U.S. patent application number 14/032975 was filed with the patent office on 2014-01-23 for treatment of cancer with complex organic-inorganic pigment compositions.
This patent application is currently assigned to Board of Regents, The University of Texas System. The applicant listed for this patent is Board of Regents, The University of Texas System. Invention is credited to Russell R. Chianelli, Siddhartha Das.
Application Number | 20140024617 14/032975 |
Document ID | / |
Family ID | 42172510 |
Filed Date | 2014-01-23 |
United States Patent
Application |
20140024617 |
Kind Code |
A1 |
Chianelli; Russell R. ; et
al. |
January 23, 2014 |
TREATMENT OF CANCER WITH COMPLEX ORGANIC-INORGANIC PIGMENT
COMPOSITIONS
Abstract
The present invention includes compositions and methods for
treating disorders of the skin or mucosa resulting from cancer
therapies comprising: identifying a patient receiving cancer
treatment; and administering to said patient an effective dose of a
treated organic/inorganic complex material with an organic dye
molecule or derivative thereof, in a pharmaceutically acceptable
carrier.
Inventors: |
Chianelli; Russell R.; (El
Paso, TX) ; Das; Siddhartha; (El Paso, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Board of Regents, The University of Texas System |
Austin |
TX |
US |
|
|
Assignee: |
Board of Regents, The University of
Texas System
Austin
TX
|
Family ID: |
42172510 |
Appl. No.: |
14/032975 |
Filed: |
September 20, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12618501 |
Nov 13, 2009 |
8563595 |
|
|
14032975 |
|
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|
61114811 |
Nov 14, 2008 |
|
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Current U.S.
Class: |
514/63 |
Current CPC
Class: |
A61K 31/404 20130101;
A61P 35/00 20180101; A61K 33/06 20130101; A61K 45/06 20130101; A61K
2300/00 20130101; A61K 31/404 20130101 |
Class at
Publication: |
514/63 |
International
Class: |
A61K 33/06 20060101
A61K033/06; A61K 45/06 20060101 A61K045/06 |
Claims
1. A method of treating cancer comprising contacting the cancer
with a composition comprising a therapeutically effective amount of
a treated organic/inorganic complex material comprising an organic
dye molecule or derivative thereof in an inorganic material, in a
pharmaceutically acceptable carrier.
2. The method of claim 1, wherein the effective amount is 10 to
3,000 mg/kg.
3. The method of claim 1, wherein the organic dye molecule
comprises Maya blue dye or derivatives thereof.
4. The method of claim 1, further comprising the step of
micronizing the composition.
5. The method of claim 1, further comprising the step of adapting
the composition for administration in an amount of from 10 mg/kg
body weight to 10,000 mg/kg body weight.
6. The method of claim 1, further comprising the step of adapting
the composition for administration orally, enterically,
intravenously, peritoneally, parenterally or subcutaneously.
7. The method of claim 1, wherein the cancer is at least one of
carcinoma, leukemia, melanoma, stomach cancer, colon cancer, breast
cancer, lung cancer, brain cancer, pancreatic cancer, ovarian
cancer, head and neck cancer, liver cancer, prostate cancer,
metastatic cancer or a solid tumor.
8. The method of claim 1, further comprising adding an effective
amount of a second chemotherapeutic agent to the composition.
9. A method for treating an animal having a tumor comprising
administering to the animal a therapeutically effective amount of a
treated organic/inorganic complex material with a Maya blue dye
molecule or derivatives thereof, in a pharmaceutically acceptable
carrier to induce necrosis in at least a portion of the tumor,
induces tumor regression or induces tumor remission.
10. The method of claim 9, wherein the effective amount is 10 to
3,000 mg/kg.
11. The method of claim 9, wherein the organic dye molecule
comprises Maya blue dye, or derivatives thereof in the form of a
prodrug thereof.
12. The method of claim 9, further comprising the step of
micronizing the composition.
13. The method of claim 9, further comprising the step of adapting
the composition for administration in an amount of from 10 mg/kg
body weight to 10,000 mg/kg body weight.
14. The method of claim 9, further comprising the step of adapting
the composition for administration orally, enterically,
intravenously, peritoneally, parenterally or subcutaneously.
15. The method of claim 9, wherein the tumor is a carcinoma,
leukemia, melanoma, stomach cancer, colon cancer, breast cancer,
lung cancer, brain cancer, pancreatic cancer, ovarian cancer, head
and neck cancer, liver cancer, prostate cancer, metastatic cancer
or a solid tumor.
16. The method of claim 9, further comprising adding an effective
amount of a second chemotherapeutic agent to the composition.
17. A method of treating a cancer comprising administering to a
subject in need thereof an effective amount of a composition
comprising a therapeutically effective amount of a treated
organic/inorganic complex material with an organic dye molecule
selected from Maya Blue dye or derivative thereof, in a
pharmaceutically acceptable carrier, wherein said cancer is
selected from the group consisting of a stomach, color or breast
cancer.
18. A method for treating disorders of the skin or mucosa resulting
from cancer therapies comprising: identifying a patient in need of
cancer treatment; and administering to the patient an effective
dose of an organic/inorganic complex of Maya Blue dye or derivative
thereof and an inorganic fibrous clay material in a
pharmaceutically acceptable carrier, wherein the Maya blue dye and
the inorganic fibrous clay material are bonded by heating the Maya
blue dye and the inorganic fibrous clay material or exposing them
to UV light to enhance a cancer suppression ability of the Maya
blue dye and the inorganic fibrous clay material.
19. A method of treating cancer comprising contacting the cancer
with a composition comprising a therapeutically effective amount of
a treated organic/inorganic complex material comprising a Maya blue
dye and an inorganic fibrous clay material, in a pharmaceutically
acceptable carrier, in which the Maya blue dye and the inorganic
fibrous clay material are connected by a stabilized organic and
inorganic bond formed by heating the Maya blue dye and the
inorganic fibrous clay material or exposing them to UV light such
that the molecular properties of the Maya blue dye and the
inorganic fibrous clay material are changed to enhance a cancer
suppression ability of the composition.
20. The method of claim 19, wherein the cancer is selected from at
least one of a carcinoma, leukemia, melanoma, stomach cancer, colon
cancer, breast cancer, lung cancer, brain cancer, pancreatic
cancer, ovarian cancer, head and neck cancer, liver cancer,
prostate cancer, metastatic cancer or a solid tumor.
21. A method for treating an animal having a cancer, comprising
administering to the animal a therapeutically effective amount of a
treated organic/inorganic complex material that comprises a Maya
blue dye and an inorganic fibrous clay material in a
pharmaceutically acceptable carrier to induce necrosis in at least
a portion of the cancer, to induce cancer regression or to induce
cancer remission, in which the Maya blue dye and the inorganic
fibrous clay material are connected by a stabilized organic and
inorganic bond formed by heating the Maya blue dye and the
inorganic fibrous clay material or exposing them to UV light such
that the molecular properties of the Maya blue dye and the
inorganic fibrous clay material are changed to enhance a cancer
suppression ability of the composition.
22. The method of claim 21, wherein the cancer is a carcinoma,
leukemia, melanoma, stomach cancer, colon cancer, breast cancer,
lung cancer, brain cancer, pancreatic cancer, ovarian cancer, head
and neck cancer, liver cancer, prostate cancer, metastatic cancer
or a solid tumor.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
12/618,501 filed on Nov. 13, 2009, entitled Treatment of Cancer
with Complex Organic-Inorganic Pigment Compositions, which claims
priority to U.S. Provisional Application Ser. No. 61/114,811, filed
Nov. 14, 2008, the entire contents of which is incorporated herein
by reference.
