U.S. patent application number 10/435319 was filed with the patent office on 2004-04-29 for intratumorally administered lactoferrin in the treatment of malignant neoplasms and other hyperproliferative diseases.
Invention is credited to Barsky, Rick, O'Malley, Bert, Petrak, Karel, Varadhachary, Atul.
Application Number | 20040082504 10/435319 |
Document ID | / |
Family ID | 29424524 |
Filed Date | 2004-04-29 |
United States Patent
Application |
20040082504 |
Kind Code |
A1 |
Varadhachary, Atul ; et
al. |
April 29, 2004 |
Intratumorally administered lactoferrin in the treatment of
malignant neoplasms and other hyperproliferative diseases
Abstract
The present invention relates to methods of treating a
hyperproliferative disease by administering a composition of
lactoferrin alone or in combination with standard anti-cancer
therapies.
Inventors: |
Varadhachary, Atul;
(Houston, TX) ; Barsky, Rick; (Houston, TX)
; Petrak, Karel; (Houston, TX) ; O'Malley,
Bert; (Houston, TX) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
1301 MCKINNEY
SUITE 5100
HOUSTON
TX
77010-3095
US
|
Family ID: |
29424524 |
Appl. No.: |
10/435319 |
Filed: |
May 9, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60379442 |
May 10, 2002 |
|
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60379441 |
May 10, 2002 |
|
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60379474 |
May 10, 2002 |
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Current U.S.
Class: |
435/6.11 ;
514/19.3; 514/2.5 |
Current CPC
Class: |
A61P 15/00 20180101;
A61K 38/40 20130101; A23V 2250/54248 20130101; A61P 1/04 20180101;
A61P 17/00 20180101; A61P 17/06 20180101; A23L 33/19 20160801; A61P
9/10 20180101; A61P 37/04 20180101; A61P 43/00 20180101; A61P 35/02
20180101; A61P 1/02 20180101; A61P 29/00 20180101; A61P 9/00
20180101; A61P 37/00 20180101; A23V 2002/00 20130101; A23V 2002/00
20130101; A61P 19/02 20180101; A61P 35/00 20180101; A61P 11/00
20180101; A61P 35/04 20180101 |
Class at
Publication: |
514/006 |
International
Class: |
A61K 038/40 |
Claims
What is claimed is:
1. A method of treating a hyperproliferative disease comprising the
step of administering intratumorally to a subject a lactoferrin
composition in an amount sufficient to provide an improvement in
the hyperproliferative disease in said subject.
2. The method of claim 1, wherein said lactoferrin composition is
dispersed in a pharmaceutically acceptable carrier.
3. The method of claim 1, wherein said lactoferrin is mammalian
lactoferrin.
4. The method of claim 3, wherein said lactoferrin is human.
5. The method of claim 3, wherein said lactoferrin is bovine.
6. The method of claim 3, wherein said lactoferrin is recombinant
lactoferrin.
7. The method of claim 1, wherein the amount of the lactoferrin
composition that is administered is about 0.1 .mu.g to about 10 g
per day.
8. The method of claim 1, wherein the hyperproliferative disease is
further defined as cancer.
9. The method of claim 8, wherein the cancer comprises a
neoplasm.
10. The method of claim 9, wherein the neoplasm is selected from
the group consisting of melanoma, non-small cell lung, small-cell
lung, lung hepatocarcinoma, retinoblastoma, astrocytoma,
gliobastoma, leukemia, neuroblastoma, squamous cell, head, neck,
gum, tongue, breast, pancreatic, prostate, renal, bone, testicular,
ovarian, mesothelioma, sarcoma, cervical, gastrointestinal,
lymphoma, brain, colon, and bladder.
11. The method of claim 1, wherein the hyperproliferative disease
is selected from the group consisting of rheumatoid arthritis,
inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas,
lipomas, hemangiomas, fibromas, vascular occlusion, restenosis,
atherosclerosis, pre-neoplastic lesions, carcinoma in situ, oral
hairy leukoplakia, and psoriasis.
12. A method of treating a hyperproliferative disease comprising
the step of supplementing a systemic immune system in a subject by
increasing the amount of lactoferrin at the site of the
hyperproliferative disease.
13. The method of claim 12, wherein said lactoferrin is recombinant
lactoferrin.
14. The method of claim 12, wherein said lactoferrin is
administered intratumorally.
15. The method of claim 12, wherein said lactoferrin stimulates the
production of interleukin-18.
16. The method of claim 12, wherein said lactoferrin stimulates the
production of GM-CSF.
17. A method of treating a hyperproliferative disease comprising
the step of supplementing the local immune system in a subject by
increasing the amount of lactoferrin at the site of the
hyperproliferative disease.
18. The method of claim 17, wherein said lactoferrin is recombinant
lactoferrin.
19. The method of claim 17, wherein said lactoferrin is
administered intratumorally.
20. The method of claim 17, wherein said lactoferrin stimulates the
production of interleukin-18.
21. The method of claim 20, wherein interleukin-18 stimulates the
production, maturation or activity of immune cells.
22. The method of claim 17, wherein said lactoferrin stimulates the
production of GM-CSF.
23. A method of enhancing a local immune response in the vicinity
of a tumor following the step of administering intratumorally to
said subject a lactoferrin composition.
24. The method of claim 23, wherein said lactoferrin is recombinant
lactoferrin.
25. The method of claim 23, wherein said lactoferrin composition
stimulates interleukin-18 in the site of injection.
26. The method of claim 23, wherein said lactoferrin composition
stimulates GM-CSF in the site of injection.
27. The method of claim 25, wherein interleukin-18 stimulates the
production, maturation or activity of immune cells.
28. The method of claim 27, wherein the immune cells are T
lymphocytes or natural killer cells.
29. The method of claim 28, wherein the T lymphocytes are selected
from the group consisting of CD4+, CD8+ and CD3+ cells.
30. The method of claim 26, wherein said GM-CSF stimulates the
production, maturation or activity of immune cells.
31. The method of claim 30, wherein the immune cells are dendritic
or other antigen presenting cells.
32. The method of claim 23, wherein said subject suffers from a
hyperproliferative disease.
33. The method of claim 1 further comprising additionally
administering chemotherapy, immunotherapy, surgery, biotherapy,
radiotherapy or a combination thereof.
34. A method of treating a hyperproliferative disease comprising
administering intratumorally to a subject a lactoferrin composition
in combination with chemotherapy, biotherapy, immunotherapy,
surgery or radiotherapy.
Description
[0001] This application claims priority to U.S. Provisional
Application No. 60/379,442 filed on May 10, 2002; U.S. Provisional
Application No. 60/379,441 filed on May 10, 2002 and U.S.
Provisional Application No. 60/379,474 filed on May 10, 2002, which
are incorporated herein by reference in their entirety.
FIELD OF INVENTION
[0002] The present invention relates to methods of treating a
hyperproliferative disease by administering a composition of
lactoferrin alone or in combination with standard anti-cancer
therapies. The lactoferrin composition may be administered orally,
intravenously, intratumorally, or topically.
BACKGROUND OF THE INVENTION
[0003] Currently, there are few effective options for the treatment
of many common cancer types. The course of treatment for a given
individual depends on the diagnosis, the stage to which the disease
has developed, and factors such as age, sex, and general health of
the patient. The most conventional options of cancer treatment are
surgery, radiation therapy, and chemotherapy. Surgery plays a
central role in the diagnosis and treatment of cancer. Typically, a
surgical approach is required for biopsy and the removal of
cancerous growth. However, if the cancer has metastasized and is
widespread, surgery is unlikely to result in a cure, and an
alternate approach must be taken. Side effects of surgery include
diminished structural or organ function and increased risk of
infection, bleeding, or coagulation related complications.
Radiation therapy, chemotherapy, biotherapy and immunotherapy are
alternatives to surgical treatment of cancer (Mayer, 1998; Ohara,
1998; Ho et al., 1998). The disadvantage of many of the alternative
therapies are the side effects, which can include myelosuppression,
skin irritation, difficulty swallowing, dry mouth, nausea,
diarrhea, hair loss, weight loss, and loss of energy (Curran, 1998;
Brizel, 1998).
[0004] Lactoferrin is a single chain metal binding glycoprotein.
Many cells types, such as monocytes, macrophages, lymphocytes, and
intestinal brush-border cells, are known to have lactoferrin
receptors. In addition to lactoferrin being an essential growth
factor for both B and T lymphocytes, lactoferrin has a wide array
of functions related to host primary defense mechanisms. For
example, lactoferrin has been reported to activate natural killer
(NK) cells, induce colony stimulating activity, activate
polymorphonuclear neutrophils (PMN), regulate granulopoeisis,
enhance antibody-dependent cell cytotoxicity, stimulate
lymphokine-activated killer (LAK) cell activity, and potentiate
macrophage toxicity.
[0005] Recently, bovine lactoferrin (bLF) was used as a prophylaxis
for tumor formation and/or established tumors. The present
invention is the first to use lactoferrin as a treatment, not a
prophylaxis, for established tumors.
BRIEF SUMMARY OF THE INVENTION
[0006] The present invention is directed to a method for treating a
hyperproliferative disease. The method of treatment involves
intratumoral administration of lactoferrin.
[0007] An embodiment of the present invention is a method of
treating a hyperproliferative disease comprising the step of
administering intratumorally to a subject a lactoferrin composition
in an amount sufficient to provide an improvement in the
hyperproliferative disease. The amount of the lactoferrin
composition that is administered is about 0.1 .mu.g to about 10
.mu.g per day.
