
repeatedly with the same tumor cells at tumor dose levels of
10.sup.5 to 10.sup.7 viable tumor cells per rat and animals were
observed for 4 months for tumor development. Of the 32 rats treated
by laser-assisted immunotherapy, eight rats were successfully
treated and tumor-free for >120 days following inoculation. In
all cured rats, metastases continued to develop after treatment,
then gradually declined and eventually disappeared without
additional treatment. Seven successfully-treated rats were
rechallenged up to three times with dose levels ranging from
10.sup.5 to 10.sup.7 viable tumor cells per injection. There was no
primary or metastatic tumor reemergence in any of these animals and
animals survived >120 days, while untreated control rats
developed primary and metastatic tumors and had an average survival
of 30 days.
Example 9
[0215] Enhancement of Laser Cancer Treatment by a Chitosan-Derived
Immunoadjuvant
[0216] The effect of the immunoadjuvant during the laser-assisted
immunotherapy treatment was evaluated in rats using four different
immunoadjuvants. Female Wistar Furth rats (6 to 8 weeks of age, 150
to 200 g) were subcutaneously inoculated with the DMBA-4
transplantable, metastatic mammary tumor (10.sup.5 viable tumor
cells) in the inguinal fat pad, 7 to 10 days before treatment. The
primary tumor generally became palpable in 5 to 7 days and the
remote inguinal and axillary metastases appeared 15 to 20 days
after inoculation. The laser-assisted immunotherapy treatment was
initiated when the primary tumor reached 0.2 to 0.5 cm.sup.3. Laser
treatment was generally performed on Day 10. The immunoadjuvants
included aqueous 1% Glycated Chitosan Solution (0.2 mL dose; n=48
rats in two experiments), 50% Complete Freund's Adjuvant (0.2 mL
dose; n=33 rats), 50% Incomplete Freund's Adjuvant (0.2 mL dose;
n=30 rats), and Corynebacterium parvum (C. parvum; 35 .mu.g/rat
dose; n=32 rats). The immunoadjuvants were mixed with 0.25%
indocyanine green and injected directly into the center of the
tumor 2 h before irradiation with the 805 nm diode laser. Animals
were anesthetized prior to irradiation and the hair overlying the
primary tumor was clipped. The laser parameters were 2 W for 10 min
with a 3 mm diameter laser treatment site, resulting in a fluence
of 96 J/cm.sup.2 for a 1 cm diameter tumor. Animals were
individually housed, observed daily, and tumor burden measurements
were collected twice a week. Data from this study was compared with
data from tumor-bearing control rats (n=38 rats) in several
different experiments. All immunoadjuvants had a statistically
significant increase in survival rate compared to control data
(p<0.05). The 1% glycated chitosan appeared to be the most
effective immunoadjuvant with a 29% long-term survival rate (see
table below). Statistical significance was observed when the
glycated chitosan adjuvant was compared to the C. parvum (p=0.009)
and Incomplete Freund's Adjuvant (p=0.03). Although not
significant, a noticeable improvement in survival was observed when
compared to Complete Freund's Adjuvant that had a comparable cure
rate (18%). A relative weak survival rate was observed following
treatment with the Incomplete Freund's Adjuvant and C. parvum.
TABLE-US-00003 Long-Term Survival Rates Following Treatment with
Four Different Immunoadjuvants Long-Term Treatment Number of Rats
Survival Rate (%) Control 38.sup.a 0 Laser + ICG + Glycated
Chitosan 48.sup.b 29 Laser + ICG + Complete 33 18 Freund's Adjuvant
Laser + ICG + Incomplete 30 7 Freund's Adjuvant Laser + ICG +
C-parvum 32 9 .sup.a= Tumor-bearing control rat data was collected
from several control groups in different studies. .sup.b= Data
collected from two separate experiments. ICG = Indocyanine
green.
Example 10
[0217] Enhancement of Photodynamic Therapy by a Chitosan-Derived
Immunoadjuvant
[0218] To evaluate photodynamic therapy as a the method for direct
tumor destruction in combination with glycated chitosan, a
combination of photofrin- and meso-substituted tetra
(meta-hydroxy-phenyl) chlorin-(mTHPC) based photodynamic therapy
and glycated chitosan injection was been studied in the EMT6
mammary sarcoma and Line 1 lung adenocarcinoma mouse models,
respectively. In each model, BALB/c mice were subcutaneously
inoculated with 10.sup.6 viable tumor cells into the lower dorsal
area. Tumors were treatment size (7 to 8 mm) after 7 days.