[0002] This application is related to U.S. Provisional Application
Ser. No. 60/390,049, filed Jun. 19, 2002; U.S. Ser. No. 10/370,288,
filed Feb. 18, 2003, now U.S. Pat. No. 7,052,541; U.S. Provisional
Application Ser. No. 60/652,105, filed Feb. 11, 2005; U.S.
Provisional Application Ser. No. 60/691,683, filed Jun. 17, 2005;
U.S. Ser. No. 11/351,577, filed Feb. 10, 2006, now U.S. Pat. No.
7,425,235; and U.S. Ser. No. 11/424,758, filed Jun. 16, 2006, the
contents of each herein incorporated by reference in their
entirety.
TECHNICAL FIELD OF THE INVENTION
[0003] The present invention relates in general to the field of
methods for the treatment of cancer, and more particularly, to
compositions and methods for the treatment of cancer, e.g., colon
and stomach cancers with a complex organic-inorganic pigment.
STATEMENT OF FEDERALLY FUNDED RESEARCH
[0004] None.
BACKGROUND OF THE INVENTION
[0005] Without limiting the scope of the invention, its background
is described in connection with organic/inorganic complexes.
[0006] In the scientific literature, the term Maya blue refers to a
"turquoise" brilliant shade of blue that is found on murals and
archaeological artifacts, for example, throughout Mesoamerica. It
is described in the literature as being composed of palygorskite
clay and indigo, that when mixed and heated, produce the stable
brilliant blue color similar to that found in Mesoamerica. The
ancient Mayan Indians would paint unlucky people blue, which was
associated with their rain deities, and throw them down a sacred
well as human sacrifices. The pigment is known to have the ability
to resist age, acid, weathering, and even modern chemical
solvents.
[0007] Indigo is a dye obtained from many plants, mainly those in
the genus Indigofera, which are native to the tropics. In ancient
times, the dye was obtained by soaking the plant leaves in water
and fermenting them in order to convert a "glycoside indican" into
the blue dye indigotin. Littman synthesized indigo-attapulgite
complexes and verified that his synthetic version was
indistinguishable from the original pigments found in the
pre-Hispanic murals and artifacts (Littman, 1980; Littman, 1982).
The prepared samples had the same physical and chemical
characteristics as the authentic Maya blue examined. Littman
concluded that the remarkable stability of the attapulgite was due
to the heat treatment the attapulgite received during the
synthesis. Others have also synthesized compounds similar to that
of Maya blue by a number of routes (Tones, 1988). They employed the
Gettens test to determine whether the laboratory synthesis of Maya
blue was indeed authentic with the same chemical resistant
properties (Gettens, 1962). The test was necessary because initial
attempts of simply mixing the palygorskite clay produced the color
of Maya blue but the mixture did not possess the same chemical
properties as the original organic/inorganic complex samples.
[0008] Since ancient times, Indigo has been used as an astringent,
emetic, stimulant, and antiseptic. In Mexico, the Anil (Indigofera
suffruticosa) is used for children's headaches by boiling leaves
until soft, then apply the leaves like a bandage to the forehead.
The Chinese would use Indigofera tinctoria L. to clean the liver,
detoxify the blood, reduce inflammation, alleviate pain, and reduce
fever. In South Africa, the powdered root of Indigofera was used to
alleviate toothaches.
[0009] U.S. Pat. No. 7,052,541 describes color compositions
comprising neutral indigo derivative pigments and dyes complexed to
the surface of inorganic clays. These materials are useful as
paints and coatings for artistic and industrial purposes, including
use in cements, plastics, papers and polymers. Upon grinding and
heating the organic and inorganic component as solid mixtures or in
aqueous solutions, the resulting color compositions have
unprecedented stability relative to the original starting
materials. Related U.S. Pat. No. 7,425,235 describes the use of
similar starting materials in methods that rely on UV-light for
preparing color compositions.
SUMMARY OF THE INVENTION
[0010] The present invention includes compositions and methods for
treating cancer, e.g., cancers of the stomach and colon. Generally,
the present invention includes the use of novel compositions
comprising organic/inorganic complexes for the treatment of
cancers. The organic dye or pigment can be used alone or complexed
with a support during use. As used in this invention, a complex is
an organic/inorganic hybrid composition wherein the organic
material and the inorganic material interact with each other at the
single molecule level or as molecular complexes.
[0011] In one embodiment, the present invention includes a method
of treating cancer comprising contacting the cancer with a
composition comprising a therapeutically effective amount of a
treated organic/inorganic complex material with an organic dye
molecule derivative thereof, in a pharmaceutically acceptable
carrier. In one aspect, the cancer is selected from the group
stomach, colon, and breast. In another aspect, the effective amount
is 10 to 3,000 mg/kg. In another aspect, the organic dye molecule
comprises Maya blue dye is in the form of a prodrug thereof. In
another aspect, the composition is micronized and is suitable for
administration to the warm blooded animal by injection. In another
aspect, the composition is administered in an amount of from 10
mg/kg body weight to 10,000 mg/kg body weight. In another aspect,
the composition is administered orally, enterically, intravenously,
peritoneally, parenterally or subcutaneously. In another aspect,
cancer is a carcinoma, leukemia, melanoma, stomach cancer, colon
cancer, breast cancer, lung cancer, brain cancer, pancreatic
cancer, ovarian cancer, head and neck cancer, liver cancer or
prostate cancer. In another aspect, the cancer involves metastasis
and/or a solid tumor. In another aspect, the composition may
further comprise a safe and effective amount of a second
chemotherapeutic agent.
[0012] Another embodiment of the present invention is a method for
treating an animal having a tumor of the stomach, color or breast,
comprising administering to the animal a therapeutically effective
amount of a treated organic/inorganic complex material with an
organic dye molecule or derivative thereof, in a pharmaceutically
acceptable carrier to induce necrosis in at least a portion of the
tumor, induces tumor regression or induces tumor remission. In
another aspect, the effective amount is 10 to 3,000 mg/kg. In
another aspect, the pigment comprises Maya blue dye is in the form
of a prodrug thereof. In another aspect, the composition is
micronized and is suitable for administration to the warm blooded
animal by injection. In another aspect, the composition is
administered in an amount of from 10 mg/kg body weight to 10,000
mg/kg body weight. In another aspect, the composition is
administered orally, enterically, intravenously, peritoneally,
parenterally or subcutaneously. In another aspect, cancer is a
carcinoma, leukemia, melanoma, stomach cancer, colon cancer, breast
cancer, lung cancer, brain cancer, pancreatic cancer, ovarian
cancer, head and neck cancer, liver cancer or prostate cancer. In
another aspect, the cancer involves metastasis and/or a solid
tumor. In another aspect, the composition may further comprise a
safe and effective amount of a second chemotherapeutic agent.
[0013] Another embodiment of the present invention includes a
method of treating a cancer comprising administering to a subject
in need thereof an effective amount of a composition comprising a
therapeutically effective amount of a treated organic/inorganic
complex material with an organic dye molecule selected from Maya
Blue or indigo dye or a derivative thereof, in a pharmaceutically
acceptable carrier, wherein said cancer is selected from the group
consisting of a stomach, color or breast cancer. In another aspect,
the effective amount is 10 to 3,000 mg/kg. In another aspect, the
pigment comprises Maya blue dye is in the form of a prodrug
thereof. In another aspect, the composition is micronized and is
suitable for administration to the warm blooded animal by
injection. In another aspect, the composition is administered in an
amount of from 10 mg/kg body weight to 10,000 mg/kg body weight. In
another aspect, the composition is administered orally,
enterically, intravenously, peritoneally, parenterally or
subcutaneously. In another aspect, cancer is a carcinoma, leukemia,
melanoma, stomach cancer, colon cancer, breast cancer, lung cancer,
brain cancer, pancreatic cancer, ovarian cancer, head and neck
cancer, liver cancer or prostate cancer. In another aspect, the
cancer involves metastasis and/or a solid tumor. In another aspect,
the composition may further comprise a safe and effective amount of
a second chemotherapeutic agent.