[0008] The lactoferrin composition is dispersed in a
pharmaceutically acceptable carrier. More particularly, the
lactoferrin is mammalian lactoferrin, for example, human or bovine.
In specific embodiments, the lactoferrin is recombinant
lactoferrin.
[0009] The hyperproliferative disease is further defined as cancer,
which comprises a neoplasm. The neoplasm is selected from the group
consisting of melanoma, non-small cell lung, small-cell lung, lung
hepatocarcinoma, retinoblastoma, astrocytoma, gliobastoma,
leukemia, neuroblastoma, squamous cell, head, neck, gum, tongue,
breast, pancreatic, prostate, renal, bone, testicular, ovarian,
mesothelioma, sarcoma, cervical, gastrointestinal, lymphoma, brain,
colon, and bladder.
[0010] Yet further, the hyperproliferative disease is selected from
the group consisting of rheumatoid arthritis, inflammatory bowel
disease, osteoarthritis, leiomyomas, adenomas, lipomas,
hemangiomas, fibromas, vascular occlusion, restenosis,
atherosclerosis, pre-neoplastic lesions, carcinoma in situ, oral
hairy leukoplakia, and psoriasis.
[0011] Another embodiment is a method of treating a
hyperproliferative disease comprising the step of supplementing a
systemic and/or local immune system in a subject by increasing the
amount of lactoferrin in the vicinity of the hyperproliferative
disease. The lactoferrin is administered intratumorally. The
lactoferrin enhances, stimulates' and/or up-regulates
interleukin-18 and Granulocyte Macrophage Colony Stimulating Factor
(GM-CSF). It is envisioned that interleukin-18 stimulates the
production or activity of immune cells, for example T lymphocytes
or natural killer cells and GM-CSF promotes the migration and
maturation of immune cells including dendritic and other antigen
presenting cells.
[0012] Another embodiment is a method of enhancing a local immune
response in the vicinity of a tumor following the step of
administering intratumorally to the subject a lactoferrin
composition. It is envisioned that the lactoferrin composition
stimulates interleukin-18 and/or GM-CSF in the site of injection,
which stimulates the production or activity of immune cells, e.g.,
T lymphocytes or natural killer cells. T lymphocytes are selected
from the group consisting of CD4+, CD8+ and CD3+ cells.
[0013] In specific embodiments, the present invention is drawn to a
method of treating a hyperproliferative disease comprising
administering intratumorally to a subject a lactoferrin composition
in combination with chemotherapy, biotherapy, immunotherapy,
surgery or radiotherapy.
[0014] The foregoing has outlined rather broadly the features and
technical advantages of the present invention in order that the
detailed description of the invention that follows may be better
understood. Additional features and advantages of the invention
will be described hereinafter which form the subject of the claims
of the invention. It should be appreciated by those skilled in the
art that the conception and specific embodiment disclosed may be
readily utilized as a basis for modifying or designing other
structures for carrying out the same purposes of the present
invention. It should also be realized by those skilled in the art
that such equivalent constructions do not depart from the spirit
and scope of the invention as set forth in the appended claims. The
novel features which are believed to be characteristic of the
invention, both as to its organization and method of operation,
together with further objects and advantages will be better
understood from the following description when considered in
connection with the accompanying figures. It is to be expressly
understood, however, that each of the figures is provided for the
purpose of illustration and description only and is not intended as
a definition of the limits of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] For a more complete understanding of the present invention,
reference is now made to the following descriptions taken in
conjunction with the accompanying drawings.
[0016] FIG. 1 shows squamous cell tumor growth with and without
oral, intravenous and intratumoral administration of recombinant
human lactoferrin.
[0017] FIG. 2 shows percent tumor growth inhibition in animals
receiving lactoferrin, cisplatin and lactoferrin in combination
with cisplatin.
[0018] FIG. 3 shows the percent tumor growth inhibition with
lactoferrin in combination with various doses of cisplatin.
[0019] FIG. 4 shows the NK activity after treatment with
lactoferrin.
[0020] FIG. 5 shows squamous cell tumor growth with and without
intratumoral administration of recombinant lactoferrin once or
twice a day.
DETAILED DESCRIPTION OF THE INVENTION
[0021] It is readily apparent to one skilled in the art that
various embodiments and modifications can be made to the invention
disclosed in this Application without departing from the scope and
spirit of the invention.
[0022] As used herein, 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."
[0023] The term "hyperproliferative disease" as used herein refers
to any disease or disorder in which the cells proliferate more
rapidly than normal tissue growth. Thus, a hyperproliferating cell
is a cell that is proliferating more rapidly than normal cells.
[0024] The term "parenteral administration" as used herein includes
any form of administration in which the compound is absorbed into
the subject without involving absorption via the intestines.
Exemplary parenteral administrations that are used in the present
invention include, but are not limited to intramuscular,
intravenous, intraperitoneal, intratumoral, intraocular, or
intraarticular administration.
[0025] The term "intravenous administration" as used herein
includes all techniques to deliver a lactoferrin composition to the
systemic circulation via an intravenous injection or infusion.
[0026] The term "intratumoral administration" as used herein
includes all techniques to deliver a lactoferrin composition to the
site of a tumor including injection, electroporation, creams,
lotions or other forms of administration.
[0027] The term "oral administration" as used herein includes oral,
buccal, enteral or intragastric administration.
[0028] The term "topical administration" as used herein includes
application to a dermal, epidermal, subcutaneous or mucosal
surface.
[0029] The term "pharmaceutically acceptable carrier" as used
herein includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutically active substances is well know in the art. Except
insofar as any conventional media or agent is incompatible with the
vectors or cells of the present invention, its use in therapeutic
compositions is contemplated. Supplementary active ingredients also
can be incorporated into the compositions.
[0030] The term "lactoferrin" or "LF" as used herein refers to
native or recombinant lactoferrin. Native lactoferrin can be
obtained by purification from mammalian milk or colostrum or from
other natural sources. Recombinant lactoferrin (rLF) can be made by
recombinant expression or direct production in genetically altered
animals, plants, fungi, bacteria, or other prokaryotic or
eukaryotic species, or through chemical synthesis.
[0031] The term "subject" as used herein, is taken to mean any
mammalian subject to which the lactoferrin composition is
administered according to the methods described herein. In a
specific embodiment, the methods of the present invention are
employed to treat a human subject. Another embodiment includes
treating a human subject suffering from a hyperproliferative
disease.
[0032] The term "therapeutically effective amount" as used herein
refers to an amount that results in an improvement or remediation
of the symptoms of the disease or condition.
[0033] The term "treating" and "treatment" as used herein refers to
administering to a subject a therapeutically effective amount of a
lactoferrin composition so that the subject has an improvement in
the disease. The improvement is any improvement or remediation of
the symptoms. The improvement is an observable or measurable
improvement. Thus, one of skill in the art realizes that a
treatment may improve the disease condition, but may not be a
complete cure for the disease. Specifically, improvements in
patients with cancer may include tumor stabilization, tumor
shrinkage, increased time to progression, increased survival or
improvements in the quality of life. Beneficial effect may also be
reflected in an improvement of the patient's immune system as
measured by the number and activity of circulating immune cells
such as CD4+ cells, CD8+ cells, NK cells and CD40+ cells.
[0034] The term "vicinity" as used herein refers to in or around
the area or site of the tumor and/or hyperproliferative disease.
For example, "vicinity of a tumor" may refer to the area in or
around the tumor or margins of the tumor. Vicinity includes the
area adjacent to the tumor, the area over the tumor, the area under
the tumor, the margin area around the tumor, or the area adjacent
the tumor margin area.
A. Pharmaceutical Compositions
[0035] The lactoferrin used according to the present invention can
be obtained through isolation and purification from natural
sources, for example, but not limited to mammalian milk. The
lactoferrin is preferably mammalian lactoferrin, such as bovine or
human lactoferrin. In preferred embodiments, the lactoferrin is
human lactoferrin produced recombinantly using genetic engineering
techniques well known and used in the art, such as recombinant
expression or direct production in genetically altered animals,
plants or eukaryotes, or chemical synthesis. See, i.e., U.S. Pat.
Nos. 5,571,896; 5,571,697 and 5,571,691, which are herein
incorporated by reference.
[0036] Administration of the lactoferrin compositions according to
the present invention will be via any common route, orally,
parenterally, or topically. Exemplary routes include, but are not
limited to oral, nasal, buccal, rectal, vaginal, intramuscular,
intraperitoneal, intravenous, intraarterial, intratumoral or
dermal. Such compositions would normally be administered as
pharmaceutically acceptable compositions as described herein.
[0037] The compositions of the present invention may be formulated
in a neutral or salt form. Pharmaceutically-acceptable salts
include the acid addition salts (formed with the free amino groups
of the protein) and which are formed with inorganic acids such as,
for example, hydrochloric or phosphoric acids, or such organic
acids as acetic, oxalic, tartaric, mandelic, and the like. Salts
formed with the free carboxyl groups can also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the
like.