[0219] In the EMT6 mammary sarcoma model, treatment groups
evaluated are detailed in the table below. Photofrin
(Mont-Saint-Hilaire, Quebec, Canada) was prepared in 5% sterile
dextrose to a 1 mg/mL concentration. A 5 mg/kg dose of photofrin
was intravenously administered 24 h prior to irradiation. Animals
were shielded from direct light immediately after the
photosensitizer injection until 3 days after photodynamic
treatment. Mice were restrained unanesthetized in holders exposing
their backs during light treatment. Light (630 nm) was delivered
through an 8 mm diameter liquid light guide. The power density was
set at 100 mW/cm.sup.2, for a total light dose of 60 J/cm.sup.2.
Immediately after light irradiation, if applicable, animals were
administered a peritumoral dose of 0.5 or 1.5% glycated chitosan.
Animals were observed for tumor emergence every 2 days up to 90
days after photodynamic treatment and changes in tumor volume was
determined 3 times a week.
TABLE-US-00004 Survival Rates After Photofrin-Based Photodynamic
and Glycated Chitosan Treatment in Mice Bearing EMT6 Mammary Tumors
Number of Long-Term Long-Term Number Surviving Survival Treatment
of Mice Mice Rate (%) Control 8 0 0.0 Non-Thermal Laser Only 8 0
0.0 Non-Thermal Laser + 1.5% GC.sup.a 8 0 0.0 Non-Thermal Laser +
Photofrin 8 3 37.5 Non-Thermal Laser + Photofrin + 8 5 62.5 0.5% GC
Non-Thermal Laser + Photofrin + 8 6 75.0 1.5% GC .sup.a= It should
be noted that the laser treatment did not result in heating the
tumor because the light absorbing agent was not used and the laser
power was not sufficient to heat the tumor. Therefore, this group
is not representative of the laser-assisted immunotherapy system.
GC = Glycated chitosan. Laser treatment with a fluence rate of 100
mW/cm.sup.2 and a total light dose of 60 J/cm.sup.2. 5 mg/kg
photofrin was intravenously administered 24 h prior to irradiation.
0.1 mL of 0.5 or 1.5% glycated chitosan was injected peritumorally
immediately after irradiation.
[0220] All photodynamic and photodynamic glycated chitosan-treated
rats had complete tumor regression by the day after treatment.
Tumor reoccurrence was generally detected within 2 weeks after
treatment. The efficacy of standard photodynamic therapy was 37.5%,
which was increased following administration of 0.5 and 1.5%
glycated chitosan with values of 62.5 and 75%, respectively.
Glycated chitosan significantly increased survival rates in
tumor-bearing mice compared to photodynamic treatment only
(p<0.05).
[0221] In the Line 1 lung tumor model, treatment groups were as
presented the table below. mTHPC was prepared in a 2:3:5 (v/v/v)
mixture of ethanol, polyethyleneglycol 400, and water for a final
0.02 mg/mL concentration. A 0.1 mg/kg dose of mTHPC was
intravenously administered 24 h prior to irradiation. Animals were
shielded from direct light immediately after the photosensitizer
injection until 3 days after photodynamic treatment. Mice were
restrained unanesthetized in holders exposing their backs during
light treatment. A 652 nm light from a 0.25 W diode laser was
delivered through an 8 mm diameter liquid light guide. The power
density was set at 110 mW/cm.sup.2, for a total light dose of 30
J/cm.sup.2. Immediately after light irradiation, if applicable,
animals were administered a peritumoral dose of 1.67% glycated
chitosan. Animals were observed for tumor emergence every 2 days up
to 90 days after photodynamic treatment and 3 times a week changes
in tumor size was determined.
TABLE-US-00005 Survival Rates After mTHPC-Based Photodynamic and
Glycated Chitosan Treatment in Mice Bearing Line 1 Lung Tumors
Number of Long- Long-Term Number Term Survival Treatment of Mice
Surviving Mice Rate (%) Control 8 0 0.0 Laser Treatment Only 8 0
0.0 Laser + GC 8 0 0.0 Laser + mTHPC 8 0 0.0 Laser + mTHPC + 1.67%
GC 8 3 37.5 GC = Glycated chitosan. mTHPC = meso-substituted tetra
(meta-hydroxy-phenyl) chlorin-based photodynamic therapy. Laser
treatment with a fluence rate of 110 mW/cm.sup.2 and a total light
dose of 30 J/cm.sup.2. 0.1 mg/kg mTHPC was intravenously
administered 24 h prior to irradiation. 0.09 mL of 1.67% glycated
chitosan was injected peritumorally immediately after
irradiation.