[0014] Yet another embodiment of the present invention is a method
for treating disorders of the skin or mucosa resulting from cancer
therapies comprising: identifying a patient receiving cancer
treatment; and administering to the patient an effective dose of a
treated organic/inorganic complex material with an organic dye
molecule or derivative thereof, in a pharmaceutically acceptable
carrier. In another aspect, the effective amount is 10 to 3,000
mg/kg. In another aspect, the pigment comprises Maya blue dye is in
the form of a prodrug thereof.
[0015] In another aspect, the composition is micronized and is
suitable for administration to the warm blooded animal by
injection. In another aspect, the composition is administered in an
amount of from 10 mg/kg body weight to 10,000 mg/kg body weight. In
another aspect, the composition is administered orally,
enterically, intravenously, peritoneally, parenterally or
subcutaneously. In another aspect, cancer is a carcinoma, leukemia,
melanoma, stomach cancer, colon cancer, breast cancer, lung cancer,
brain cancer, pancreatic cancer, ovarian cancer, head and neck
cancer, liver cancer or prostate cancer. In another aspect, the
cancer involves metastasis and/or a solid tumor. In another aspect,
the composition may further comprise a safe and effective amount of
a second chemotherapeutic agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] For a more complete understanding of the features and
advantages of the present invention, reference is now made to the
detailed description of the invention along with the accompanying
figures and in which:
[0017] FIG. 1 shows HT-29 cells treated with H.sub.2O.sub.2.
[0018] FIG. 2 shows HT-29 cells treated with 5 uL of an
Organic/Inorganic Complex Materials with Maya Blue (suspended in
water).
[0019] FIG. 3 shows HT-29 cells treated with 10 uL of an
Organic/Inorganic Complex Materials with Maya Blue (suspended in
water).
[0020] FIG. 4 shows HT-29 cells treated with 30 uL of an
Organic/Inorganic Complex Materials with Maya Blue (suspended in
water).
[0021] FIG. 5 shows HT-29 cells treated with 40 uL of an
Organic/Inorganic Complex Materials with Maya Blue (suspended in
water).
[0022] FIG. 6 shows HT-29 cells treated with 50 uL of an
Organic/Inorganic Complex Materials with Maya Blue (suspended in
water).
[0023] FIG. 7 shows HT-29 cells treated with Indigo (suspended in
water).
[0024] FIG. 8 shows HT-29 cells treated with clay.
[0025] FIG. 9 shows NIH-3T3 cells treated with H.sub.2O.sub.2.
[0026] FIG. 10 shows NIH-3T3 cells treated with Indigo (suspended
in water).
[0027] FIG. 11 shows NIH-3T3 cells treated with different
concentrations of Organic/Inorganic Complex Materials Mayan Blue
(suspended in water).
[0028] FIG. 12 shows MCF-7 cells treated with H.sub.2O.sub.2.
[0029] FIG. 13 shows MCF-7 cells treated with Indigo (suspended in
water).
[0030] FIG. 14 shows MCF-7 cells treated with 5 uL of an
Organic/Inorganic Complex Materials with Maya Blue (suspended in
water).
[0031] FIG. 15 shows MCF-7 cells treated with 10 uL of an
Organic/Inorganic Complex Materials with Maya Blue (suspended in
water).
[0032] FIG. 16 shows MCF-7 cells treated with 25 uL of an
Organic/Inorganic Complex Materials with Maya Blue (suspended in
water).
[0033] FIG. 17 shows MCF-7 cells treated with 50 uL of an
Organic/Inorganic Complex Materials with Maya Blue (suspended in
water).
[0034] FIG. 18 shows MCF-7 cells treated with clay.
[0035] FIG. 19 is a 1.5% agarose gel of cells treated with controls
and Mayan Blue (suspended in water) after 4 hours.
[0036] FIG. 20 is a 1.5% agarose gel of cells treated with controls
and an Organic/Inorganic Complex Materials with Maya Blue
(suspended in water) after 24 hours.
[0037] FIG. 21 is a Fluorescence Activated Cell Sorter (FACS) graph
of Propidium iodide and Annexin V of control cells.
[0038] FIG. 22 is a Fluorescence Activated Cell Sorter (FACS) graph
of Propidium iodide and Annexin V of cells treated with
H.sub.2O.sub.2.
[0039] FIG. 23 is a Fluorescence Activated Cell Sorter (FACS) graph
of Propidium iodide and Annexin V of cells treated with Clay
alone.
[0040] FIG. 24 is a Fluorescence Activated Cell Sorter (FACS) graph
of Propidium iodide and Annexin V of cells treated with Indigo.
[0041] FIG. 25 is a Fluorescence Activated Cell Sorter (FACS) graph
of Propidium iodide and Annexin V of cells treated with an
Organic/Inorganic Complex Materials with Maya Blue at 2.5 uL.
[0042] FIG. 26 is a Fluorescence Activated Cell Sorter (FACS) graph
of Propidium iodide and Annexin V of cells treated with an
Organic/Inorganic Complex Materials with Maya Blue at 12.5 uL.
[0043] FIG. 27 is a graph that shows the relative effect of various
conditions on cell viability.
DETAILED DESCRIPTION OF THE INVENTION
[0044] While the making and using of various embodiments of the
present invention are discussed in detail below, it should be
appreciated that the present invention provides many applicable
inventive concepts that can be embodied in a wide variety of
specific contexts. The specific embodiments discussed herein are
merely illustrative of specific ways to make and use the invention
and do not delimit the scope of the invention.
[0045] To facilitate the understanding of this invention, a number
of terms are defined below. Terms defined herein have meanings as
commonly understood by a person of ordinary skill in the areas
relevant to the present invention. Terms such as "a", "an" and
"the" are not intended to refer to only a singular entity, but
include the general class of which a specific example may be used
for illustration. The terminology herein is used to describe
specific embodiments of the invention, but their usage does not
delimit the invention, except as outlined in the claims.
[0046] The present invention includes a method for treating cancer
cells that uses a composition of matter comprising organic pigments
and/or dyes complexed with a variety of solid supports based on the
Organic/Inorganic Complex Materials with Maya Blue and derivatives
thereof. Various Organic/Inorganic Complex Materials with Maya Blue
and derivatives thereof may be used with the present invention,
including those taught in U.S. Pat. No. 7,052,541, relevant
portions incorporated herein by reference. Various methods for
making these compositions also are provided and include blending,
grinding, heating, and/or treating with light, such as ultraviolet
(UV) light or microwave. Alteration of properties during synthesis,
such as pH, particle size and support type, will change the color
of the final composition. Because of the unique chemical nature of
the resulting product, the compositions remain stable over time and
under a variety of environmental conditions.
[0047] As used herein, the term "therapeutically effective amount"
refers to an amount of an Organic/Inorganic Complex Materials with
Maya Blue or indigo, or derivatives thereof, effective to yield the
desired therapeutic response. For example, an amount effective to
delay the growth of or to cause a cancer, either a sarcoma or
lymphoma, to shrink or not metastasize. The specific safe and
effective amount or therapeutically effective amount will vary with
such factors as the particular condition being treated, the
physical condition of the patient, the type of mammal being
treated, the duration of the treatment, the nature of concurrent
therapy (if any), and the specific formulations employed and the
structure of the compounds or its derivatives.
[0048] As used herein, a "pharmaceutically acceptable" refers to a
component that is suitable for use with humans and/or animals
without undue adverse side effects (such as toxicity, irritation,
and allergic response) commensurate with a reasonable benefit/risk
ratio.