[0038] Sterile injectable solutions are prepared by incorporating
the lactoferrin in the required amount in the appropriate solvent
with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0039] Further in accordance with the present invention, the
inventive composition suitable for oral administration is provided
in a pharmaceutically acceptable carrier with or without an inert
diluent. The carrier should be assimilable or edible and includes
liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar
as any conventional media, agent, diluent or carrier is detrimental
to the recipient or to the therapeutic effectiveness of a
lactoferrin preparation contained therein, its use in an orally
administrable lactoferrin for use in practicing the methods of the
present invention is appropriate. Examples of carriers or diluents
include fats, oils, water, saline solutions, lipids, liposomes,
resins, binders, fillers and the like, or combinations thereof.
[0040] In accordance with the present invention, the composition is
combined with the carrier in any convenient and practical manner,
i.e., by solution, suspension, emulsification, admixture,
encapsulation, microencapsulation, absorption and the like. Such
procedures are routine for those skilled in the art.
[0041] In a specific embodiment of the present invention, the
composition in powder form is combined or mixed thoroughly with a
semi-solid or solid carrier. The mixing can be carried out in any
convenient manner such as grinding. Stabilizing agents can be also
added in the mixing process in order to protect the composition
from loss of therapeutic activity through, i.e., denaturation in
the stomach. Examples of stabilizers for use in an orally
administrable composition include buffers, antagonists to the
secretion of stomach acids, amino acids such as glycine and lysine,
carbohydrates such as dextrose, mannose, galactose, fructose,
lactose, sucrose, maltose, sorbitol, mannitol, etc., proteolytic
enzyme inhibitors, and the like. More preferably, for an orally
administered composition, the stabilizer can also include
antagonists to the secretion of stomach acids.
[0042] Further, the composition for oral administration which is
combined with a semi-solid or solid carrier can be further
formulated into hard or soft shell gelatin capsules, tablets, or
pills. More preferably, gelatin capsules, tablets, or pills are
enterically coated. Enteric coatings prevent denaturation of the
composition in the stomach or upper bowel where the pH is acidic.
See, i.e., U.S. Pat. No. 5,629,001. Upon reaching the small
intestines, the basic pH therein dissolves the coating and permits
the composition to be released and absorbed by specialized cells,
i.e., epithelial enterocytes and Peyer's patch M cells.
[0043] In another embodiment, a powdered composition is combined
with a liquid carrier such as, i.e., water or a saline solution,
with or without a stabilizing agent.
[0044] A specific formulation that may be used in the present
invention is a solution of lactoferrin in a hypotonic phosphate
based buffer that is free of potassium where the composition of the
buffer is as follows: 6 mM sodium phosphate monobasic monohydrate,
9 mM sodium phosphate dibasic heptahydrate, 50 mM sodium chloride,
pH 7.0.+-.0.1. The concentration of lactoferrin in a hypotonic
buffer may range from 10 microgram/ml to 100 milligram/ml. This
formulation may be administered via any route of administration,
for example, but not limited to intratumoral administration.
[0045] Further, a composition for topical administration which is
combined with a semi-solid carrier can be further formulated into a
gel ointment. A preferred carrier for the formation of a gel
ointment is a gel polymer. Preferred polymers that are used to
manufacture a gel composition of the present invention include, but
are not limited to carbopol, carboxymethyl-cellulose, and pluronic
polymers. Specifically, a powdered lactoferrin composition is
combined with an aqueous gel containing an polymerization agent
such as Carbopol 980 at strengths between 0.5% and 5% wt/volume for
application to the skin for treatment of hyperproliferative disease
on or beneath the skin.
[0046] Upon formulation, solutions are administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective to result in an improvement or
remediation of the symptoms. The formulations are easily
administered in a variety of dosage forms such as ingestible
solutions, drug release capsules and the like. Some variation in
dosage can occur depending on the condition of the subject being
treated. The person responsible for administration can, in any
event, determine the appropriate dose for the individual subject.
Moreover, for human administration, preparations meet sterility,
general safety and purity standards as required by FDA Office of
Biologics standards.
B. Treatment of Hyperproliferative Diseases
[0047] In accordance with the present invention, a lactoferrin
composition provided in any of the above-described pharmaceutical
carriers is administered to a subject suspected of or having a
hyperproliferative disease. One of skill in the art can determine
the therapeutically effective amount of human lactoferrin to be
administered to a subject based upon several considerations, such
absorption, metabolism, method of delivery, age, weight, disease
severity and response to the therapy.
[0048] The route of administration will vary, naturally, with the
location and nature of the lesion, and include, for example
intradermal, transdermal, parenteral, intravenous, intramuscular,
intranasal, subcutaneous, percutaneous, intratracheal,
intraperitoneal, intratumoral, perfusion, lavage, direct injection,
and oral administration.
[0049] Oral administration of the lactoferrin composition includes
oral, buccal, enteral or intragastric administration. It is also
envisioned that the composition may be used as a food additive. For
example, the composition is sprinkled on food or added to a liquid
prior to ingestion.
[0050] Intratumoral administration of the lactoferrin composition
includes intratumoral injection, electroporation, or surgical or
endoscopic implantation. Intratumoral injection, or injection into
the tumor vasculature is specifically contemplated for discrete,
solid, accessible tumors. Local, regional or systemic
administration also may be appropriate.
[0051] The hyperproliferative disease, includes but is not limited
to neoplasms. A neoplasm is an abnormal tissue growth, generally
forming a distinct mass that grows by cellular proliferation more
rapidly than normal tissue growth. Neoplasms show partial or total
lack of structural organization and functional coordination with
normal tissue. These can be broadly classified into three major
types. Malignant neoplasms arising from epithelial structures are
called carcinomas, malignant neoplasms that originate from
connective tissues such as muscle, cartilage, fat or bone are
called sarcomas and malignant tumors affecting hematopoietic
structures (structures pertaining to the formation of blood cells)
including components of the immune system, are called leukemias,
lymphomas and myelomas. A tumor is the neoplastic growth of the
disease cancer. As used herein, a "neoplasm", also referred to as a
"tumor", is intended to encompass hematopoietic neoplasms as well
as solid neoplasms. Examples of neoplasms include, but are not
limited to melanoma, non-small cell lung, small-cell lung, lung,
hepatocarcinoma, retinoblastoma, astrocytoma, gliobastoma, gum,
tongue, leukemia, neuroblastoma, head, neck, breast, pancreatic,
prostate, renal, bone, testicular, ovarian, mesothelioma, sarcoma,
cervical, gastrointestinal, lymphoma, brain, colon, bladder,
myeloma, or other malignant or benign neoplasms.
[0052] Other hyperproliferative diseases include, but are not
limited to neurofibromatosis, rheumatoid arthritis, Waginer's
granulomatosis, Kawasaki's disease, lupus erathematosis, midline
granuloma, inflammatory bowel disease, osteoarthritis, leiomyomas,
adenomas, lipomas, hemangiomas, fibromas, vascular occlusion,
restenosis, atherosclerosis, pre-neoplastic lesions, carcinoma in
situ, oral hairy leukoplakia, or psoriasis, and pre-leukemias,
anemia with excess blasts, and myelodysplastic syndrome.
[0053] Particular neoplasms of interest in the present invention
include, but are not limited to hematopoietic neoplasms. For
example, a hematopoietic neoplasm may include acute myelogenous
leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome,
chronic myelomonocytic leukemia, juvenile myelomonocyte leukemia,
multiple myeloma, chronic lymphocytic leukemia or other malignancy
of hematologic origin.
[0054] In a preferred embodiment of the present invention, the
lactoferrin compositions are administered in an effective amount to
decrease, reduce, inhibit or abrogate the growth of a tumor. The
amount may vary from about 0.1 .mu.g to about 100 g of the
lactoferrin composition. Preferably, the lactoferrin composition is
orally administered in the range of 1 mg to 100 g per day, more
preferably about 20 mg to about 10 g per day with the most
preferred dose being 4.5 g per day. Intravenously administered
lactoferrin can be in the range of 0.1 .mu.g to about to 10 g per
day, more preferably about 0.1 .mu.g to about 1 mg with the most
preferred dose being 250 mg per day. Preferably, a lactoferrin
composition is intratumorally administered in the range of 0.1
.mu.g to 10 g per day with the most preferred dose being 100 .mu.g
per day. Topically, the amount of lactoferrin may vary from about 1
.mu.g to about 100 g of lactoferrin. Preferably, the topical gel,
solution, capsule or tablet comprises a lactoferrin concentration
of about 0.01% to about 20%. More preferably, the topical gel,
solution, capsule or tablet may comprise a lactoferrin
concentration of about 1% to about 8.5%.
[0055] Treatment regimens may vary as well, and often depend on
tumor type, tumor location, disease progression, and health and age
of the patient. Obviously, certain types of tumor will require more
aggressive treatment, while at the same time, certain patients
cannot tolerate more taxing protocols. The clinician will be best
suited to make such decisions based on the known efficacy and
toxicity (if any) of the therapeutic formulations.
[0056] In certain embodiments, the tumor being treated may not, at
least initially, be resectable. Treatments with the lactoferrin
composition may increase the resectability of the tumor due to
shrinkage at the margins or by elimination of certain particularly
invasive portions. Following treatments, resection may be possible.
Additional treatments subsequent to resection will serve to
eliminate microscopic residual disease at the tumor site.
[0057] Alternatively, the present invention may be used at the time
of surgery, and/or thereafter, to treat residual or metastatic
disease. For example, a resected tumor bed may be injected or
perfused with a formulation comprising the lactoferrin composition.
The perfusion may be continued post-resection, for example, by
leaving a catheter implanted at the site of the surgery. Periodic
post-surgical treatment is also envisioned.