[0222] Tumor reoccurrences were observed in all mice within 3
weeks. Following mTHPC-based photodynamic therapy, administration
of 1.67% glycated chitosan resulted in a 37.5% survival rate, while
other combinations were not effective. The Line 1 lung tumor model
was considered a poorly immunogenic tumor model. The effect of
tumor-localized glycated chitosan treatment on the response of the
mouse Line 1 tumors to mTHPC-based photodynamic therapy is
presented in FIG. 6.
[0223] The results of these studies indicate that an active
immunological stimulation is needed to augment the efficiency of
phototherapy.
Example 11
[0224] Effect of Different Components of Laser-Assisted
Immunotherapy in Treatment of Metastatic Tumors in Rats
[0225] Various combinations of three components of the
laser-assisted immunotherapy system were evaluated in this study
utilizing female and male rats bearing metastatic breast and
prostate tumors, respectively. The laser-assisted immunotherapy
system consisted of a near-infrared laser diode laser with a
maximum output of 25 W, the laser-absorbing dye, indocyanine green;
and the immunoadjuvant, glycated chitosan. When the primary tumor
was 0.2 to 0.5 cm.sup.3, treatment was initiated in the
tumor-bearing rats. A solution of 0.2 mL of GC and/or ICG was
injected into the center of the primary tumor in all groups. In
rats receiving laser treatment, the injections occurred 2 h before
irradiation, with animals anesthetized and the hair overlaying the
tumor clipped. The laser settings were 2 W and 10 min, with the
laser fiber tip maintained a distance of 4 mm from the overlying
skin and the laser energy directed to the treatment sites through
optical fibers. The animals were individually housed following
treatment. In the survival studies, the breast or prostate
tumor-bearing rats were observed daily and the three dimensions of
each tumor were measured weekly. Female Wistar Furth rats (5 to 6
weeks of age, 100 to 125 g) were subcutaneously inoculated with the
DMBA-4 transplantable, metastatic mammary tumor (10.sup.5 viable
tumor cells) into one inguinal fat pad of each rat. The primary
tumor emerged 7 to 10 days after inoculation. Metastatic tumors
along the lymphatics and at remote sites usually became palpable in
approximately 2 weeks. Without treatment, tumor-bearing rats have
an average survival time of 35 days. Eight groups of metastatic
breast tumor-bearing rats were treated with the different
components of the laser-assisted immunotherapy system, as detailed
in the table below. The survival rate and primary and metastatic
tumor profiles were determined for the individual components and
various combinations of the components. In addition, three groups
of female rats (n=16/group) were treated with 0.5, 1.0, and 2.0%
glycated chitosan to evaluate the impact of the immunoadjuvant
concentration on rat survival.
TABLE-US-00006 Treatment Parameters of Different Laser-Assisted
Immunotherapy Components in Female Metastatic Breast Tumor-Bearing
Rats Number of Group Laser Dye/Adjuvant Rats Control -- -- 35.sup.a
ICG Injection Only -- 0.25% ICG.sup.b 12 GC Injection Only -- 1.0%
GC.sup.b 12 Laser Only 2 W, 10 min -- 12 Laser + ICG 2 W, 10 min
0.25% ICG.sup.b 12 Laser + GC 2 W, 10 min 1.0% GC.sup.b 12 ICG + GC
-- 0.25% ICG/1.0% GC.sup.b 12 Laser + ICG + GC 2 W, 10 min 0.25%
ICG/1.0% GC.sup.b 31.sup.a .sup.a= Data collected from 2 separate
experiments. .sup.b= The injection volume (0.2 mL) was injected
directly to the center of the primary tumor. GC = Glycated
chitosan. ICG = Indocyanine green. -- = Not applicable.
[0226] In the metastatic breast tumor-bearing rats, single
component treatment resulted in all rats in the indocyanine green
and laser-only groups dying, with average survival times similar to
the control group. Two rats in the glycated chitosan group
survived, with one rat considered a long-term survivor and the
other rat considered a prolonged survivor (>120 days). Following
treatment with two components, 1 and 2 long-term survivors were
observed in the laser plus glycated chitosan and indocyanine green
plus glycated chitosan groups, respectively. There was no
statistical significance in the survival time when the single- or
two-component treatment groups were compared to the control group.