[0049] As used herein, the term "safe and effective amount" refers
to the quantity of a component that is sufficient to yield a
desired therapeutic response without undue adverse side effects
(such as toxicity, irritation, or allergic response) commensurate
with a reasonable benefit/risk ratio when used in the manner of
this invention.
[0050] As used herein, a "pharmaceutical salts" refers to a salt
for making an acid or base salt of a compound. Examples of
pharmaceutically acceptable salts include, but are not limited to,
mineral or organic acid salts of basic residues such as amines;
alkali or organic salts of acidic residues such as phenols.
Preferably the salts are made using an organic or inorganic acid.
These preferred acid salts are chlorides, bromides, sulfates,
nitrates, phosphates, sulfonates, formates, tartrates, maleates,
malates, citrates, benzoates, salicylates, ascorbates, and the
like. The preferred phenolate salts are the alkaline earth metal
salts, sodium, potassium or lithium.
[0051] As used herein, a "pharmaceutical carrier" refers to a
pharmaceutically acceptable solvent, suspending agent or vehicle,
for delivering the Maya Blue dye compound to the animal or human.
The carrier may be liquid or solid and is selected with the planned
manner of administration in mind. Liposomes are also a
pharmaceutical carrier.
[0052] As used herein, "cancer" refers to all types of cancer or
neoplasm or malignant tumors found in mammals, including carcinomas
and sarcomas. Non-limiting examples of cancers are cancer of the
brain, breast, cervix, colon, head & neck, kidney, lung,
non-small cell lung, melanoma, mesothelioma, ovary, sarcoma,
stomach, uterus and medulloblastoma.
[0053] As used herein, the phrase "Organic/Inorganic Complex
Materials (OICM)" refers to a combination of organic dye molecules
(e.g., indigo and its derivatives) and an inorganic material such
as palygorskite clay. When the OICM are stabilized using, e.g.,
heat or exposure to UV light, the organic and inorganic bond is
stabilized and both the dye and the clay become more stable. The
treatment changes the molecular configuration of the inorganic
molecule as seen by a color change upon forming the OICM, thereby
changing the properties of the OICM from the two original organic
and inorganic molecules. The treatment also results in properties
that are superior or different from the original organic molecule,
e.g., cancer suppression with indigo based OICM that is larger than
with the original indigo molecule alone. Also, the inorganic part
may be different from palygorskite that is a fibrous clay. For
example, Sepiolite may be used as a fibrous clay, as are layered
(Montmorilanite, etc.), Zeolites and other silica alumina
materials.
[0054] Dosages. A dosage unit for use of the Organic/Inorganic
Complex Materials of the present invention, may be a single
compound or mixtures thereof with other compounds, e.g., a
potentiator. The compounds may be mixed together, form ionic or
even covalent bonds. The Maya Blue dye of the present invention may
be administered in oral, intravenous (bolus or infusion),
intraperitoneal, subcutaneous, or intramuscular form, all using
dosage forms well known to those of ordinary skill in the
pharmaceutical arts. Depending on the particular location or method
of delivery, different dosage forms, e.g., tablets, capsules,
pills, powders, granules, elixirs, tinctures, suspensions, syrups,
and emulsions may be used to provide the Maya Blue dye of the
present invention to a patient in need of therapy that has or is
suspected of having a cancer. The Maya Blue dye may also be
administered as any one of known salt forms.
[0055] The Organic/Inorganic Complex Materials of the present
invention may be administered in admixture with suitable
pharmaceutical salts, buffers, diluents, extenders, excipients
and/or carriers (collectively referred to herein as a
pharmaceutically acceptable carrier or carrier materials) selected
based on the intended form of administration and as consistent with
conventional pharmaceutical practices. Depending on the best
location for administration, the Organic/Inorganic Complex
Materials may be formulated to provide, e.g., maximum and/or
consistent dosing for the particular form for oral, rectal,
topical, intravenous injection or parenteral administration. While
the Organic/Inorganic Complex Materials may be administered alone,
it will generally be provided in a stable salt form mixed with a
pharmaceutically acceptable carrier. The carrier may be solid or
liquid, depending on the type and/or location of administration
selected.
[0056] Techniques and compositions for making useful dosage forms
using the present invention are described in one or more of the
following references: Ansel, Introduction to Pharmaceutical Dosage
Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th
ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in
Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds.,
1992); Advances in Pharmaceutical Sciences Vol 7. (David Ganderton,
Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric
Coatings for Pharmaceutical Dosage Forms (Drugs and the
Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989);
Pharmaceutical Particulate Carriers: Therapeutic Applications:
Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed.,
1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood
Books in the Biological Sciences. Series in Pharmaceutical
Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.);
Modern Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40
(Gilbert S. Banker, Christopher T. Rhodes, Eds.), and the like,
relevant portions incorporated herein by reference.
[0057] For example, the Organic/Inorganic Complex Materials may be
included in a tablet. Tablets may contain, e.g., suitable binders,
lubricants, disintegrating agents, coloring agents, flavoring
agents, flow-inducing agents and/or melting agents. For example,
oral administration may be in a dosage unit form of a tablet,
gelcap, caplet or capsule, the active drug component being combined
with an non-toxic, pharmaceutically acceptable, inert carrier such
as lactose, gelatin, agar, starch, sucrose, glucose, methyl
cellulose, magnesium stearate, dicalcium phosphate, calcium
sulfate, mannitol, sorbitol, mixtures thereof, and the like.
Suitable binders for use with the present invention include:
starch, gelatin, natural sugars (e.g., glucose or beta-lactose),
corn sweeteners, natural and synthetic gums (e.g., acacia,
tragacanth or sodium alginate), carboxymethylcellulose,
polyethylene glycol, waxes, and the like. Lubricants for use with
the invention may include: sodium oleate, sodium stearate,
magnesium stearate, sodium benzoate, sodium acetate, sodium
chloride, mixtures thereof, and the like. Disintegrators may
include: starch, methyl cellulose, agar, bentonite, xanthan gum,
mixtures thereof, and the like.
[0058] Organic/Inorganic Complex Materials may be administered in
the form of liposome delivery systems, e.g., small unilamellar
vesicles, large unilamallar vesicles, and multilamellar vesicles,
whether charged or uncharged. Liposomes may include one or more:
phospholipids (e.g., cholesterol), stearylamine and/or
phosphatidylcholines, mixtures thereof, and the like.
[0059] Organic/Inorganic Complex Materials may also be coupled to
one or more soluble, biodegradable, bioacceptable polymers as drug
carriers or as a prodrug. Such polymers may include:
polyvinylpyrrolidone, pyran copolymer,
polyhydroxylpropylmethacrylamide-phenol,
polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysine
substituted with palmitoyl residues, mixtures thereof, and the
like. Furthermore, the Organic/Inorganic Complex Materials may be
coupled one or more biodegradable polymers to achieve controlled
release of the Organic/Inorganic Complex Materials, biodegradable
polymers for use with the present invention include: polylactic
acid, polyglycolic acid, copolymers of polylactic and polyglycolic
acid, polyepsilon caprolactone, polyhydroxy butyric acid,
polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates,
and crosslinked or amphipathic block copolymers of hydrogels,
mixtures thereof, and the like.
[0060] In one embodiment, gelatin capsules (gelcaps) may include
the Organic/Inorganic Complex Materials and powdered carriers, such
as lactose, starch, cellulose derivatives, magnesium stearate,
stearic acid, and the like. Like diluents may be used to make
compressed tablets. Both tablets and capsules may be manufactured
as immediate-release, mixed-release or sustained-release
formulations to provide for a range of release of medication over a
period of minutes to hours. Compressed tablets may be sugar coated
or film coated to mask any unpleasant taste and protect the tablet
from the atmosphere. An enteric coating may be used to provide
selective disintegration in, e.g., the gastrointestinal tract.