[0058] Continuous administration also may be applied where
appropriate, for example, where a tumor is excised and the tumor
bed is treated to eliminate residual, microscopic disease. Delivery
via syringe or catherization is preferred. Such continuous
perfusion may take place for a period from about 1-2 hours, to
about 6-12 hours, to about 12-24 hours, to about 1-2 days, to about
1-2 weeks or longer following the initiation of treatment.
Generally, the dose of the therapeutic composition via continuous
perfusion will be equivalent to that given by a single or multiple
injections, adjusted over a period of time during which the
perfusion occurs. It was further contemplated that limb perfusion
may be used to administer therapeutic compositions of the present
invention, particularly in the treatment of melanomas and
sarcomas.
[0059] In specific embodiments, the lactoferrin composition is
given in a single dose or multiple doses. The single dose may be
administered daily, or multiple times a day, or multiple times a
week, or monthly or multiple times a month. In a further
embodiment, the lactoferrin composition is given in a series of
doses. The series of doses may be administered daily, or multiple
times a day, weekly, or multiple times a week, or monthly, or
multiple times a month.
[0060] A further embodiment of the present invention is a method of
treating a hyperproliferative disease comprising the step of
supplementing a mucosal immune system by increasing the amount of
lactoferrin in the gastrointestinal tract. Preferably, the
lactoferrin is administered orally.
[0061] Still yet, a further embodiment is a method of enhancing a
mucosal immune response in the gastrointestinal tract in a subject
comprising the step of administering orally to said subject a
lactoferrin composition, preferably human lactoferrin. It is
envisioned that lactoferrin stimulates interleukin-18 and GM-CSF in
the gastrointestinal tract, which enhance immune cells. For
example, interleukin-18 enhances T lymphocytes or natural killer
cells and GM-CSF promotes maturation and migration of immune cells
including dendritic and other antigen presenting cells. In specific
embodiments, interleukin-18 (IL-18) enhances CD4+, CD8+ and CD3+
cells. It is known by those of skill in the art that IL-18 is a Th1
cytokine that acts in synergy with interleukin-12 and interleukin-2
in the stimulation of lymphocyte IFN-gamma production. Other
cytokines may also be enhanced for example, but not limited to
IL-1b or, IL-12 or IFN-gamma. It is also envisioned that
lactoferrin stimulates interleukin-18 following oral
administration, which inhibits angiogenesis and thereby has
activity against tumor cells which are dependent on
neovascularization.
[0062] A further embodiment of the present invention is a method of
treating a hyperproliferative disease comprising the step of
supplementing the systemic immune system by increasing the amount
of lactoferrin in the systemic circulation. Preferably, the
lactoferrin composition is administered intravenously. It is
envisioned that lactoferrin stimulates interleukin-18 and GM-CSF in
the tissue, which enhance immune cells. For example, interleukin-18
enhances T lymphocytes or natural killer cells and GM-CSF promotes
maturation and migration of immune cells including dendritic and
other antigen presenting cells. In specific embodiments,
interleukin-18 (IL-18) enhances CD4+, CD8+ and CD3+ cells. It is
known by those of skill in the art that IL-18 is a Th1 cytokine
that acts in synergy with interleukin-12 and interleukin-2 in the
stimulation of lymphocyte IFN-gamma production. Other cytokines may
also be enhanced for example, but not limited to IL-1b or, IL-12 or
IFN-gamma. It is also envisioned that lactoferrin stimulates
interleukin- 18 following intravenous administration, which
inhibits angiogenesis and thereby has activity against tumor cells
which are dependent on neovascularization.
[0063] A further embodiment of the present invention is a method of
treating a hyperproliferative disease comprising the step of
supplementing a local or systemic immune system by increasing the
amount of lactoferrin in the vicinity of the tumor. Vicinity of the
tumor refers to the general area of the tumor, for example the
lactoferrin can be administered directly into or on the tumor, or
in the general area of the tumor, but not directly into the tumor.
The general area may include the margin area or near or adjacent
the margin area of the tumor. Preferably, the lactoferrin
composition is administered intratumorally. It is envisioned that
lactoferrin stimulates interleukin-18 and GM-CSF in the local
tissue, which enhances immune cells. For example, interleukin-18
enhances T lymphocytes or natural killer cells and GM-CSF promotes
maturation and migration of immune cells including dendritic and
other antigen presenting cells. In specific embodiments,
interleukin-18 (IL-18) enhances CD4+, CD8+ and CD3+ cells. It is
known by those of skill in the art that IL-18 is a Th1 cytokine
that acts in synergy with interleukin-12 and interleukin-2 in the
stimulation of lymphocyte IFN-gamma production. Other cytokines may
also be enhanced for example, but not limited to IL-1b or, IL12 or
IFN-gamma. It is also envisioned that lactoferrin stimulates
interleukin-18 following intratumoral administration, which
inhibits angiogenesis and thereby has activity against tumor cells
which are dependent on neovascularization.
[0064] A further embodiment of the present invention is a method of
treating a hyperproliferative disease comprising the step of
supplementing a local or systemic immune system by increasing the
amount of lactoferrin in the skin in the vicinity of the tumor.
Preferably, the lactoferrin composition is administered topically.
As above, administration in the vicinity of the tumor includes
administration near or adjacent to the margins of the tumor or
directly in the margin area of the tumor. It is envisioned that
lactoferrin stimulates interleukin-18 and GM-CSF in the local
tissue (e.g., keratinocytes), which enhances immune cells. For
example, interleukin-18 enhances T lymphocytes or natural killer
cells and GM-CSF promotes maturation and migration of immune cells
including dendritic and other antigen presenting cells. In specific
embodiments, interleukin-18 (IL-18) enhances CD4+, CD8+ and CD3+
cells. It is known by those of skill in the art that IL-18 is a Th1
cytokine that acts in synergy with interleukin-12 and interleukin-2
in the stimulation of lymphocyte IFN-gamma production. Other
cytokines may also be enhanced for example, but not limited to
EL-1b or, IL-12 or IFN-gamma. It is also envisioned that
lactoferrin stimulates interleukin-18 following intratumoral
administration, which inhibits angiogenesis and thereby has
activity against tumor cells which are dependent on
neovascularization.
C. Combination Treatments
[0065] In order to increase the effectiveness of the human
lactoferrin composition of the present invention, it may be
desirable to combine the composition of the present invention with
other agents effective in the treatment of hyperproliferative
disease, such as anti-cancer agents, or with surgery. An
"anti-cancer" agent is capable of negatively affecting cancer in a
subject, for example, by killing cancer cells, inducing apoptosis
in cancer cells, reducing the growth rate of cancer cells, reducing
the incidence or number of metastases, reducing tumor size,
inhibiting tumor growth, reducing the blood supply to tumor or
cancer cells, promoting an immune response against cancer cells or
a tumor, preventing or inhibiting the progression of cancer, or
increasing the lifespan of a subject with cancer. Anti-cancer
agents include biological agents (biotherapy), chemotherapy agents,
and radiotherapy agents. More generally, these other compositions
would be provided in a combined amount effective to kill or inhibit
proliferation of the cell. This process may involve administering
the human lactoferrin composition of the present invention and the
agent(s) or multiple factor(s) at the same time. This may be
achieved by administering a single composition or pharmacological
formulation that includes both agents, or by administering two
distinct compositions or formulations, at the same time, or at
times close enough so as to result in an overlap of this effect,
wherein one composition includes the human lactoferrin composition
and the other includes the second agent(s).
[0066] Alternatively, the lactoferrin composition of the present
invention may precede or follow the other anti-cancer agent
treatment by intervals ranging from minutes to weeks. In
embodiments where the other anti-cancer agent and lactoferrin
composition are administered or applied separately to the cell, one
would generally ensure that a significant period of time did not
expire between the time of each delivery, such that the agent and
lactoferrin composition would still be able to exert an
advantageously combined effect on the cell. In such instances, it
is contemplated that one may contact the cell with/administer both
modalities within about 1-14 days of each other and, more
preferably, within about 12-24 hours of each other. In some
situations, it may be desirable to extend the time period for
treatment significantly, however, where several days (2, 3, 4, 5, 6
or 7) to several weeks (2, 3, 4, 5, 6, 7 or 8) lapse between the
respective administrations.
1. Chemotherapy
[0067] Cancer therapies also include a variety of chemical based
treatments. Some examples of chemotherapeutic agents include
without limitation antibiotic chemotherapeutics such as
Doxorubicin, Daunorubicin, Adriamycin, Mitomycin (also known as
mutamycin and/or mitomycin-C), Actinomycin D (Dactinomycin),
Bleomycin, Plicomycin, plant alkaloids such as Taxol, Vincristine,
Vinblastine, miscellaneous agents such as platinum based agents
(e.g., Cisplatin (CDDP)), etoposide (VP16), Tumor Necrosis Factor,
and alkylating agents such as, Carmustine, Melphalan (also known as
alkeran, L-phenylalanine mustard, phenylalanine mustard, L-PAM, or
L-sarcolysin, (a phenylalanine derivative of nitrogen mustard),
Cyclophosphamide, Chlorambucil, Busulfan (also known as myleran),
taxane based agents (e.g., docetaxel) and Lomustine.