Nine rats had long-term survival after the three-component
laser-assisted immunotherapy (i.e., photothermal application
combined with glycated chitosan) treatment, resulting in an
approximate 30% cure rate in two separate experiments of 31 rats. A
significant difference (p<0.0001) in median survival time of the
treated rats was observed compared to the control rats. The
survival rate of rats following the treatment with one, two, or
three components of the laser-assisted immunotherapy system is
presented in FIGS. 7A-7C. Metastatic tumors usually emerged 2 weeks
after the inoculation of the primary tumor and reached a peak size
before the regression.
Example 12
[0227] Antitumor Immunity Induced by Laser-Assisted Immunotherapy
and its Adoptive Transfer
[0228] To investigate the mechanism of the antitumor immunity
induced by laser-assisted immunotherapy, adoptive transfer using
immune spleen cells was performed. Female Wistar Furth rats were
subcutaneously inoculated with the DMIBA-4 transplantable,
metastatic mammary tumor (10.sup.5 viable tumor cells) into one
inguinal fat pad of each rat, 7 to 10 days prior to laser-assisted
immunotherapy treatment. Without treatment, tumor-bearing rats
survived an average of approximately 30 days. Laser treated rats
were administered 0.2 mL of a solution containing both 0.25%
indocyanine green and 1% glycated chitosan directly into the
primary tumor before laser treatment. An 805 nm laser at 2 W for 10
min was used for irradiation. The protective ability of induced
immunity was evaluated in several groups of successfully treated
tumor-bearing rats that were challenged repeatedly with increased
inoculation doses of viable tumor cells. In addition, resistance to
tumor challenges after laser-assisted immunotherapy and the
inhibition of tumor growth were evaluated in naive rats.
[0229] Fifteen rats that had been successfully treated by
laser-assisted immunotherapy were rechallenged with 10.sup.6 viable
tumor cells 120 days after initial inoculation. Eighteen naive
age-matched rats (25 weeks of age) were inoculated with 10.sup.6
viable tumor cells for comparative purposes. All of the
successfully treated rats showed total resistance to the challenge,
with neither primary tumors nor metastasis observed; however, the
age-matched control rats developed primary and metastatic tumors
and died within 30 days after inoculation. A separate group of
young rats (approximately 8 weeks of age) were inoculated with 10'
viable tumor cells. Survival appeared to be dependent on the tumor
dose, with control rats inoculated with 10' and 10.sup.6 viable
tumor cells surviving on average 33 and 28 days, respectively.
Successfully treated rats usually experienced a gradual regression
in both treated primary tumor and untreated metastasis.
[0230] After the first rechallenge, the rats from several
experimental groups were followed by two subsequent challenges in a
time interval from 1 to 5 months, with the 10.sup.6 viable tumor
cells. The rats successfully treated by laser-assisted
immunotherapy were totally refractory to three tumor challenges.
This data is presented in the table below. In contrast, the
age-matched control tumor-bearing rats developed multiple
metastases in remote inguinal and axillary areas and died within 35
days. Multiple metastases developed in all 20 control rats
inoculated with 10' viable tumor cells; however, these rats had a
slightly increased survival time compared with the age-matched
control rats that were inoculated with the higher 10.sup.6 viable
tumor cell dose. The resistance to tumor rechallenge in
successfully treated rats strongly suggests tumor-selective
immunity.
TABLE-US-00007 Tumor Rechallenge in Rats Previously Cured with
Laser-Assisted Immunotherapy Number Number of Tumor Death Rate
Death Rate Survival Group of Rats Tumor Cells Rate 30 Days 40 Days
(Days) Cured Rats.sup.a 15 10.sup.6 0% 0% 0% >120 Cured
Rats.sup.b 15 10.sup.6 0% 0% 0% >120 Cured Rats.sup.c 15
10.sup.6 0% 0% 0% >120 Age-Matched Tumor 18 10.sup.6 100% 83%
100% 28.2 .+-. 2.8 Control Rats.sup.d Young Tumor Control
Rats.sup.e 20 10.sup.5 100% 20% 100% 32.7 .+-. 3.5 .sup.a= First
challenge. Tumor-bearing rats cured by laser-assisted
immunotherapy, rechallenged with viable tumor cells 120 days after
the initial inoculation. .sup.b= Second challenge. Tumor-bearing
rats cured by laser-assisted immunotherapy, rechallenged with
viable tumor cells a second time after the first challenge. .sup.c=
Third challenge. Tumor-bearing rats cured by laser-assisted
immunotherapy, rechallenged with viable tumor cells a third time
after the second challenge. .sup.d= Untreated rats the same age as
the cured rats at time of inoculation (no previous tumor exposure).