[0061] For oral administration in a liquid dosage form, the oral
drug components may be combined with any oral, non-toxic,
pharmaceutically acceptable inert carrier such as ethanol,
glycerol, water, and the like. Examples of suitable liquid dosage
forms include solutions or suspensions in water, pharmaceutically
acceptable fats and oils, alcohols or other organic solvents,
including esters, emulsions, syrups or elixirs, suspensions,
solutions and/or suspensions reconstituted from non-effervescent
granules and effervescent preparations reconstituted from
effervescent granules. Such liquid dosage forms may contain, for
example, suitable solvents, preservatives, emulsifying agents,
suspending agents, diluents, sweeteners, thickeners, and melting
agents, mixtures thereof, and the like.
[0062] Liquid dosage forms for oral administration may also include
coloring and flavoring agents that increase patient acceptance and
therefore compliance with a dosing regimen. In general, water, a
suitable oil, saline, aqueous dextrose (e.g., glucose, lactose and
related sugar solutions) and glycols (e.g., propylene glycol or
polyethylene glycols) may be used as suitable carriers for
parenteral solutions. Solutions for parenteral administration
include generally, a water soluble salt of the active ingredient,
suitable stabilizing agents, and if necessary, buffering salts.
Antioxidizing agents such as sodium bisulfite, sodium sulfite
and/or ascorbic acid, either alone or in combination, are suitable
stabilizing agents. Citric acid and its salts and sodium EDTA may
also be included to increase stability. In addition, parenteral
solutions may include pharmaceutically acceptable preservatives,
e.g., benzalkonium chloride, methyl- or propyl-paraben, and/or
chlorobutanol. Suitable pharmaceutical carriers are described in
Remington's Pharmaceutical Sciences, Mack Publishing Company, a
standard reference text in this field, relevant portions
incorporated herein by reference.
[0063] For direct delivery to the nasal passages, sinuses, mouth,
throat, esophagous, tachea, lungs and alveoli, the
Organic/Inorganic Complex Materials may also be delivered as an
intranasal form via use of a suitable intranasal vehicle. For
dermal and transdermal delivery, the Organic/Inorganic Complex
Materials may be delivered using lotions, creams, oils, elixirs,
serums, transdermal skin patches and the like, as are well known to
those of ordinary skill in that art. Parenteral and intravenous
forms may also include pharmaceutically acceptable salts and/or
minerals and other materials to make them compatible with the type
of injection or delivery system chosen, e.g., a buffered, isotonic
solution. Examples of useful pharmaceutical dosage forms for
administration of Organic/Inorganic Complex Materials may include
the following forms.
[0064] Capsules. Capsules may be prepared by filling standard
two-piece hard gelatin capsules each with 10 to 500 milligrams of
powdered active ingredient, 5 to 150 milligrams of lactose, 5 to 50
milligrams of cellulose and 6 milligrams magnesium stearate and the
Organic/Inorganic Complex Materials.
[0065] Soft Gelatin Capsules. A mixture of Organic/Inorganic
Complex Materials is dissolved in a digestible oil such as soybean
oil, cottonseed oil or olive oil. The active ingredient is prepared
and injected by using a positive displacement pump into gelatin to
form soft gelatin capsules containing, e.g., 100-500 milligrams of
the active ingredient. The capsules are washed and dried.
[0066] Tablets. A large number of tablets are prepared by
conventional procedures so that the dosage unit was 100-500
milligrams of Organic/Inorganic Complex Materials, 0.2 milligrams
of colloidal silicon dioxide, 5 milligrams of magnesium stearate,
50-275 milligrams of microcrystalline cellulose, 11 milligrams of
starch and 98.8 milligrams of lactose. Appropriate coatings may be
applied to increase palatability or delay absorption.
[0067] To provide an effervescent tablet appropriate amounts of,
e.g., monosodium citrate and sodium bicarbonate, are blended
together and then roller compacted, in the absence of water, to
form flakes that are then crushed to give granulates. The
granulates are then combined with the Organic/Inorganic Complex
Materials and/or salt thereof, conventional beading or filling
agents and, optionally, sweeteners, flavors and lubricants.
[0068] Injectable solution. A parenteral composition suitable for
administration by injection is prepared by stirring 1% to 10% by
weight of Organic/Inorganic Complex Materials in deionized water
and mixed with, e.g., up to 10% by volume propylene glycol and
water. The solution is made isotonic with sodium chloride and
sterilized using, e.g., ultrafiltration.
[0069] Suspension. An aqueous suspension of Organic/Inorganic
Complex Materials is prepared for oral administration so that each
5 ml contain 100 mg of finely divided active ingredient, 200 mg of
sodium carboxymethyl cellulose, 5 mg of sodium benzoate, 1.0 g of
sorbitol solution, U.S.P., and 0.025 ml of vanillin.
[0070] For mini-tablets, the Organic/Inorganic Complex Materials is
compressed into a hardness in the range 6 to 12 Kp. The hardness of
the final tablets is influenced by the linear roller compaction
strength used in preparing the granulates, which are influenced by
the particle size of, e.g., the monosodium hydrogen carbonate and
sodium hydrogen carbonate. For smaller particle sizes, a linear
roller compaction strength of about 15 to 20 KN/cm may be used.
[0071] Kits. The present invention also includes pharmaceutical
kits useful, for example, for the treatment of cancer, which
comprise one or more containers containing a pharmaceutical
composition comprising a therapeutically effective amount of
Organic/Inorganic Complex Materials. Such kits may further include,
if desired, one or more of various conventional pharmaceutical kit
components, such as, for example, containers with one or more
pharmaceutically acceptable carriers, additional containers, etc.,
as will be readily apparent to those skilled in the art. Printed
instructions, either as inserts or as labels, indicating quantities
of the components to be administered, guidelines for
administration, and/or guidelines for mixing the components, may
also be included in the kit. It should be understood that although
the specified materials and conditions are important in practicing
the invention, unspecified materials and conditions are not
excluded so long as they do not prevent the benefits of the
invention from being realized.
[0072] The Organic/Inorganic Complex Materials and other
chemotherapeutic agents, e.g., cancer treatment potentiators, are
typically mixed with a pharmaceutically acceptable carrier. This
carrier can be a solid or liquid and the type is generally chosen
based on the type of administration being used. The active agent
can be coadministered in the form of a tablet or capsule, liposome,
as an agglomerated powder or in a liquid form. Examples of suitable
solid carriers include lactose, sucrose, gelatin and agar. Capsule
or tablets can be easily formulated and can be made easy to swallow
or chew; other solid forms include granules, and bulk powders.
Tablets may contain suitable binders, lubricants, diluents,
disintegrating agents, coloring agents, flavoring agents,
flow-inducing agents, and melting agents. Examples of suitable
liquid dosage forms include solutions or suspensions in water,
pharmaceutically acceptable fats and oils, alcohols or other
organic solvents, including esters, emulsions, syrups or elixirs,
suspensions, solutions and/or suspensions reconstituted from
non-effervescent granules and effervescent preparations
reconstituted from effervescent granules. Such liquid dosage forms
may contain, for example, suitable solvents, preservatives,
emulsifying agents, suspending agents, diluents, sweeteners,
thickeners, and melting agents. Oral dosage forms optionally
contain flavorants and coloring agents. Parenteral and intravenous
forms may also include minerals and other materials to make them
compatible with the type of injection or delivery system
chosen.