[0068] Some examples of other agents include, but are not limited
to, Carboplatin, Procarbazine, Mechlorethamine, Irinotecan,
Topotecan, Ifosfamide, Nitrosurea, Etoposide (VP16), Tamoxifen,
Raloxifene, Toremifene, Idoxifene, Droloxifene, TAT-59,
Zindoxifene, Trioxifene, ICI 182,780, EM-800, Estrogen Receptor
Binding Agents, Gemcitabinen, Navelbine, Farnesyl-protein
transferase inhibitors, Transplatinum, 5-Fluorouracil, hydrogen
peroxide, and Methotrexate, Temazolomide (an aqueous form of DTIC),
Mylotarg, Dolastatin-10, Bryostatin, or any analog or derivative
variant of the foregoing.
2. Radiotherapeutic Agents
[0069] Radiotherapeutic agents and factors include radiation and
waves that induce DNA damage for example, .gamma.-irradiation,
X-rays, UV-irradiation, microwaves, electronic emissions,
radioisotopes, and the like. Therapy may be achieved by irradiating
the localized tumor site with the above described forms of
radiations. It is most likely that all of these factors effect a
broad range of damage to DNA, the precursors of DNA, the
replication and repair of DNA, and the assembly and maintenance of
chromosomes.
[0070] Dosage ranges for X-rays range from daily doses of 50 to 200
roentgens for prolonged periods of time (3 to 4 weeks), to single
doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes
vary widely, and depend on the half-life of the isotope, the
strength and type of radiation emitted, and the uptake by the
neoplastic cells.
3. Surgery
[0071] Approximately 60% of persons with cancer will undergo
surgery of some type, which includes preventative, diagnostic or
staging, curative and palliative surgery. Curative surgery is a
cancer treatment that may be used in conjunction with other
therapies, such as the treatment of the present invention,
chemotherapy, radiotherapy, hormonal therapy, gene therapy,
immunotherapy and/or alternative therapies.
[0072] Curative surgery includes resection in which all or part of
cancerous tissue is physically removed, excised, and/or destroyed.
Tumor resection refers to physical removal of at least part of a
tumor. In addition to tumor resection, treatment by surgery
includes laser surgery, cryosurgery, electrosurgery, and
miscopically controlled surgery (Mohs' surgery). It is further
contemplated that the present invention may be used in conjunction
with removal of superficial cancers, precancers, or incidental
amounts of normal tissue.
[0073] Upon excision of part of all of cancerous cells, tissue, or
tumor, a cavity may be formed in the body. Treatment may be
accomplished by perfusion, direct injection or local application of
the area with an additional anti-cancer therapy. Such treatment may
be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or
every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, or 12 months. These treatments may be of varying dosages as
well.
4. Other Biotherapy Agents
[0074] It is contemplated that other biological agents may be used
in combination with the present invention to improve the
therapeutic efficacy of treatment. These additional agents include,
without limitation, agents that affect the upregulation of cell
surface receptors and GAP junctions, cytostatic and differentiation
agents, inhibitors of cell adhesion, agents that increase the
sensitivity of the hyperproliferative cells to apoptotic inducers,
or other biological agents, as well as biotherapy such as for
example, hyperthermia.
[0075] Hyperthermia is a procedure in which a patient's tissue is
exposed to high temperatures (up to 106.degree. F). External or
internal heating devices may be involved in the application of
local, regional, or whole-body hyperthermia. Local hyperthermia
involves the application of heat to a small area, such as a tumor.
Heat may be generated externally with high-frequency waves
targeting a tumor from a device outside the body. Internal heat may
involve a sterile probe, including thin, heated wires or hollow
tubes filled with warm water, implanted microwave antennae, or
radiofrequency electrodes.
[0076] A patient's organ or a limb is heated for regional therapy,
which is accomplished using devices that produce high energy, such
as magnets. Alternatively, some of the patient's blood may be
removed and heated before being perfused into an area that will be
internally heated. Whole-body heating may also be implemented in
cases where cancer has spread throughout the body. Warm-water
blankets, hot wax, inductive coils, and thermal chambers may be
used for this purpose.
[0077] Hormonal therapy may also be used in conjunction with the
present invention. The use of hormones may be employed in the
treatment of certain cancers such as breast, prostate, ovarian, or
cervical cancer to lower the level or block the effects of certain
hormones such as testosterone or estrogen and this often reduces
the risk of metastases.
[0078] Adjuvant therapy may also be used in conjunction with the
present invention. The use of adjuvants or immunomodulatory agents
include, but are not limited to tumor necrosis factor; interferon
alpha, beta, and gamma; IL-2 and other cytokines; F42K and other
cytokine analogs; or MIP-1, MIP-1beta, MCP-1, RANTES, and other
chemokines.
5. Immunotherapy
[0079] Immunotherapeutics, generally, rely on the use of immune
effector cells and molecules to target and destroy cancer cells.
The immune effector may be, for example, an antibody specific for
some marker on the surface of a tumor cell. The antibody alone may
serve as an effector of therapy or it may recruit other cells to
actually effect cell killing. The antibody also may be conjugated
to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain,
cholera toxin, pertussis toxin, etc.) and serve merely as a
targeting agent. Alternatively, the effector may be a lymphocyte
carrying a surface molecule that interacts, either directly or
indirectly, with a tumor cell target. Various effector cells
include cytotoxic T cells and NK cells.
[0080] It is contemplated that vaccines that are used to treat
cancer may be used in combination with the present invention to
improve the therapeutic efficacy of the treatment. Such vaccines
include peptide vaccines or dendritic cell vaccines. Peptide
vaccines may include any tumor-specific antigen that is recognized
by cytolytic T lymphocytes. Yet further, one skilled in the art
realizes that dendritic cell vaccination comprises dendritic cells
that are pulsed with a peptide or antigen and the pulsed dendritic
cells are administered to the patient.
[0081] Examples of tumor-specific antigens that are being used as
vaccines in melanoma include, but are not limited to gp100 or
MAGE-3. These antigens are being administered as peptide vaccines
and/or as dendritic cell vaccines.
D. EXAMPLES
[0082] The following examples are included to demonstrate preferred
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 in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. 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
Inhibition of Tumor Growth by rhLF
[0083] Human squamous cell carcinoma (O12) was used. The cells were
injected into the right flank of athymic nude mice. rhLF was
administered either intratumorally (49 animals, 7 doses ranging
from 0.05 .mu.g to 125 .mu.g per dose), intravenously (7 animals,
125 ug/dose) or orally (7 animals 20 mg/dose). Control animals were
treated with only the vehicle; no rhLF was administered to the
control animals. rhLF was administered twice a day for either five
days (intravenous group) or eight days (all other groups) starting
11 days after inoculation with tumor cells to allow forrnation of
established tumors.
[0084] The efficacy of treatment was evaluated by measuring the
solid tumor size during and at the end of the experiment; the body
weights were also determined at the time of tumor measurements. As
seen in FIG. 1 and Table 1, treatment with rhLF reduced rates of
tumor growth relative to the control by 46% to 80%. In fact, oral
treatment with 20 mg rhLF most significantly reduced the tumor
growth, by 80% compared to the control (p=0.0073).
1TABLE 1 Summary of tumor growth inhibition by rhLF in O12 tumor
model in mice Inhibition Relative to Placebo P value Day 19* Day
28* Day 19* Day 28* Intratumoral 28% 46% 0.139 0.0263** N = 49
Intravenous 55% 65% 0.0666 0.0233** N = 7 Oral 76% 80% 0.0175**
0.0073*** N = 7 *following start of treatment **statistically
significant (p < 0.05) ***highly statistically significant (p
< 0.01)
[0085] Results from this study showed that rhLF administered by
multiple routes significantly inhibited tumor growth in a squamous
cell tumor model in mice, with oral administration being the most
effective. Based upon these results, it was further contemplated
that oral lactoferrin affects the tumor by enhancing immune cell
activity.
Example 2
Evaluation of rhLF in Tumor Types
[0086] Tumor cells from a broad range of tumor types are injected
into the right flank of athymic nude mice. Animals are administered
either rhLF, native hLF or bovine LF orally. Control animals are
treated with only the vehicle, no rhLF is administered to the
control animals. rhLF is administered either once or twice a day
for either one, five, seven or fourteen days or eight days starting
approximately eleven days after inoculation with tumor cells to
allow formation of established tumors or at such other time as is
generally done with standard or published regimens.
[0087] The efficacy of treatment is evaluated by measuring the
solid tumor size during and at the end of the experiment; the body
weights are also determined at the time of tumor measurements. The
immune response is measured by measuring the amount of cytokines,
T-cells and NK cells in circulation and in the intestine.
Example 3
Effect of Oral Administration of rhLF and bLF
[0088] Recombinant human lactoferrin and bovine lactoferrin were
orally administered to mice, and the production of IL-18 in the
small intestine was measured.
[0089] Mice were treated for three days daily with 65 mg/kg/day of
rhLF, 300 mg/kg/day of rhLF or 300 mg/kg/day of bLF. For a control,
mice were only administered the pharmaceutical carrier. Twenty-four
hours following administration of the LF or control for 3 days,
animals were weighed and blood and serum were collected. Serum was
used for cytokine ELISA assays.
[0090] Also, at these time points, animals were sacrificed and the
small intestinal tissue was removed for further analysis. Small
intestinal epithelium was homogenized using a lysis buffer
consisting of PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and
0.1% sodium dodecyl sulphate containing 10 .mu.g/ml
PhenylMetheylsulfonyl fluoride. Homogenate was centrifuiged at
15,000 rpm for 10 minutes and the supernatant stored at -80 C. till
it was tested for IL-18 levels.