.sup.e= Untreated rats that were 8 weeks of age at the time of
inoculation (no previous tumor exposure).
[0231] For the adoptive immunization experiment, viable tumor
tissue harvested from live DMBA-4 tumor-bearing rats was dispersed
to a single-cell suspension by grinding in a loose-fitting ground
glass homogenizer. The long-surviving rats were sacrificed 28 days
after tumor rechallenge with the 10.sup.6 viable tumor cells, and
their spleens were dissected free of fat. Two separate experiments
were conducted using the splenocytes from control tumor-bearing
rats. The spleen cells were harvested 22 and 39 days after tumor
inoculation in the first and second experiment, respectively. Cell
suspensions were prepared by mechanical disruption into medium with
10% fetal calf serum. Spleen cells were also collected from a naive
rat without prior exposure to the tumor cells. Spleen cells and
viable tumor cells were counted on a hemocytometer before mixing to
a 400:1 spleen:tumor cell ratio. Naive rats were inoculated with
the mixture containing 4.times.10; spleen cells and 10.sup.5 viable
tumor cells in a volume of 0.2 mL. For the adoptive immunity
transfer experiments, 4 groups of naive female Wistar Furth rats
were inoculated with tumor cells. The treatment groups were Group A
tumor-bearing control rats inoculated with 10.sup.5 viable tumor
cells without treatment; Group B rats inoculated with the tumor
cells mixed with the spleen cells from a control tumor-bearing rat;
Group C rats inoculated with the tumor cells mixed with the spleen
cells from a tumor-bearing rat successfully treated by
laser-assisted immunotherapy, 28 days after tumor rechallenge; and
Group D rats inoculated with the tumor cells mixed with the spleen
cells from a naive rat without prior tumor exposure. The experiment
was performed in duplicate and the survival of rats from both
experiments was combined and is presented in FIG. 8. There were no
primary or metastatic tumors observed in Group C rats indicating
that the spleen cells from laser-assisted immunotherapy
successfully treated rats by providing 100% protection to the
recipients. Multiple metastases and death within 35 days of tumor
inoculation were observed in all Group A tumor-bearing control
rats. There was no protection provided by the spleen cells from a
healthy rat in Group D. A single rat out of 10 rats in Group B
survived; however, this rat later developed both primary tumor and
metastases. All Group C rats were rechallenged 60 days after the
adoptive immunity transfer, and all withstood the challenge. The
immune spleen cells of the rats in Group C were collected and mixed
with tumor cells in the same ratio as in the first adoptive
transfer to evaluate the ability of these animals' spleen cells in
protecting a subsequent cohort of normal Wistar Furth recipient
rats (n=6) that were inoculated with this admixture. The immune
cells from the Group C rats protected 5 of 6 naive rats, as neither
primary nor metastatic tumors were observed. The rat that died had
a prolonged survival time (60 versus 30 days) and a delayed tumor
emergence after inoculation (37 versus 7 to 10 days), in comparison
with the control group.
[0232] The resistance of successfully treated rats when tumor
rechallenged strongly suggests that the tumor-selective immunity
has a long-lasting effect.
Example 13
[0233] Combination of Laser-Assisted Immunotherapy and Low-Dose
Chemotherapy
[0234] In one exemplary clinical study, two breast cancer patients
received cyclophosphamide weekly (after inCVAX treatment) at a dose
of between 150 and 200 mg/m.sup.2. The patients initially responded
well to the treatment with tumor shrinkage and minimal adverse
reactions. After a few months the response slowed, so the
oncologist changed the low-dose chemotherapy to a weekly regimen of
Paclitaxel at 75 mg/m.sup.2, and again the response was very good
with shrinking tumors. No new metastases appeared. The patients
continued to receive the low dose chemotherapy. A third patient
became operable following the low dose chemotherapy, and a
mastectomy was performed, so a combination with surgery was also an
option.
* * * * *