[0073] As used herein, the term "potentiator" refers to a compound
that accentuates or potentiates a cytotoxic activity upon a cell,
e.g., a cell that is targeted using the Organic/Inorganic Complex
Materials. The potentiator many be a procodazole, triprolidine,
propionic acid, monensin, an anti-sense inhibitor of the RAD51
gene, bromodeoxyuridine, dipyridamole, indomethacin, a monoclonal
antibody, an anti-transferrin receptor immunotoxin, metoclopramide,
7-thia-8-oxoguanosine,
N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl)ethylenediamine,
N-[4[(4-fluorphenyl)sulfonyl]phenyl]acetamide, leucovorin, heparin,
heparin sulfate, cimetidine, a radiosensitizer, a chemosensitizer,
a hypoxic cell cytotoxic agent, muramyl dipeptide, vitamin A,
2'-deoxycoformycin, a bis-diketopiperazine derivative having
potentiator activity, dimethyl sulfoxide or mixtures thereof. The
potentiator may be added in conjunction with the Organic/Inorganic
Complex Materials. The potentiator may be added before, during or
after a dose of Organic/Inorganic Complex Materials and may even be
conjugated directly with the Organic/Inorganic Complex Materials,
either covalently or ionically. In one example, the
Organic/Inorganic Complex Materials and the potentiator are mixed
with a biodegradable resin or matrix that releases the
Organic/Inorganic Complex Materials and the potentiator at the same
or different rates, at the same or disparate times and combinations
thereof.
[0074] Dyes. As discussed above, the selection of Organic/Inorganic
Complex Materials is based on its ability to kill cancer cells.
Thus, in one embodiment, the dye/pigment may be represented by
Formula I:
##STR00001##
[0075] Supports. Compositions of the present invention may also
include a support. Though the supports may take a variety of
different forms, one feature of the present invention is that the
dyes and pigments undergo a chemical or atomic interaction with the
support to form a complex. The complex formed is an
organic/inorganic hybrid composition. The interactions include
coordinate covalent bonding, covalent bonding and/or hydrogen
bonding.
[0076] Clays. As used herein, the term "clay" refers to fibrous
clays. The fibrous clay is preferentially a palygorskite clay, a
sepiolite clay, or a mixture of palygorskite and sepiolite clays.
The mixture may be in any ratio. For example, it may be 50%
palygorskite and 50% sepiolite or it may be 10%/90%, 20%/80%,
30%/70%, 40%/60%, 60%/40%, 70%/30%, 80%/20%, or 90%/10%
(palygorskite/sepiolite). As used herein the term palygorskite and
attapulgite are used interchangeably to refer to the same type of
clay.
[0077] Previous work has shown that synthesis of comparable
organic/inorganic complex paints using clays with plate-like
structures, including kaolinite, bentonite, nontronite, and
mordenite all produce a blue colored pigment, but without the
stability of the indigo derivative/palygorskite complex (Olphen,
1966a; Olphen, 1966b). This indicates that the possible stability
of the Maya blue pigment is due to the fiber-like structure of the
clays used since the use of plate-like clays had not been shown to
yield a stable pigment (Olphen, 1966a; Olphen, 1966b; Littman,
1980; Littman, 1982).
[0078] The particle size of the clay may be varied. It is
preferentially between about 0.01 .mu.m and about 40 .mu.m, about
0.05 .mu.m and about 10 .mu.m, or more preferably between about 0.1
.mu.m and about 4 .mu.m. As the color changes with the size of the
particles, varying the particle size allows for greater control of
color.
[0079] Polymers, Binding Agents and Modifiers. In addition to the
supports described above, one or more binding agents or modifiers
may be added to the compositions to increase stability, uniformity,
spreadability, adhesion, coating thickness, etc. Binding agents and
modifiers are well known in the art of paint formulation and may be
included in the composition. Binding agents such as
solvent-containing binding agents (acryl, cyclized rubber, butyl
rubber, hydrocarbon resin, .alpha.-methylstyrene-acrylonitrile
copolymers, polyester imide, acryl acid butyl esters, polyacrylic
acid esters, polyurethanes, aliphatic polyurethanes and chloro
sulphonated polyethylene), may be added to the composition.
[0080] General Methods for Producing Compositions. The general
method for producing compositions according to certain embodiments
of the present invention comprises providing a dye or pigment as
described herein. The amount of dye or pigment used can be in the
range of about, at least about, or at most about 0.01%, 0.02%,
0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%,
0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%,
6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 11.0%, 12.0%, 13.0%, 14.0%, 15.0%,
16.0%, 17.0%, 18.0%, 19.0%, 20.0%, 21%, 22%, 23%, 24% or 25% by
weight based on the total weight of the composition or more
preferably about 0.1% to about 25% by weight based on the total
weight of the composition. The amount of dye or pigment used can be
in the range of about 0.01% to about 25% by weight or more
preferably about 0.1% to about 25% by weight based on the total
weight of the composition. In certain embodiments of the invention
the amount of dye or pigment is at least 8% or more by weight based
on the total weight of the composition.
[0081] The next step comprises combining the dye/pigment with a
support, such as a clay, as described herein. When combining with a
support, this next step optionally comprises the grinding or mixing
of the dye or pigment with the support, for example, in a blender,
industrial blender, industrial mixer, shear blender, or a precise
solid state blender. The support and the dye/pigment may be ground
separately and then ground together or they may be combined and
ground to both mix the two components in order to obtain the
preferred ratio. Techniques for grinding and blending the
dye/pigment and support compositions are found in Mixing of Solids
(Weinekotter and Gericke, 2000), Powder and Bulk Solids Handling
Processes (Iinoya et al., 1988), or Bulk Solids Mixing (Gyenis and
Gyenis, 1999).
[0082] The support-containing mixture should be ground to obtain
particles of between about 0.005 .mu.m and about 50 .mu.m. In
certain embodiments the range of particle sizes is between about
0.01 .mu.m and about 20 .mu.m. In certain embodiments the range of
particle sizes is between about 0.05 .mu.m and about 10 .mu.m. In
certain embodiments the range of particle sizes is between about
0.1 .mu.m and about 8 .mu.m. A range of particles sizes is
expected, but in certain embodiments over 60% of the particles are
within the desired range of particle sizes. In certain embodiments
over 80% of the particles are within the desired range of particle
sizes. In certain embodiments over 90% of the particles are within
the desired range of particle sizes. In certain embodiments over
95% of the particles are within the desired range of particle
sizes. In certain embodiments over 99% of the particles of the
particles are within the desired range of particle sizes.
[0083] The next step may comprise heating the composition of
ingredients. The heating may comprise heating at a temperature of
about 170.degree. C. to about 300.degree. C. The heating may
comprise heating at a temperature of about 100.degree. C.,
110.degree. C., 115.degree. C., 120.degree. C., 125.degree. C.,
130.degree. C., 135.degree. C., 140.degree. C., 145.degree. C.,
150.degree. C., 155.degree. C., 160.degree. C., 165.degree. C.,
170.degree. C., 175.degree. C., 180.degree. C., 185.degree. C.,
190.degree. C., 195.degree. C., 200.degree. C., 205.degree. C.,
210.degree. C., 215.degree. C., 220.degree. C., 225.degree. C.,
230.degree. C., 235.degree. C., 240.degree. C., 245.degree. C.,
250.degree. C., 255.degree. C., 260.degree. C., 265.degree. C.,
270.degree. C., 275.degree. C., 280.degree. C., 285.degree. C.,
290.degree. C., 295.degree. C. or 300.degree. C., or more. In
certain embodiments, the composition is heated to a temperature
between about 115.degree. C. and about 200.degree. C. The heating
may be for several hours, or may last up to one day. The heating
can be carried out in, but not limited to, a batch oven, a drying
oven, an infrared oven, or a powder coating oven.