[0091] As seen in Table 2 and Table 3, administration of rhLF at
both doses significantly enhanced the amounts of IL-18 in both the
serum and in the intestinal extract. Bovine LF caused a lesser
increase in the intestinal IL-18 levels and did not increase the
serum levels of IL-18.
2TABLE 2 Effect of rhLF and bLF on IL-18 levels in the gut and
serum Intestinal Extract (pg) Serum (pg) Control 955 141 300 mg/kg
bLF 4,515 134 65 mg/kg rhLF 7,879 259 300 mg/kg rhLF 8,350 328
[0092]
3TABLE 3 Stimulation by rhLF and bLF of IL-18 levels in the gut and
serum Intestinal Extract Serum % Increase P-value % Increase
P-value Increase Over Control 300 mg/kg bLF 373% 0.0086 -5% 0.5411
65 mg/kg rhLF 725% 0.0034 84% 0.0132 300 mg/kg rhLF 775% 0.0001
132% 0.0007 Increase Over Blf 65 mg/kg rhLF 75% 0.1490 94% 0.0366
300 mg/kg rhLF 85% 0.0617 145% 0.0084
Example 4
Effect of Oral rhLF on NK Activity in vivo
[0093] Balb/c naive mice were treated orally with rhLF or placebo
once a day for 3 days (see Table 4).
4TABLE 4 Treatment Regimen Treatment* N Dose (mg/kg) Route Schedule
Group 1 Placebo 6 0 -- -- Group 2 RhLF 7 300 mg/kg/day Oral 3
days
[0094] On day 4, mice were sacrificed and spleens were collected.
NK cells were separated using a magnetic bead cell sorting assay
(MACS anti-NK-DX5) and counted. Cells were then tested in vitro for
NK-activity against YAC targets using a lactate dehydrogenase (LDH)
release test.
[0095] Table 5 shows that oral rhLF treatment resulted in a
significant increase of NK activity ex-vivo against YAC-target
cells (10%@30:1 versus 2.8% of ctrl group). No significant change
in NK activity was observed in placebo treated mice.
5TABLE 5 NK activity in mice treated with oral rhLF Control Low
Medium High 0.056 0.09 0.407 Placebo RhLF-treated Raw data Raw data
E:T % Cyto- E:T % Cytotoxicity* E:T cell E cell toxicity* cell E
cell Increased ratio mix ctrl Final mix ctrl Final over ctrl 30:1
0.281 0.215 2.86 0.358 0.267 9.81 7** 15:1 0.176 0.110 2.85 0.214
0.143 4.12 1.3 7.5:1 0.117 0.054 2.21 0.131 0.074 0.44 0 3.7:1
0.086 0.030 0.19 0.096 0.042 0 0 *% Cytotoxicity =
[(Effector:target cell mix - effectors cell ctrl)] - low
ctrl/[(high ctrl - low ctrl)] .times. 100 **p < 0.05 ((2-tailed
p value)
Example 5
Effect of Oral Administration of rhLF on GM-CSF in vivo
[0096] Recombinant human lactoferrin or placebo were orally
administered to mice, and the production of GM-CSF in the small
intestine was measured.
[0097] Mice (5 animals per group) were treated for three days daily
with 300 mg/kg/day of rhLF. For a control, mice were only
administered the pharmaceutical carrier. Twenty-four hours
following administration of the LF or placebo for 3 days, animals
were and the small intestinal tissue was removed for further
analysis. Small intestinal epithelium was homogenized using a lysis
buffer consisting of PBS, 1% Nonidet P-40, 0.5% sodium
deoxycholate, and 0.1% sodium dodecyl sulphate containing 10
.mu.g/ml PhenylMetheylsulfonyl fluoride. Homogenate was centrifuged
at 15,000 rpm for 10 minutes and the supernatant stored at -80 C.
till it was tested for GM-CSF levels using an ELISA kit.
[0098] As shown in Table 6, treatment with rhLF increased the
production of a key immunostimulatory cytokine, GM-CSF, in the
small intestine relative to the placebo treated animals.
6TABLE 6 Effect of rhLF on GM-CSF levels in the gut and serum Mean
(SEM) in pg Increase over Placebo Placebo 6.48 (0.32) -- 300 mg/kg
rhLF 7.74 (0.19) 19.4% (p < 0.01)
Example 6
Combination of hLF with Chemotherapy
[0099] A murine squamous carcinoma cell line (SCCVII) was injected
into the floor of the mouth through the neck skin of
immunocompetent C3H mice (Day 0). Five days after tumor cell
implantation (Day 5), a skin incision was made in the lower neck
and surgical dissection revealed the established tumors. Tumors
were measured in three dimensions with calipers.
[0100] Tumor-bearing mice were randomized into a control group and
seven groups receiving rhLF and/or cisplatin. RhLF (4 mg; 200
mg/Kg) was administered once daily by oral gavage for 8 days on
days 5 through 12. Cisplatin was administered as a single dose of 5
mg/Kg given intraperitoneally either at the start of rhLF (day 5),
in the middle (day 8) or at the end (day 12) of rhLF therapy.
Animals were sacrificed on day 12 post-implantation and the
residual tumor masses were measured and processed for later
additional analyses.
7TABLE 7 Experimental Groups Description N RhLF mg/kg Cisplatin
Group A Placebo 5 0 (Placebo) 0 Group B RhLF Alone 5 200 mg/kg 0
Group C CP Day 5 5 0 5 mg/kg on day 5* Group D CP Day 8 5 0 5 mg/kg
on day 8* Group E CP Day 12 4 0 5 mg/kg on day 12 Group F RhLF/CP-5
5 200 mg/kg 5 mg/kg on day 5* Group G RhLF/CP-8 5 200 mg/kg 5 mg/kg
on day 8* Group H RhLF/CP-12 5 200 mg/kg 5 mg/kg on day 12*
[0101] Mice treated either with rhLF alone, cisplatin alone or both
agents, showed a tumor growth inhibition (TGI) relative to the
placebo animals. The maximum inhibition was observed in the group
receiving both therapies (Table 7 and FIG. 2).
[0102] In all cases, the animals receiving rhLF+cisplatin showed a
TGI relative to the relevant group receiving cisplatin alone. When
pooled for analysis, animals receiving rhLF+cisplatin showed a 77%
TGI relative to the placebo animals (P<0.0001), a 66% TGI
relative to rhLF alone (P<0.01) and a 63% TGI relative to
cisplatin alone (P<0.01).
[0103] Cisplatin dosing immediately prior to the start of rhLF
(RhLF+CP-5) or during the period of rhLF administration (RhLF+CP-8)
provided greater incremental benefit than when cisplatin was
administered following completion of rhLF therapy (RhLF+CP-12).
However, only the straddling regimen (RhLF+CP-8) provided a
statistically significant improvement (P<0.01) TGI of 77% over
cisplatin alone (CP Day 8).
8TABLE 8 Tumor Growth Inhibition (TGI) by Treatment Group Growth
Relative to Placebo* Group (SEM) TGI (%) P-value A (Placebo) 741
(79) -- -- B (RhLF alone) 496 (155) 33% 0.0989 C (CP Day 5) 240
(137) 68% 0.0066 D (CP Day 8) 693 (146) 6% 0.3898 E (CP Day 12) 433
(175) 42% 0.0634 F (RhLF + CP-5) 14 (5) 98% <0.0001 G (RhLF +
CP-8) 159 (48) 79% 0.0001 H (RhLF + CP-12) 331 (47) 55% 0.0011 C to
E (All CP) 457 (96) 38% 0.0564 F to H (All rhLF/CP) 168 (40) 77%
<0.0001 *Inhibition and 1-tailed P-value relative to the placebo
group ** Inhibition and 1-tailed P-value compared to the respective
Cisplatin groups
Example 7
Dose Dependence of Combining hLF with Chemotherapy
[0104] A murine squamous carcinoma cell line (SCCVII) was injected
into the floor of the mouth through the neck skin of
immunocompetent C3H mice (Day 0) as described in Example 6. On Day
5 days after initial implantation, tumors were measured for the
baseline, then treated with either cisplatin (Day 8, i.p., 5 mg/kg)
alone or cisplatin plus three doses of oral rhLF (daily by gavage
for 7-8 days on days 5 through 11/12). Animals were sacrificed on
Day 11/12 and tumors measured. There was a dose dependent
inhibition of tumor growth in the animals receiving both rhLF and
cisplatin as compared to the animals receiving cisplatin alone as
shown in FIG. 3.
Example 8
Combination of hLF with Docetaxel
[0105] A murine squamous carcinoma cell line (SCCVII) was injected
into the floor of the mouth through the neck skin of
immunocompetent C3H mice (Day 0) as described in Example 7. On Day
5 after initial implantation, tumors were measured for the
baseline, then treated with either oral placebo alone (once daily
from days 5 to 12; 6 animals), placebo and docetaxel (i.v. bolus of
31.3 mg/kg docetaxel on Day 8; 9 animals), or docetaxel plus oral
rhLF (200 mg/kg, administered once daily by gavage from days 5 to
12; 9 animals). Animals were sacrificed on Day 14 and tumors
measured. Docetaxel alone caused an inhibition of tumor growth
relative to placebo and the combination of rhLF and docetaxel
induced a further growth inhibition. Inhibition and p-values
(1-tailed) are shown in Table 9.
9TABLE 9 Tumor Growth Inhibition (TGI) by Treatment Group Growth
Relative to Placebo* Relative to Docetaxel Group (SEM) TGI (%)
P-value TGI (%) P-value Placebo 5,157 (497) -- -- -- -- Docetaxel
2,103 (209) 59% <0.0001 -- -- Docetaxel + 1,288 (286) 75%
<0.0001 39% 0.0175 rhLF
Example 9
Combination of hLF with Radiotherapy
[0106] A murine squamous carcinoma cell line (SCCVI) was injected
into the floor of mouth through the neck skin of immunocompetent
C3H mice (Day 0). Five days after tumor cell implantation (Day 5),
a skin incision was made in the lower neck and surgical dissection
revealed the established tumors. Tumors were measured in three
dimensions with calipers.
[0107] Tumor-bearing mice were randomized into six groups receiving
rhLF (200 mg/Kg) and/or radiotherapy as described below. RhLF (4
mg; 200 mg/kg) was administered by oral gavage once daily for 8
days on days 5 through 12. Radiotherapy was administered as single
dose of 2 Gray given at the begirning (day 5) or at during (day 8)
rhLF-therapy. Animals were sacrificed on day 14 post-treatment and
the residual tumor masses were measured and processed for later
additional analyses.
10TABLE 10 Experimental Groups Description N RhLF mg/kg* Radiation
Group A Placebo 10 0 (Placebo) None Group B RhLF Alone 10 200 mg/kg
None Group C Radiation Day 5 8 0 2 Gray on day 5 Group D RhLF/Rad 5
10 200 mg/Kg 2 Gray on day 5 Group E Radiation Day 8 10 0 2 Gray on
day 8 Group F RhLF/Rad 8 10 200 mg/kg 2 Gray on day 8 *RhLF/placebo
was administered once daily by oral gave from Days 5 to 12.
[0108] Mice receiving rhLF alone, radiotherapy alone, or
combination therapy showed a significant tumor growth inhibition
(TGI) relative to placebo treated mice. The mice receiving both
rhLF and radiation showed a modest increase in TGI over monotherapy
with rhLF (28%, P<0.05) and radiation (15%, P=0.1207).
11TABLE 11 Tumor Growth Inhibition (TGI) by Treatment Group: Growth
Relative to Placebo Group (SEM) Inhibition* P-value* A (Placebo)
2348 (395) -- B (rhLF alone) 1074 (163) 54% 0.0040 C (Radiation Day
5) 827 (105) 65% 0.0021 D (rhLF/Rad 5) 750 (125) 68% 0.0006 E
(Radiation Day 8) 977 (112) 58% 0.0018 F (rhLF/Rad 8) 797 (119) 66%
0.0007 C/E (Both Radiation) 911 (78) 61% <0.0001 D/F (Both
rhLF/Rad) 774 (84) 67% <0.0001 *Inhibition and 1-tailed P-value
compared to the placebo group
[0109] Thus, lactoferrin stimulated the immune system. Still
further, lactoferrin in combination with cisplatin, docetaxel and
radiation resulted in inhibition of tumor growth.
Example 10
Oral Administration of hLF in Humans
[0110] Recombinant human lactoferrin was orally administered to
human patients with a range of metastatic cancer types that had
failed standard chemotherapy in two different studies conducted in
multiple centers in four countries (Argentina, Brazil, Chile, U.S.)
RhLF was administered at doses of 1.5 to 9 grams daily in two
divided doses in cycles of 14 each with a 14 day gap.
[0111] Tumor size progression was monitored through CT scans and
tumor markers where available. CT scans were performed at baseline
and after each 8-week period once treatment was initiated, and also
compared with a pre-baseline scan conducted prior to enrollment in
the study. Tumor markers are measured every 4 weeks. Blood samples
were collected to measure subclasses of circulating lymphocytes and
NK cell activity. Plasma, serum and blood cell extract samples were
collected to measure circulating IL-18, IL-1, IL-2, and IL-4, IL-5,
IL-10, IL-12 and IFN-.gamma..
[0112] Out of nineteen evaluable patients (those with a baseline CT
scan and at least one post-treatment scan), nine patients (47%)
exhibited stable disease by the RECIST criteria at the time of the
first post-treatment scan. Patients with a broad range of tumor
types showed a benefit from lactoferrin administration.
[0113] Table 12 shows the tumor response of five individual
patients with different tumor types. In all cases, the percent
growth of the tumor size prior to treatment of rhLF (the relevant
duration of time is shown in parentheses) and the growth of the
tumor in the ensuing two time periods, as measured by CT, showed a
diminution in their rate of tumor growth or an actual
shrinkage.
12TABLE 12 Tumor Response of Patients Receiving Oral rhLF for
treatment of Metastatic Cancer Pretreatment Post Treatment 1 Post
Treatment 2 % Growth % Growth % Growth Patient# Cancer (Weeks)
(Weeks) (Weeks) # 204 Breast 40% (8) 0% (10) 0% (6.5) # 106
Melanoma 24% (19) -18% (11) Not yet done # 104 Gastric 25% (5.5)
10% (10) -5% (7) # 102 Ovarian 30% (21) -5% (10.5) -7% (8.5) # 007
Lung 160% (5.5) 13% (7.5) 12% (8.5)
Example 11
Combination Therapy with Oral hLF in Humans
[0114] Recombinant human lactoferrin is orally administered to
human patients to inhibit tumor growth either alone or in
combination with standard anti-cancer regimens.
[0115] Briefly, rhLF is administered using the optimum regimen and
doses identified in Example 10 and the standard anti-cancer
regimen(s) for the selected tumor type is used as part of the
combination therapy. The route of administration and regimen of the
additional anti-cancer therapy is as approved by the FDA for that
indication or as described in a peer reviewed publication.
[0116] Tumor size progression is monitored through CT scans and
tumor markers where available. CT scans are performed at baseline
and after each 8-week period once treatment is initiated. Tumor
markers are measured every 4 weeks once treatment is initiated.
Blood samples are collected to measure subclasses of circulating
lymphocytes and NK cell activity. Plasma, serum and blood cell
extract samples are collected to measure circulating IL-18, IL-1,
IL-2, IL-4, IL-5, IL-10, and IL-12 and IFN-.gamma..
Example 12
Immunostimulation Following Administration of Oral rhLF
[0117] Balb/c nave mice were treated orally with rhLF or placebo
once a day for 3 days. One day later (day 4), mice were sacrificed
and spleens collected. NK cells were separated using a magnetic
bead cell sorting assay (MACS anti-NK-DX5) and counted. Cells were
then tested in vitro for NK-activity against YAC targets using a
lactate dehydrogenase (LDH) release test.
[0118] As shown in FIG. 4, oral rhLF treatment resulted in a
significant increase of NK activity ex-vivo against YAC-target
cells. At a 30:1 E:T ratio rhLF administration resulted in a 243%
relative increase over placebo-treated animals (from 2.86% to
9.81%; p<0.05).
Example 13
Effect of Intravenous Administration
[0119] Recombinant lactoferrin, bovine lactoferrin and native
lactoferrin are intravenously administered to animals, preferably
rats, and the production of IL-18, IL-1, IL-2, IL-4, IL-5, IL-10,
IL-12 and IFN-gamma in the plasma, serum and blood packed cells are
measured.
[0120] Briefly, rats are treated for fourteen consecutive days with
0.05 .mu.g to 1000 .mu.g per dose. For a control, rats are only
administered the pharmaceutical carrier. At specific time points
following administration of the LF or control for 0 days, 2 days, 3
days, 5 days, 9 days and 14 days, animals are weighed and blood and
serum are collected. The levels of CD4+, CD8+ and NK cells are
counted from the blood that was collected. Plasma, serum and an
extract of the blood cells are used for cytokine ELISA assays.
[0121] Also, at 24 day time point, animals are sacrificed and
tissues are removed for further analysis. Tissues are homogenized
using a lysis buffer consisting of PBS, 1% Nonidet P-40, 0.5%
sodium deoxycholate, and 0.1% sodium dodecyl sulphate containing 10
.mu.g/ml PhenylMetheylsulfonyl fluoride. Homogenate is centrifuged
at 15,000 rpm for 10 minutes and the supernatant stored at -80 C.
till it is tested for the cytokines IL-18, IL-1, IL-2, IL-4, IL-5,
IL-10, IL-12 and IFN-gamma.
Example 14
Combination Chemotherapy of Intravenously rhLF with other
Agents
[0122] Tumor cells to be tested are injected into the right flank
of athymic nude mice. Animals are administered rhLF intravenously
alone and in combination with other anti-cancer regimens as
described in Example 13. Control animals are treated with only the
vehicle; no rhLF is administered to the control animals. rhLF is
administered using regimens identified as being optimal in the
trials described in Example 13. Anti-cancer therapy is administered
using standard or published regimens. Therapy starts approximately
11 days after inoculation with tumor cells to allow formation of
established tumors or at such other time as is generally done with
standard or published regimens.
[0123] The efficacy of individual and combination treatments are
evaluated by measuring the solid tumor size during and at the end
of the experiment; the body weights are also determined at the time
of tumor measurements.
Example 15
Intravenous Administration of hLF in Humans
[0124] Recombinant lactoferrin is intravenously administered to
patients to inhibit tumor growth.
[0125] Briefly, rhLF at a dose of 500 mg per day for eight days to
patients suffering from unresectable or metastatic cancer.