[0084] An alternative to heating comprises treating the composition
with radiation, including ultraviolet. Light radiation in the range
from about 10 nm to about 500 nm can be used in certain embodiments
of the present invention, particularly about 200-400 nm (i.e., near
UV). Treatment times will vary from very brief--as short as one
minute--to several hours (e.g., about 1-48 hours, such as about, at
least about, or at most about 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12,
18, 24, 36, 48 or more hours). In certain embodiments the radiation
treatment can range from about 1 to about 48 hours. Suitable
devices for providing UV exposure exist including chambers and
reactor vessels.
[0085] Next, the pH of the composition may be adjusted to an acidic
or neutral pH, depending on the final color desired. Exemplary
acids that may be used to adjust the pH comprise: any protonic
acid, H.sub.2SO.sub.4, HClO.sub.4, HClO.sub.3, H.sub.3PO.sub.4,
HNO.sub.3, HCN, HF, HBr, HI, H.sub.3O.sup.+, or CH.sub.3COOH, or
more preferably HCl. Exemplary bases that may be used to adjust the
pH comprise: LiOH, NaOH, KOH, RbOH, CsOH, Ca(OH).sub.2,
Sr(OH).sub.2, Ba(OH).sub.2 or more preferably NaOH. The pH of the
composition can range from about 1-12, including about 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, or 12. The pH of the system can be monitored
with a pH meter that is calibrated with buffers that range from a
pH of about 1-12, including about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, or 12.
[0086] Additional steps in making a final composition may comprise:
treating the composition with acid such as but not limited to any
protonic acid, H.sub.2SO.sub.4, HClO.sub.4, HClO.sub.3,
H.sub.3PO.sub.4, HNO.sub.3, HCN, HF, HBr, HI, H.sub.3O.sup.+, or
CH.sub.3COOH, or more preferably HCl, to remove impurities from the
support; applying the composition to a surface; blending the
composition with a polymer, plastic or organic binder as discussed
in Encyclopedia of Polymer Science and Engineering, 2nd ed.
(Herman, 1990) and Paint and Surface Coatings: Theory and Practice,
2.sup.nd ed. (Lambourne and Strivens, 1999).
[0087] Certain embodiments of the present invention may comprise
hydrogen and/or coordinate covalent bonding prior to any mixing,
blending, heating, irradiation, and/or pH alteration. Compositions
may form hydrogen and/or coordinate covalent bonds at any point
during combining, mixing, blending, heating, irradiation and/or pH
alteration.
[0088] The following examples are included to demonstrate certain
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well. However, those of skill in the art should, in light
of the present disclosure, appreciate that many changes can be made
in the specific embodiments which are disclosed and still obtain a
like or similar result without departing from the spirit and scope
of the invention.
Example 1
Killing of Cancer Cells with Maya Blue
[0089] An Organic/Inorganic Complex Pigment was used to test the
effect of the Organic/Inorganic Complex Materials with Maya Blue
dye or Ingido dye was used to test for killing of various cancer
cell types. Three different cancer cells were targeted for killing,
a human colon cancer cell line, a breast cancer cell line and a
lung cancer cell line.
[0090] Briefly, an Organic/Inorganic Complex Materials with Maya
Blue was used against the following cancer cell lines: HT-29: Human
colon adenocarcinoma; MCF-7: Breast Cancer; and CRL: Lung Cancer. A
positive control for killing of the cancer cells was
H.sub.2O.sub.2. The vehicle control used was H.sub.2O and tests
were performed in a 96-well plate. For the determination of cell
viability, MTS dye (Promega, Wis.) was used and tested using a
spectrophotometer.
[0091] The Organic/Inorganic Complex Materials with Maya Blue was
made into a slurry at 10 mg/mL of water. The cells, 10,000
cells/well, were plated in the 96 well plate and grow overnight in
2004 of media; keep one lane without cells, only media and that is
control for the spectrophotometer. The following day, various
concentrations of Organic/Inorganic Complex Materials with Maya
Blue were added (at 5 .mu.L, 10 .mu.L, 30 .mu.L, 40 .mu.L, 50 .mu.L
along) with the vehicle and positive controls, followed by the MTS
assay. Briefly, 12 .mu.L of MTS solution were added to each well
and incubated for 40-45 minutes (at 37.degree. C.), or until the
color changes to brown in the control. The 96-well plate was reader
at 495 nm.
[0092] FIG. 1 shows HT-29 cells treated with the positive control
for cell killing: H.sub.2O.sub.2. FIG. 2 shows HT-29 cells treated
with different concentrations of Mayan Blue (suspended in water).
FIG. 3 shows HT-29 cells treated with different concentrations of
Mayan Blue (suspended in water). FIG. 4 shows HT-29 cells treated
with different concentrations of Mayan Blue (suspended in water).
FIG. 5 shows HT-29 cells treated with different concentrations of
Mayan Blue (suspended in water). FIG. 6 shows HT-29 cells treated
with different concentrations of Mayan Blue (suspended in water).
FIG. 7 shows HT-29 cells treated with Indigo (suspended in water).
FIG. 8 shows HT-29 cells treated with clay.
[0093] FIG. 9 shows NIH-3T3 cells treated with H.sub.2O.sub.2. FIG.
10 shows NIH-3T3 cells treated with Indigo (suspended in water).
FIG. 11 shows NIH-3T3 cells treated with different concentrations
of Organic/Inorganic Complex Materials with Maya Blue (suspended in
water). FIG. 12 shows MCF-7 cells treated with H.sub.2O.sub.2. FIG.
13 shows MCF-7 cells treated with Indigo (suspended in water). FIG.
14 shows MCF-7 cells treated with 5 uL of Mayan Blue (suspended in
water). FIG. 15 shows MCF-7 cells treated with 10 uL of Mayan Blue
(suspended in water). FIG. 16 shows MCF-7 cells treated with 25 uL
of Mayan Blue (suspended in water). FIG. 17 shows MCF-7 cells
treated with 50 uL of Mayan Blue (suspended in water). FIG. 18
shows MCF-7 cells treated with clay. FIG. 19 is a 1.5% agarose gel
of cells treated with controls and Mayan Blue (suspended in water)
after 4 hours. FIG. 20 is a 1.5% agarose gel of cells treated with
controls and Mayan Blue (suspended in water) after 24 hours.
[0094] FIG. 21 is a Fluorescence Activated Cell Sorter (FACS) graph
of Propidium iodide and Annexin V of control cells. FIG. 22 is a
Fluorescence Activated Cell Sorter (FACS) graph of Propidium iodide
and Annexin V of cells treated with H.sub.2O.sub.2. FIG. 23 is a
Fluorescence Activated Cell Sorter (FACS) graph of Propidium iodide
and Annexin V of cells treated with Clay alone. FIG. 24 is a
Fluorescence Activated Cell Sorter (FACS) graph of Propidium iodide
and Annexin V of cells treated with Indigo. FIG. 25 is a
Fluorescence Activated Cell Sorter (FACS) graph of Propidium iodide
and Annexin V of cells treated with Mayan Blue at 2.5 uL. FIG. 26
is a Fluorescence Activated Cell Sorter (FACS) graph of Propidium
iodide and Annexin V of cells treated with Mayan Blue at 12.5 uL.
FIG. 27 is a graph that shows the relative effect of various
conditions on cell viability.
[0095] Cell Culture--The human colon cancer cell line HT-29 were
obtained from the American Type Culture Collection. They were
cultured in T-25 flasks with McCoy's 5A Medium supplemented with
10% FBS and 10% PenStrep. The cells were then harvested from the
flask and 100,000 cells were seeded in each well of a 12 well plate
with 1 mL of media.