Alternatively, rhLF is administered for one to eight days to
patients suffering from metastatic cancer in daily doses of 0.1, 1,
10, 100, and 1000 mg. The dose is administered intravenously.
[0126] Tumor size progression is monitored through CT scans and
tumor markers where available. CT scans are performed at baseline
and after each 8-week period once treatment is initiated. Tumor
markers are measured every 4 weeks. Blood samples are collected to
measure subclasses of circulating lymphocytes and NK cell activity.
Plasma, serum and blood cell extract samples are collected to
measure circulating IL-18, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12 and
IFN-.gamma..
Example 16
Combination Therapy with Intravenous hLF
[0127] Recombinant lactoferrin is intravenously administered to
patients to inhibit tumor growth either alone or in combination
with standard anti-cancer regimens.
[0128] Briefly, rhLF is administered using the optimum regimen and
doses identified in Example 15 and the standard anti-cancer
regimen(s) for the selected tumor type is used as part of the
combination therapy. The route of administration and regimen of the
additional anti-cancer therapy is as approved by the FDA for that
indication or as described in a peer reviewed publication.
[0129] Tumor size progression is monitored through CT scans and
tumor markers where available. CT scans are performed at baseline
and after each 8-week period once treatment is initiated. Tumor
markers are measured every 4 weeks once treatment is initiated.
Blood samples are collected to measure subclasses of circulating
lymphocytes and NK cell activity. Plasma, serum and blood cell
extract samples are collected to measure circulating IL-18, IL-1,
IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-.gamma..
Example 17
Activity of Intratumoral rhLF
[0130] O12 human oropharyngeal squamous cell carcinoma tumor cells
were injected to the right flank of athymic nude mice. Recombinant
human lactoferrin and vehicle controls were dosed via intratumoral
injection. Each animal was administered different concentrations of
rhLF in 50 .mu.L doses consisting of four separate injections of
approximately 12.5 .mu.L of the dose, at different directions and
angles (approximately S/N/E/W) to ensure that the dose was
distributed evenly throughout the tumor (fanning).
13TABLE 13 Treatment schedule of intratumor injections of
recombinant human lactoferrin in O12 human squamous carcinoma cell
tumors in nude mice Dose of rhLF per animal in group Group Regimen
0 1 2 3 4 5 E. A Once on day 1, 0 100 .mu.g 250 .mu.g 500 .mu.g 250
na kill 8 days later .mu.g* (nude mice) C Twice/day for 0 25 .mu.g
50 .mu.g 125 .mu.g 250 500 8 days starting .mu.g .mu.g on Day 11
after inoculation, kill on Day 20 (nude mice)
[0131] Table 13 shows the regimen followed for each experimental
group and the dose of rhLF per injection for each animal per group.
In this study, rhLF was administered directly into the tumor. Each
animal was tracked daily for tumor growth by external caliper
measurements of the protruding tumor.
[0132] Using this model, significant reduction of tumor growth was
evident in both rhLF treated groups relative to the control
animals. Compared to the median tumor size for the pooled placebo
samples from groups A and C, the rates of tumor growth in animals
receiving a single dose of rhLF (Group A) were reduced by 50% on
day 11 after the administration of rhLF (p<0.05). The rates of
tumor growth in animals dosed twice daily (Group C) were reduced by
56% when compared to the pooled control group (p<0.01) (See FIG.
5).
Example 18
Immune Stimulation Following Intratumoral rhLF
[0133] Normal C3H/HeJ mice were implanted with one of two mouse
tumors following the methodology described in Example 17. Tumors
used were SCCVII and RIF mouse tumor cell lines. Following
establishment of the tumors in the mice, tumors were injected
intratumorally daily for 4 days with 250 or 500 .mu.g rhLF per dose
or with vehicle control. Twenty four hours following the last
intratumoral injection, animals were sacrificed and the blood
examined for lymphocyte populations. The number of circulating
lymphocytes were increased by 34% to 56% relative to the placebo
treated control animals (Table 14).
14TABLE 14 Increase in circulating lymphocytes following
intratumoral administration of rhLF CD3+ CD4+ CD8+ Number of Cells
Placebo 2104 1800 785 rhLF treated 3291 2621 1054 Increase with
rhLF 56% 46% 34%
Example 19
Combination Chemotherapy of hLF with other Agents
[0134] Tumor cells to be tested are injected into the right flank
of athymic nude mice. Animals are administered rhLF intratumorally
alone and in combination with other anti-cancer regimens as
described in Example 1 or Example 17. Control animals are treated
with only the vehicle; no rhLF is administered to the control
animals. Anti-cancer therapy is administered using standard or
published regimens. Therapy starts approximately 11 days after
inoculation with tumor cells to allow formation of established
tumors or at such other time as is generally done with standard or
published regimens.
[0135] The efficacy of individual and combination treatments are
evaluated by measuring the solid tumor size during and at the end
of the experiment; the body weights are also determined at the time
of tumor measurements.
Example 20
Intratumoral Administration of hLF
[0136] Recombinant lactoferrin is intratumorally administered to
patients to inhibit tumor growth.
[0137] Briefly, rhLF at a dose of 1000 .mu.g per day for eight days
to patients suffering from unresectable or metastatic cancer.
Alternatively, rhLF is administered for one to eight days to
patients suffering from metastatic cancer in daily doses of 10, 50,
100, 500 and 1000 .mu.g. The dose is administered
intratumorally.
[0138] Tumor size progression is monitored through CT scans and
tumor markers where available. CT scans are performed at baseline
and after each 8-week period once treatment is initiated. Tumor
markers are measured every 4 weeks. Blood samples are collected to
measure subclasses of circulating lymphocytes and NK cell activity.
Plasma, serum and blood cell extract samples are collected to
measure circulating IL-18, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12 and
IFN-.gamma..
Example 21
Combination Therapy with Intratumoral hLF
[0139] Recombinant lactoferrin is intratumorally administered to
patients to inhibit tumor growth either alone or in combination
with standard anti-cancer regimens.
[0140] Briefly, rhLF is administered using the optimum regimen and
doses identified in Example 20 and the standard anti-cancer
regimen(s) for the selected tumor type is used as part of the
combination therapy. The route of administration and regimen of the
additional anti-cancer therapy is as approved by the FDA for that
indication or as described in a peer reviewed publication.
[0141] Tumor size progression is monitored through CT scans and
tumor markers where available. CT scans are performed at baseline
and after each 8-week period once treatment is initiated. Tumor
markers are measured every 4 weeks once treatment is initiated.
Blood samples are collected to measure subclasses of circulating
lymphocytes and NK cell activity. Plasma, serum and blood cell
extract samples are collected to measure circulating IL-18, IL-1,
IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-.gamma..
Example 22
Topical Administration of hLF in Humans
[0142] Recombinant lactoferrin in a gel formulation is administered
to patients to inhibit tumor growth.
[0143] Briefly, rhLF gel at strengths of 1%, 2.5% or 8.5% is
applied twice a day to a skin or subcutaneous cancerous lesion in a
patient with metastatic disease. Application of rhLF gel continues
till tumor progression.
[0144] Size progression of the metastatic disease is monitored
through CT scans and tumor markers where available. CT scans are
performed at baseline and after each 8-week period once treatment
is initiated. Tumor markers are measured every 4 weeks. Blood
samples are collected to measure subclasses of circulating
lymphocytes and NK cell activity. Plasma, serum and blood cell
extract samples are collected to measure circulating IL-18, IL-1,
IL-2, IL-4, IL-5, IL-10, IL-12 and IFN-.gamma..
Example 23
Combination Therapy with Topical hLF
[0145] Recombinant lactoferrin in a gel formulation is administered
to patients to inhibit tumor growth either alone or in combination
with standard anti-cancer regimens.
[0146] Briefly, rhLF is administered using the optimum regimen and
doses identified in Examples 22 and the standard anti-cancer
regimen(s) for the selected tumor type is used as part of the
combination therapy. The route of administration and regimen of the
additional anti-cancer therapy is as approved by the FDA for that
indication or as described in a peer reviewed publication.
[0147] Size progression of the metastatic disease is monitored
through CT scans and tumor markers where available. CT scans are
performed at baseline and after each 8-week period once treatment
is initiated. Tumor markers are measured every 4 weeks once
treatment is initiated. Blood samples are collected to measure
subclasses of circulating lymphocytes and NK cell activity. Plasma,
serum and blood cell extract samples are collected to measure
circulating IL-18, IL-1, IL-2, IL-4, IL-5, IL-10, IL-12 and
IFN-.gamma..
References Cited
[0148] All patents and publications mentioned in the specifications
are indicative of the levels of those skilled in the art to which
the invention pertains. All patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
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[0178] Although the present invention and its advantages have been
described in detail, it should be understood that various changes,
substitutions and alterations can be made herein without departing
from the spirit and scope of the invention as defined by the
appended description. Moreover, the scope of the present
application is not intended to be limited to the particular
embodiments of the process, machine, manufacture, composition of
matter, means, methods and steps described in the specification. As
one of ordinary skill in the art will readily appreciate from the
disclosure of the present invention, processes, machines,
manufacture, compositions of matter, means, methods, or steps,
presently existing or later to be developed that perform
substantially the same function or achieve substantially the same
result as the corresponding embodiments described herein may be
utilized according to the present invention. Accordingly, the
appended descriptions are intended to include within their scope
such processes, machines, manufacture, compositions of matter,
means, methods, or steps.
* * * * *