[0096] Cell Treatment--The purpose of treating the cells was to
determine whether the Mayan Blue pigments had any effect in
changing the morphology of the cells. Indigo and clay were also
tested because the Organic/Inorganic Complex Materials with Maya
Blue is composed of both clay and indigo. After the cells acquired
an elongated morphology, they were treated with 3% Hydrogen
Peroxide (positive control), 20 uL Dimethyl sulfoxide (second
positive control), 20 uL clay (10 mg/mL DMSO), 5 uM, 10 uM, 25 uM,
and 50 uM of indigo (7.6 mM), and 2 uL, 5 uL, 15 uL, 25 uL of
Organic/Inorganic Complex Materials with Maya Blue (slurry was made
10 mg/mL of DMSO).
[0097] Cell Morphology Study--The purpose of looking at the cell
morphology was to determine if there were any signs of apoptosis
within the treated cells, as opposed to the control. After treating
the cells for different time points (4 hours, 12 hours, 24 hours)
they were observed under a light microscope and pictures were
taken.
[0098] Fluorescence-activated cell sorting (FACS Assay)/Flow
Cytometry Assay--The purpose of the Flow Cytometry Assay is not
only to quantify the cells, but also to separate by analyzing the
cell surface, characterizing cell types (apoptotic, necrotic, late
apoptotic, and viable) in cell populations, and analyzing cell
size.
[0099] The HT-29 cells were grown in two 12-well plates at a
concentration of 100,000 cells/mL. They were incubated for 24 hours
with different concentrations of Organic/Inorganic Complex
Materials with Maya Blue or indigo. The supernatants from
individual wells were then taken out and placed in FACS tubes
("flow tubes"). Then 300 uL of trypsin were added to each well and
incubated for 5 minutes. Then detached cells with trypsin were
collected and placed in the respective FACS tubes. The tubes were
then centrifuged at 1400 rpm for 5 minutes at 4.degree. C. to
obtain a pellet. The media/trypsin solution was then discarded. The
pellet was washed twice with 1.times.PBS, centrifugation process
was repeated and the supernatant was discarded. Then 100 uL of
Annexin V Binding Buffer was added to each pellet along with 5 uL
of Annexin-V FITC and 5 uL of propidium iodide. (The purpose of
Annexin-V FITC is to detect the cells that die apoptotically, and
the purpose of propidium iodide is to detect the cells that die
necrotically). Due to the photosensitivity of the Annexin-V FITC
and the propidium iodide, this process must be performed in the
dark.
[0100] Purification of Total DNA from cultured animal cells. The
HT-29 cells were grown in two 6-well plates at a concentration of
1,000,000 cells/mL, they were treated with 3% Hydrogen Peroxide
(positive control), 20 uL dimethyl sulfoxide (second positive
control), 20 uL clay (10 mg/mL DMSO), and 2 uL, 5 uL, 15 uL, 20 uL,
40 uL and 60 uL of Mayan Blue pigments (slurry was made 10 mg/mL of
DMSO) for 24 hours. Treated HT-29 cells were centrifuged in DNeasy
Mini Spin Columns for 5 minutes at 300.times.g. The maximum number
of cells was no greater than 5.times.10.sup.6. Freezing the cells
overnight is optional. The thawed cells were resuspended in 200 uL
of Phosphate Buffered Saline (0.8 g NaCl, 0.2 g KCl, 1.44 g
Na.sub.2HPO.sub.4, 0.24 g KH.sub.2PO.sub.4 in 800 ml of distilled
H.sub.2O). Then 20 uL of Proteinase K and 200 uL of Buffer AL were
added to the sample, making sure not to add Proteinase K directly
to Buffer AL. The solution was vortexed immediately and incubated
at 70.degree. C. for 10 minutes. Following this, 200 uL of ethanol
(96-100%) were added to the sample and vortexed to yield a
homogenous solution. If a white precipitate formed, it was added
with solution. The homogenous solution was pipetted into DNeasy
Mini Spin column and placed in a new 2 mL collection tube. Buffer
AW1 was added (500 uL), then centrifuged for 1 minute at 8000 rpm.
Flow through and collection tubes were discarded. Then 500 uL of
Buffer AW2 were added and centrifuged for 3 minutes at 14000 rpm to
dry DNeasy membrane. Flow through and collection tubes were
discarded. DNeasy Mini Spin column were placed in a clean 1.5 mL/2
mL microcentrifuge tube. Buffer AE (200 uL) were pipetted directly
into the DNeasy membrane and incubated at room temperature for 1
minute. The tubes were then centrifuged for 1 minute at 8000 rpm to
elute.
[0101] Table 1 shows the effect of Organic/Inorganic Complex
Pigment with Mayan Blue on tumors. The listed tumors were injected
into animals and the animals were treated with treated with the
concentrations of the various pigments listed. The percent
inhibition demonstrates the effect of the OICM of the present
invention is listed.
TABLE-US-00001 TABLE 1 Effect of Organic/Inorganic Complex Pigment
with Mayan Blue on Tumors. Tumor Dose No. of Size Inhibition
Statistic Test Tumor Group (mg/kg .times. d) animals X .+-. SD (%)
(ST test) LLC control -- 10 3.5 .+-. 0.44 0 LLC Indirubin 100
.times. 9 10 2.58 .+-. 0.21 26.3 .+-. 2.8 P < 0.05 LLC
Meisoindigo 106 .times. 9 10 1.80 .+-. 0.15 48.6 .+-. 4.1 P <
0.01 Walker control -- 10 9.7 .+-. 1.02 0 256 Walker Indirubin 100
.times. 9 10 3.94 .+-. 0.71 59.4 .+-. 2.9 P < 0.01 256 Walker
Meisoindigo 106 .times. 9 10 2.10 .+-. 0.17 71.6 .+-. 3.1 P <
0.01 256
[0102] It is contemplated that any embodiment discussed in this
specification can be implemented with respect to any method, kit,
reagent, or composition of the invention, and vice versa.
Furthermore, compositions of the invention can be used to achieve
methods of the invention.
[0103] It will be understood that particular embodiments described
herein are shown by way of illustration and not as limitations of
the invention. The principal features of this invention can be
employed in various embodiments without departing from the scope of
the invention. Those skilled in the art will recognize, or be able
to ascertain using no more than routine experimentation, numerous
equivalents to the specific procedures described herein. Such
equivalents are considered to be within the scope of this invention
and are covered by the claims.
[0104] All publications and patent applications mentioned in the
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All publications and
patent applications are herein incorporated by reference to the
same extent as if each individual publication or patent application
was specifically and individually indicated to be incorporated by
reference.
[0105] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one." The use of
the term "or" in the claims is used to mean "and/or" unless
explicitly indicated to refer to alternatives only or the
alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for
the device, the method being employed to determine the value, or
the variation that exists among the study subjects.
[0106] As used in this specification and claim(s), the words
"comprising" (and any form of comprising, such as "comprise" and
"comprises"), "having" (and any form of having, such as "have" and
"has"), "including" (and any form of including, such as "includes"
and "include") or "containing" (and any form of containing, such as
"contains" and "contain") are inclusive or open-ended and do not
exclude additional, unrecited elements or method steps.
[0107] The term "or combinations thereof" as used herein refers to
all permutations and combinations of the listed items preceding the
term. For example, "A, B, C, or combinations thereof" is intended
to include at least one of: A, B, C, AB, AC, BC, or ABC, and if
order is important in a particular context, also BA, CA, CB, CBA,
BCA, ACB, BAC, or CAB. Continuing with this example, expressly
included are combinations that contain repeats of one or more item
or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so
forth. The skilled artisan will understand that typically there is
no limit on the number of items or terms in any combination, unless
otherwise apparent from the context.
[0108] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. All such similar substitutes and modifications apparent
to those skilled in the art are deemed to be within the spirit,
scope and concept of the invention as defined by the appended
claims.
REFERENCES
[0109] www.mexicanmercados.com/produce/mediherb.html [0110]
www.hort.purdue.edu/newcrop/med-aro/factsheets/INDIGO.html
* * * * *
References